@article {1562906, title = {Enhanced Protein-Protein Interaction Discovery via AlphaFold-Multimer}, journal = {bioRxiv}, year = {2024}, month = {2024/01/01}, pages = {2024.02.19.580970}, abstract = {Accurately mapping protein-protein interactions (PPIs) is critical for elucidating cellular functions and has significant implications for health and disease. Conventional experimental approaches, while foundational, often fall short in capturing direct, dynamic interactions, especially those with transient or small interfaces. Our study leverages AlphaFold-Multimer (AFM) to re-evaluate high-confidence PPI datasets from Drosophila and human. Our analysis uncovers a significant limitation of the AFM-derived interface pTM (ipTM) metric, which, while reflective of structural integrity, can miss physiologically relevant interactions at small interfaces or within flexible regions. To bridge this gap, we introduce the Local Interaction Score (LIS), derived from AFM\&$\#$039;s Predicted Aligned Error (PAE), focusing on areas with low PAE values, indicative of the high confidence in interaction predictions. The LIS method demonstrates enhanced sensitivity in detecting PPIs, particularly among those that involve flexible and small interfaces. By applying LIS to large-scale Drosophila datasets, we enhance the detection of direct interactions. Moreover, we present FlyPredictome, an online platform that integrates our AFM-based predictions with additional information such as gene expression correlations and subcellular localization predictions. This study not only improves upon AFM\&$\#$039;s utility in PPI prediction but also highlights the potential of computational methods to complement and enhance experimental approaches in the identification of PPI networks.Competing Interest StatementThe authors have declared no competing interest.}, url = {http://biorxiv.org/content/early/2024/02/21/2024.02.19.580970.abstract}, author = {Ah-Ram Kim and Yanhui Hu and Aram Comjean and Jonathan Rodiger and Stephanie E Mohr and Norbert Perrimon} } @article {1544076, title = {Finding information about uncharacterized Drosophila melanogaster genes}, journal = {Genetics}, year = {2023}, abstract = {

Genes that have been identified in the genome but remain uncharacterized with regards to function offer an opportunity to uncover novel biological information. Novelty is exciting but can also be a barrier. If nothing is known, how does one start planning and executing experiments? Here, we provide a recommended information-mining workflow and a corresponding guide to accessing information about uncharacterized Drosophila melanogaster genes, such as those assigned only a systematic coding gene identifier. The available information can provide insights into where and when the gene is expressed, what the function of the gene might be, whether there are similar genes in other species, whether there are known relationships to other genes, and whether any other features have already been determined. In addition, available information about relevant reagents can inspire and facilitate experimental studies. Altogether, mining available information can help prioritize genes for further study, as well as provide starting points for experimental assays and other analyses.

}, author = {Mohr, Stephanie E and Kim, Ah-Ram and Hu, Yanhui and Perrimon, Norbert} } @article {1548676, title = {Genetic manipulation of an Ixodes scapularis cell line}, journal = {bioRxiv}, year = {2023}, month = {2023/01/01}, pages = {2023.09.08.556855}, abstract = {Although genetic manipulation is one of the hallmarks in model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the blacklegged tick Ixodes scapularis, an arthropod that serves as a vector of several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology dependent recombination. Targeting the E3 ubiquitin ligase X-linked inhibitor of apoptosis (xiap) and its substrate p47 led to alteration in molecular signaling within the immune deficiency (IMD) network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for genetic engineering of ticks in vivo and aid in circumventing the long-life cycle of I. scapularis, of which limits efficient and scalable molecular genetic screens.Importance Genetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of I. scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent A. phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the blacklegged tick Ixodes scapularis.Competing Interest StatementThe authors have declared no competing interest.}, url = {http://biorxiv.org/content/early/2023/09/10/2023.09.08.556855.abstract}, author = {Nisha Singh and Agustin Rolandelli and Anya J. O{\textquoteright}Neal and L. Rainer Butler and Sourabh Samaddar and Hanna J. Laukaitis-Yousey and Matthew Butnaru and Stephanie E. Mohr and Norbert Perrimon and Joao H. F. Pedra} } @article {1517996, title = {Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for research}, journal = {Elife}, volume = {12}, year = {2023}, month = {2023 Jul 20}, abstract = {

Expression of activated Ras, Ras, provides cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted Ras expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad Ras expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Genomic Resource Center.

}, issn = {2050-084X}, doi = {10.7554/eLife.85814}, author = {Coleman-Gosser, Nikki and Hu, Yanhui and Raghuvanshi, Shiva and Stitzinger, Shane and Chen, Weihang and Luhur, Arthur and Mariyappa, Daniel and Josifov, Molly and Zelhof, Andrew and Mohr, Stephanie E and Perrimon, Norbert and Simcox, Amanda} } @article {1518016, title = {Next-generation large-scale binary protein interaction network for Drosophila melanogaster}, volume = {14}, year = {2023}, month = {2023/04/15}, pages = {2162}, abstract = {Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of \> 10,000 Drosophila proteins result in the {\textquoteleft}FlyBi{\textquoteright} dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.}, isbn = {2041-1723}, url = {https://doi.org/10.1038/s41467-023-37876-0}, author = {Tang, Hong-Wen and Spirohn, Kerstin and Hu, Yanhui and Hao, Tong and Kov{\'a}cs, Istv{\'a}n A. and Gao, Yue and Binari, Richard and Yang-Zhou, Donghui and Wan, Kenneth H. and Bader, Joel S. and Balcha, Dawit and Bian, Wenting and Booth, Benjamin W. and Cot{\'e}, Atina G. and de Rouck, Steffi and Desbuleux, Alice and Goh, Kah Yong and Kim, Dae-Kyum and Knapp, Jennifer J. and Lee, Wen Xing and Lemmens, Irma and Li, Cathleen and Li, Mian and Li, Roujia and Lim, Hyobin Julianne and Liu, Yifang and Luck, Katja and Markey, Dylan and Pollis, Carl and Rangarajan, Sudharshan and Rodiger, Jonathan and Schlabach, Sadie and Shen, Yun and Sheykhkarimli, Dayag and TeeKing, Bridget and Roth, Frederick P. and Tavernier, Jan and Calderwood, Michael A. and Hill, David E. and Celniker, Susan E. and Vidal, Marc and Perrimon, Norbert and Mohr, Stephanie E.} } @article {1498791, title = {Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in cells}, journal = {Elife}, volume = {12}, year = {2023}, month = {2023 Apr 20}, abstract = {

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (), a member of the lipocalin protein family (), and a zinc finger C2H2 transcription factor (). Mechanistically, we demonstrate that overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in cells.

}, keywords = {Animals, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Drosophila, genome, Sirolimus}, issn = {2050-084X}, doi = {10.7554/eLife.85542}, author = {Xia, Baolong and Viswanatha, Raghuvir and Hu, Yanhui and Mohr, Stephanie E and Perrimon, Norbert} } @article {1548671, title = {Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness}, journal = {bioRxiv}, year = {2023}, month = {2023/01/01}, pages = {2023.08.31.555785}, abstract = {Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.Competing Interest StatementThe authors have declared no competing interest.}, url = {http://biorxiv.org/content/early/2023/09/03/2023.08.31.555785.abstract}, author = {Agustin Rolandelli and Hanna J. Laukaitis-Yousey and Haikel N. Bogale and Nisha Singh and Sourabh Samaddar and Anya J. O{\textquoteright}Neal and Camila R. Ferraz and Matthew Butnaru and Enzo Mameli and Baolong Xia and M. Tays Mendes and L. Rainer Butler and Liron Marnin and Francy E. Cabrera Paz and Luisa M. Valencia and Vipin S. Rana and Ciaran Skerry and Utpal Pal and Stephanie E. Mohr and Norbert Perrimon and David Serre and Joao H.F. Pedra} } @article {1448324, title = {NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization}, journal = {Nucleic Acids Res}, volume = {50}, year = {2022}, month = {2022 08 12}, pages = {7906-7924}, abstract = {Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.}, keywords = {Animals, Chromatin, Chromosomal Proteins, Non-Histone, DNA, Drosophila, drosophila melanogaster, Drosophila Proteins, Insulator Elements, Microtubule-Associated Proteins, Nuclear Proteins, Nucleosomes}, issn = {1362-4962}, doi = {10.1093/nar/gkac600}, author = {Chen, Shue and Rosin, Leah F and Pegoraro, Gianluca and Moshkovich, Nellie and Murphy, Patrick J and Yu, Guoyun and Lei, Elissa P} } @article {1442944, title = {FlyPhoneDB: an integrated web-based resource for cell-cell communication prediction in Drosophila}, journal = {Genetics}, volume = {220}, year = {2022}, month = {2022 03 03}, abstract = {Multicellular organisms rely on cell-cell communication to exchange information necessary for developmental processes and metabolic homeostasis. Cell-cell communication pathways can be inferred from transcriptomic datasets based on ligand-receptor expression. Recently, data generated from single-cell RNA sequencing have enabled ligand-receptor interaction predictions at an unprecedented resolution. While computational methods are available to infer cell-cell communication in vertebrates such a tool does not yet exist for Drosophila. Here, we generated a high-confidence list of ligand-receptor pairs for the major fly signaling pathways and developed FlyPhoneDB, a quantification algorithm that calculates interaction scores to predict ligand-receptor interactions between cells. At the FlyPhoneDB user interface, results are presented in a variety of tabular and graphical formats to facilitate biological interpretation. To illustrate that FlyPhoneDB can effectively identify active ligands and receptors to uncover cell-cell communication events, we applied FlyPhoneDB to Drosophila single-cell RNA sequencing data sets from adult midgut, abdomen, and blood, and demonstrate that FlyPhoneDB can readily identify previously characterized cell-cell communication pathways. Altogether, FlyPhoneDB is an easy-to-use framework that can be used to predict cell-cell communication between cell types from single-cell RNA sequencing data in Drosophila.}, keywords = {Animals, Cell Communication, Drosophila, Internet, Ligands, Sequence Analysis, RNA, Single-Cell Analysis, Transcriptome}, issn = {1943-2631}, doi = {10.1093/genetics/iyab235}, author = {Liu, Yifang and Li, Joshua Shing Shun and Rodiger, Jonathan and Comjean, Aram and Attrill, Helen and Antonazzo, Giulia and Brown, Nicholas H and Hu, Yanhui and Perrimon, Norbert} } @article {1467176, title = {NanoTag Nanobody Tools for Drosophila In Vitro and In Vivo Studies}, journal = {Curr Protoc}, volume = {2}, year = {2022}, month = {2022 Dec}, pages = {e628}, abstract = {

Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. {\textcopyright} 2022 Wiley Periodicals LLC. Basic Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression and purification in bacterial cells Basic Protocol 3: Immunostaining with nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a method to alter protein localization Support Protocol: CRISPR-mediated tagging of endogenous genes with NanoTags.

}, keywords = {Animals, Drosophila, Protein Binding, Protein Transport, Single-Domain Antibodies}, issn = {2691-1299}, doi = {10.1002/cpz1.628}, author = {Kim, Ah-Ram and Xu, Jun and Cheloha, Ross and Mohr, Stephanie E and Zirin, Jonathan and Ploegh, Hidde L and Perrimon, Norbert} } @article {1447492, title = {CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor}, journal = {Nature}, volume = {610}, year = {2022}, month = {2022 Oct}, pages = {349-355}, abstract = {Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.}, keywords = {Animals, Bacterial Toxins, Biological Control Agents, Drosophila, drosophila melanogaster, Humans, Mucins, Photorhabdus, Proline, Serine, threonine, Virulence Factors}, issn = {1476-4687}, doi = {10.1038/s41586-022-05250-7}, author = {Xu, Ying and Viswanatha, Raghuvir and Sitsel, Oleg and Roderer, Daniel and Zhao, Haifang and Ashwood, Christopher and Voelcker, Cecilia and Tian, Songhai and Raunser, Stefan and Perrimon, Norbert and Dong, Min} } @article {1447493, title = {A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress}, journal = {PLoS Genet}, volume = {18}, year = {2022}, month = {2022 Sep}, pages = {e1010430}, abstract = {Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually causes CDGs. While both in vivo models ostensibly cause cellular stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.}, keywords = {Clustered Regularly Interspaced Short Palindromic Repeats, Congenital Disorders of Glycosylation, Fructose, genome, glycoproteins, Humans, Mannosyltransferases, N-Acetylglucosaminyltransferases}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1010430}, author = {Dalton, Hans M and Viswanatha, Raghuvir and Brathwaite, Roderick and Zuno, Jae Sophia and Berman, Alexys R and Rushforth, Rebekah and Mohr, Stephanie E and Perrimon, Norbert and Chow, Clement Y} } @article {1443472, title = {Identification of two pathways mediating protein targeting from ER to lipid droplets}, journal = {Nature Cell Biol}, year = {2022}, month = {2022}, abstract = {Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD{\textendash}ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.}, isbn = {1476-4679}, url = {https://doi.org/10.1038/s41556-022-00974-0}, author = {Jiunn Song and Arda Mizrak and Chia-Wei Lee and Marcelo Cicconet and Zon Weng Lai and Tang, Wei-Chun and Chieh-Han Lu and Mohr, Stephanie E. and Farese, Robert V. and Tobias C. Walther} } @article {1447591, title = {Modeling exercise using optogenetically contractible Drosophila larvae}, journal = {BMC Genomics}, volume = {23}, year = {2022}, month = {2022 Aug 30}, pages = {623}, abstract = {The pathophysiological effects of a number of metabolic and age-related disorders can be prevented to some extent by exercise and increased physical activity. However, the molecular mechanisms that contribute to the beneficial effects of muscle activity remain poorly explored. Availability of a fast, inexpensive, and genetically tractable model system for muscle activity and exercise will allow the rapid identification and characterization of molecular mechanisms that mediate the beneficial effects of exercise. Here, we report the development and characterization of an optogenetically-inducible muscle contraction (OMC) model in Drosophila larvae that we used to study acute exercise-like physiological responses. To characterize muscle-specific transcriptional responses to acute exercise, we performed bulk mRNA-sequencing, revealing striking similarities between acute exercise-induced genes in flies and those previously identified in humans. Our larval muscle contraction model opens a path for rapid identification and characterization of exercise-induced factors.}, keywords = {Animals, Drosophila, Humans, Larva, Muscle Contraction, Muscle, Skeletal, Physical Conditioning, Animal}, issn = {1471-2164}, doi = {10.1186/s12864-022-08845-6}, author = {Ghosh, Arpan C and Hu, Yanhui and Tattikota, Sudhir Gopal and Liu, Yifang and Comjean, Aram and Perrimon, Norbert} } @article {https://doi.org/10.1002/cpz1.628, title = {NanoTag Nanobody Tools for Drosophila In Vitro and In Vivo Studies}, journal = {Current Protocols}, volume = {2}, number = {12}, year = {2022}, pages = {e628}, abstract = {Abstract Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. {\textcopyright} 2022 Wiley Periodicals LLC. Basic Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression and purification in bacterial cells Basic Protocol 3: Immunostaining with nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a method to alter protein localization Support Protocol: CRISPR-mediated tagging of endogenous genes with NanoTags}, keywords = {Crispr, Drosophila, knock-in, nanobody, NanoTag}, doi = {https://doi.org/10.1002/cpz1.628}, url = {https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/cpz1.628}, author = {Kim, Ah-Ram and Xu, Jun and Cheloha, Ross and Mohr, Stephanie E. and Zirin, Jonathan and Ploegh, Hidde L. and Perrimon, Norbert} } @article {1447664, title = {Optimal transport analysis of single-cell transcriptomics directs hypotheses prioritization and validation}, journal = {bioRxiv}, year = {2022}, month = {2022/01/01}, pages = {2022.06.27.497786}, abstract = {The explosive growth of regulatory hypotheses from single-cell datasets demands accurate prioritization of hypotheses for in vivo validation, but current computational methods fail to shortlist a high-confidence subset that can be feasibly tested. We present Haystack, an algorithm that combines active learning and optimal transport theory to identify and prioritize transient but causally-active transcription factors in cell lineages. We apply Haystack to single-cell observations, guiding efficient and cost-effective in vivo validations that reveal causal mechanisms of cell differentiation in Drosophila gut and blood lineages.Competing Interest StatementThe authors have declared no competing interest.}, url = {http://biorxiv.org/content/early/2022/09/19/2022.06.27.497786.abstract}, author = {Singh, Rohit and Li, Joshua Shing Shun and Tattikota, Sudhir Gopal and Liu, Yifang and Xu, Jun and Hu, Yanhui and Perrimon, Norbert and Berger, Bonnie} } @inbook {1442045, title = {Prime Editing for Precise Genome Engineering in Drosophila}, booktitle = {Drosophila: Methods and Protocols}, year = {2022}, month = {2022}, pages = {113 - 134}, publisher = {Springer US}, organization = {Springer US}, address = {New York, NY}, abstract = {Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3\ months.}, isbn = {978-1-0716-2541-5}, url = {https://doi.org/10.1007/978-1-0716-2541-5_5}, author = {Bosch, Justin A. and Perrimon, Norbert}, editor = {Dahmann, Christian} } @article {1427734, title = {Protein visualization and manipulation in through the use of epitope tags recognized by nanobodies}, journal = {Elife}, volume = {11}, year = {2022}, month = {2022 Jan 25}, abstract = {Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (\<15 aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.}, issn = {2050-084X}, doi = {10.7554/eLife.74326}, author = {Xu, Jun and Kim, Ah-Ram and Cheloha, Ross W and Fischer, Fabian A and Li, Joshua Shing Shun and Feng, Yuan and Stoneburner, Emily and Binari, Richard and Mohr, Stephanie E and Zirin, Jonathan and Ploegh, Hidde L and Perrimon, Norbert} } @article {1442945, title = {State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila}, journal = {Trends Genet}, volume = {38}, year = {2022}, month = {2022 05}, pages = {437-453}, abstract = {For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered\ regularly\ interspaced\ short\ palindromic\ repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.}, keywords = {Animals, CRISPR-Cas Systems, Drosophila, drosophila melanogaster, Gene Editing, Mutagenesis, Recombinational DNA Repair}, issn = {0168-9525}, doi = {10.1016/j.tig.2021.11.006}, author = {Zirin, Jonathan and Bosch, Justin and Viswanatha, Raghuvir and Mohr, Stephanie E and Perrimon, Norbert} } @article {1423083, title = {A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress}, journal = {BioRxiv}, year = {2021}, abstract = {Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1 CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.}, url = {https://www.biorxiv.org/content/10.1101/2021.12.03.471178v1}, author = {Dalton, Hans M. and Viswanatha, Raghuvir and Brathwaite Jr., Ricky and Zuno, Jae Sophia and Mohr, Stephanie E and Perrimon, Norbert and Chow, Clement Y.} } @article {1422874, title = {Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos}, journal = {Nat Commun}, volume = {12}, year = {2021}, month = {2021 Nov 24}, pages = {6825}, abstract = {Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.}, issn = {2041-1723}, doi = {10.1038/s41467-021-27129-3}, author = {Viswanatha, Raghuvir and Mameli, Enzo and Rodiger, Jonathan and Merckaert, Pierre and Feitosa-Suntheimer, Fabiana and Colpitts, Tonya M and Mohr, Stephanie E and Hu, Yanhui and Perrimon, Norbert} } @article {1398205, title = {Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies}, journal = {bioRxiv}, year = {2021}, month = {2021-01-01 00:00}, abstract = {Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags installed on proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we combine the advantages of short epitopes (NanoTags) and nanobodies specific for them by characterizing two short (\<15 aa) tags, 127D01 and VHH05, which are high-affinity targets of nanobodies. We demonstrate that these NanoTags and the nanobodies that recognize them can be used in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.Competing Interest StatementThe authors have declared no competing interest.}, author = {Xu, Jun and Kim, Ah-Ram and Cheloha, Ross W. and Fischer, Fabian A. and Li, Joshua Shing Shun and Feng, Yuan and Stoneburner, Emily and Binari, Richard and Mohr, Stephanie E. and Zirin, Jonathan and Ploegh, Hidde and Perrimon, Norbert} } @article {1415897, title = {Identification of two pathways mediating protein targeting from ER to lipid droplets [NOTE: a modified final version was published in Nat Cell Biol and is now available]}, year = {2021}, abstract = {Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER.}, url = {https://www.biorxiv.org/content/10.1101/2021.09.14.460330v1}, author = {Jiunn Song and Arda Mizrak and Chia-Wei Lee and Marcelo Cicconet and Zon Weng Lai and Chieh-Han Lu and Mohr, Stephanie E. and Robert V. Farese, Jr and Tobias C. Walther} } @article {Viswanatha2021.03.29.437496, title = {Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos [NOTE: A modified final version was published in Nat Comm and is now available.]}, journal = {bioRxiv}, year = {2021}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.Competing Interest StatementThe authors have declared no competing interest.}, doi = {10.1101/2021.03.29.437496}, url = {https://www.biorxiv.org/content/early/2021/03/30/2021.03.29.437496}, author = {Viswanatha, Raghuvir and Mameli, Enzo and Rodiger, Jonathan and Merckaert, Pierre and Feitosa-Suntheimer, Fabiana and Colpitts, Tonya M. and Mohr, Stephanie E. and Hu, Yanhui and Perrimon, Norbert} } @article {1401910, title = {DRscDB: A single-cell RNA-seq resource for data mining and data comparison across species}, journal = {Comput Struct Biotechnol J}, volume = {19}, year = {2021}, month = {2021}, pages = {2018-2026}, abstract = {With the advent of single-cell RNA sequencing (scRNA-seq) technologies, there has been a spike in studies involving scRNA-seq of several tissues across diverse species including Drosophila. Although a few databases exist for users to query genes of interest within the scRNA-seq studies, search tools that enable users to find orthologous genes and their cell type-specific expression patterns across species are limited. Here, we built a new search database, DRscDB (https://www.flyrnai.org/tools/single_cell/web/), to address this need. DRscDB serves as a comprehensive repository for published scRNA-seq datasets for Drosophila and relevant datasets from human and other model organisms. DRscDB is based on manual curation of Drosophila scRNA-seq studies of various tissue types and their corresponding analogous tissues in vertebrates including zebrafish, mouse, and human. Of note, our search database provides most of the literature-derived marker genes, thus preserving the original analysis of the published scRNA-seq datasets. Finally, DRscDB serves as a web-based user interface that allows users to mine gene expression data from scRNA-seq studies and perform cell cluster enrichment analyses pertaining to various scRNA-seq studies, both within and across species.}, issn = {2001-0370}, doi = {10.1016/j.csbj.2021.04.021}, author = {Hu, Yanhui and Tattikota, Sudhir Gopal and Liu, Yifang and Comjean, Aram and Gao, Yue and Forman, Corey and Kim, Grace and Rodiger, Jonathan and Papatheodorou, Irene and Dos Santos, Gilberto and Mohr, Stephanie E and Perrimon, Norbert} } @article {1411212, title = {Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila}, journal = {Insect Sci}, year = {2021}, month = {2021 Aug 05}, abstract = {Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3(Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr and Abd-A showed the potential complex mechanism of kkv transcription are regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis. This article is protected by copyright. All rights reserved.}, issn = {1744-7917}, doi = {10.1111/1744-7917.12954}, author = {Yue, Xiangzhao and Liang, Yongkang and Wei, Zhishuang and Lv, Jun and Cai, Yongjin and Fan, Xiaobin and Zhang, Wenqing and Chen, Jie} } @article {1447590, title = {Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11}, journal = {Genet Med}, volume = {23}, year = {2021}, month = {2021 10}, pages = {1889-1900}, abstract = {PURPOSE: Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. METHODS: We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. RESULTS: Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients{\textquoteright} variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants. CONCLUSION: GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.}, keywords = {Animals, Bone Morphogenetic Proteins, Craniofacial Abnormalities, Growth Differentiation Factors, Humans, Mutation, Missense, Phenotype, Spine, zebrafish}, issn = {1530-0366}, doi = {10.1038/s41436-021-01216-8}, author = {Ravenscroft, Thomas A and Phillips, Jennifer B and Fieg, Elizabeth and Bajikar, Sameer S and Peirce, Judy and Wegner, Jeremy and Luna, Alia A and Fox, Eric J and Yan, Yi-Lin and Rosenfeld, Jill A and Zirin, Jonathan and Kanca, Oguz and Diseases Network, Undiagnosed and Benke, Paul J and Cameron, Eric S and Strehlow, Vincent and Platzer, Konrad and Jamra, Rami Abou and Kl{\"o}ckner, Chiara and Osmond, Matthew and Licata, Thomas and Rojas, Samantha and Dyment, David and Chong, Josephine S C and Lincoln, Sharyn and Stoler, Joan M and Postlethwait, John H and Wangler, Michael F and Yamamoto, Shinya and Krier, Joel and Westerfield, Monte and Bellen, Hugo J} } @article {1401907, title = {Methods and tools for spatial mapping of single-cell RNAseq clusters in Drosophila}, journal = {Genetics}, volume = {217}, year = {2021}, month = {2021 Apr 15}, abstract = {Single-cell RNA sequencing (scRNAseq) experiments provide a powerful means to identify clusters of cells that share common gene expression signatures. A major challenge in scRNAseq studies is to map the clusters to specific anatomical regions along the body and within tissues. Existing data, such as information obtained from large-scale in situ RNA hybridization studies, cell type specific transcriptomics, gene expression reporters, antibody stainings, and fluorescent tagged proteins, can help to map clusters to anatomy. However, in many cases, additional validation is needed to precisely map the spatial location of cells in clusters. Several approaches are available for spatial resolution in Drosophila, including mining of existing datasets, and use of existing or new tools for direct or indirect detection of RNA, or direct detection of proteins. Here, we review available resources and emerging technologies that will facilitate spatial mapping of scRNAseq clusters at high resolution in Drosophila. Importantly, we discuss the need, available approaches, and reagents for multiplexing gene expression detection in situ, as in most cases scRNAseq clusters are defined by the unique coexpression of sets of genes.}, issn = {1943-2631}, doi = {10.1093/genetics/iyab019}, author = {Mohr, Stephanie E and Tattikota, Sudhir Gopal and Xu, Jun and Zirin, Jonathan and Hu, Yanhui and Perrimon, Norbert} } @article {1401902, title = {Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes}, journal = {Nat Commun}, volume = {12}, year = {2021}, month = {2021 05 20}, pages = {2960}, abstract = {Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex\ quinquefasciatus and Drosophila\ melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.}, keywords = {Animals, Animals, Genetically Modified, CRISPR-Cas Systems, Culex, drosophila melanogaster, Female, Gene Drive Technology, Insecticide Resistance, Male, Mosquito Control, Mosquito Vectors, Mutagenesis, Site-Directed, RNA, Guide}, issn = {2041-1723}, doi = {10.1038/s41467-021-23239-0}, author = {Feng, Xuechun and L{\'o}pez Del Amo, V{\'\i}ctor and Mameli, Enzo and Lee, Megan and Bishop, Alena L and Perrimon, Norbert and Gantz, Valentino M} } @article {1390346, title = {Precise genome engineering in Drosophila using prime editing}, journal = {Proc Natl Acad Sci U S A}, volume = {118}, number = {1}, year = {2021}, note = {Bosch, Justin ABirchak, GabrielPerrimon, NorbertengP41 GM132087/GM/NIGMS NIH HHS/R24 OD030002/OD/NIH HHS/Proc Natl Acad Sci U S A. 2021 Jan 5;118(1). pii: 2021996118. doi: 10.1073/pnas.2021996118.}, month = {Jan 5}, edition = {2021/01/15}, abstract = {Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36\% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.}, keywords = {Crispr, Drosophila, genome engineering, pegRNA, prime editing}, isbn = {1091-6490 (Electronic)0027-8424 (Linking)}, author = {Bosch, J. A. and Birchak, G. and Perrimon, N.} } @article {1399112, title = {Proteomics of protein trafficking by in vivo tissue-specific labeling}, journal = {Nat Commun}, volume = {12}, year = {2021}, month = {2021 04 22}, pages = {2382}, abstract = {Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.}, keywords = {Animals, Animals, Genetically Modified, Biotin, Biotinylation, Carbon-Nitrogen Ligases, Cell Line, Disease Models, Animal, Drosophila, Embryonic Stem Cells, Escherichia coli Proteins, Female, Humans, Male, Mice, Protein Engineering, Protein Transport, proteomics, Repressor Proteins, Staining and Labeling, Tandem Mass Spectrometry, Teratoma}, issn = {2041-1723}, doi = {10.1038/s41467-021-22599-x}, author = {Droujinine, Ilia A and Meyer, Amanda S and Wang, Dan and Udeshi, Namrata D and Hu, Yanhui and Rocco, David and McMahon, Jill A and Yang, Rui and Guo, JinJin and Mu, Luye and Carey, Dominique K and Svinkina, Tanya and Zeng, Rebecca and Branon, Tess and Tabatabai, Areya and Bosch, Justin A and Asara, John M and Ting, Alice Y and Carr, Steven A and McMahon, Andrew P and Perrimon, Norbert} } @article {1442946, title = {TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data}, journal = {Nucleic Acids Res}, volume = {49}, year = {2021}, month = {2021 07 02}, pages = {W641-W653}, abstract = {Uncovering how transcription factors regulate their targets at DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) and assign mechanisms in normal and diseased states. RNA-seq is a standard method measuring gene regulation using an established set of analysis stages. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time-series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods integrating ordered RNA-seq data with protein-DNA binding data to distinguish direct from indirect interactions are urgently needed. We present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time-series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to determine causal regulatory mechanism networks by leveraging time-series RNA-seq, motif analysis, protein-DNA binding data, and protein-protein interaction networks. TIMEOR{\textquoteright}s user-catered approach helps non-coders generate new hypotheses and validate known mechanisms. We used TIMEOR to identify a novel link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at https://github.com/ashleymaeconard/TIMEOR.git and http://timeor.brown.edu.}, keywords = {Circadian Rhythm, Gene Expression Regulation, Gene Regulatory Networks, Genomics, Humans, Insulin, Internet, Protein Interaction Mapping, RNA-seq, Software, Transcription Factors}, issn = {1362-4962}, doi = {10.1093/nar/gkab384}, author = {Conard, Ashley Mae and Goodman, Nathaniel and Hu, Yanhui and Perrimon, Norbert and Singh, Ritambhara and Lawrence, Charles and Larschan, Erica} } @article {1390357, title = {CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells}, journal = {BioRxiv}, year = {2020}, month = {01 Nov, 2020}, abstract = {Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.}, author = {Viswanatha, R. and Zaffagni, M. and Zirin, J. and Perrimon, N. and Kadener, S.} } @article {1391780, title = {TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data [NOTE: A modified final version was published in NAR and is now available]}, journal = {BioRxiv}, year = {2020}, month = {Sept 2020}, abstract = {Uncovering how transcription factors (TFs) regulate their targets at the DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) in normal and diseased states. RNA-seq has become a standard method to measure gene regulation using an established set of analysis steps. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods which integrate ordered RNA-seq data with transcription factor binding data are urgently needed. Here, we present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to predict causal regulatory mechanism networks between TFs from time series multi-omics data. We used TIMEOR to identify a new link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at https://github.com/ashleymaeconard/TIMEOR.git.}, url = {https://doi.org/10.1101/2020.09.14.296418 }, author = {Conard, A.M. and Goodman, N. and Hu, Y, and Perrimon, N. and Singh, R. and Lawrence, C. and Larschan, E.} } @article {1427766, title = {Polarized sorting of Patched enables cytoneme-mediated Hedgehog reception in the Drosophila wing disc}, journal = {EMBO J}, volume = {39}, year = {2020}, month = {2020 06 02}, pages = {e103629}, abstract = {Hedgehog (Hh) signal molecules play a fundamental role in development, adult stem cell maintenance and cancer. Hh can signal at a distance, and we have proposed that its graded distribution across Drosophila epithelia is mediated by filopodia-like structures called cytonemes. Hh reception by Patched (Ptc) happens at discrete sites along presenting and receiving cytonemes, reminiscent of synaptic processes. Here, we show that a vesicle fusion mechanism mediated by SNARE proteins is required for Ptc placement at contact sites. Transport of Ptc to these sites requires multivesicular bodies (MVBs) formation via ESCRT machinery, in a manner different to that regulating Ptc/Hh lysosomal degradation after reception. These MVBs include extracellular vesicle (EV) markers and, accordingly, Ptc is detected in the purified exosomal fraction from cultured cells. Blockage of Ptc trafficking and fusion to basolateral membranes result in low levels of Ptc presentation for reception, causing an extended and flattened Hh gradient.}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Endosomal Sorting Complexes Required for Transport, Hedgehog Proteins, Imaginal Discs, Protein Transport, Receptors, Cell Surface, SNARE Proteins, Wings, Animal}, issn = {1460-2075}, doi = {10.15252/embj.2019103629}, author = {Gonz{\'a}lez-M{\'e}ndez, Laura and Gradilla, Ana-Citlali and S{\'a}nchez-Hern{\'a}ndez, David and Gonz{\'a}lez, Esperanza and Aguirre-Tamaral, Adri{\'a}n and Jim{\'e}nez-Jim{\'e}nez, Carlos and Guerra, Milagros and Aguilar, Gustavo and Andr{\'e}s, Germ{\'a}n and Falc{\'o}n-P{\'e}rez, Juan M and Guerrero, Isabel} } @article {1378223, title = {BioLitMine: Advanced Mining of Biomedical and Biological Literature About Human Genes and Genes from Major Model Organisms}, journal = {G3 (Bethesda)}, year = {2020}, month = {2020 Oct 07}, abstract = {The accumulation of biological and biomedical literature outpaces the ability of most researchers and clinicians to stay abreast of their own immediate fields, let alone a broader range of topics. Although available search tools support identification of relevant literature, finding relevant and key publications is not always straightforward. For example, important publications might be missed in searches with an official gene name due to gene synonyms. Moreover, ambiguity of gene names can result in retrieval of a large number of irrelevant publications. To address these issues and help researchers and physicians quickly identify relevant publications, we developed BioLitMine, an advanced literature mining tool that takes advantage of the medical subject heading (MeSH) index and gene-to-publication annotations already available for PubMed literature. Using BioLitMine, a user can identify what MeSH terms are represented in the set of publications associated with a given gene of the interest, or start with a term and identify relevant publications. Users can also use the tool to find co-cited genes and a build a literature co-citation network. In addition, BioLitMine can help users build a gene list relevant to a MeSH terms, such as a list of genes relevant to "stem cells" or "breast neoplasms." Users can also start with a gene or pathway of interest and identify authors associated with that gene or pathway, a feature that makes it easier to identify experts who might serve as collaborators or reviewers. Altogether, BioLitMine extends the value of PubMed-indexed literature and its existing expert curation by providing a robust and gene-centric approach to retrieval of relevant information.}, issn = {2160-1836}, doi = {10.1534/g3.120.401775}, author = {Hu, Yanhui and Chung, Verena and Comjean, Aram and Rodiger, Jonathan and Nipun, Fnu and Perrimon, Norbert and Mohr, Stephanie E} } @article {1378219, title = {CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells}, journal = {Nat Protoc}, volume = {15}, year = {2020}, month = {2020 Oct}, pages = {3478-3498}, abstract = {Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.}, issn = {1750-2799}, doi = {10.1038/s41596-020-0383-8}, author = {Xia, Baolong and Amador, Gabriel and Viswanatha, Raghuvir and Zirin, Jonathan and Mohr, Stephanie E and Perrimon, Norbert} } @article {1379402, title = {FlyRNAi.org-the database of the Drosophila RNAi screening center and transgenic RNAi project: 2021 update}, journal = {Nucleic Acids Res}, year = {2020}, month = {2020 Oct 26}, abstract = {The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) provides a suite of online resources that facilitate functional genomics studies with a special emphasis on Drosophila melanogaster. Currently, the database provides: gene-centric resources that facilitate ortholog mapping and mining of information about orthologs in common genetic model species; reagent-centric resources that help researchers identify RNAi and CRISPR sgRNA reagents or designs; and data-centric resources that facilitate visualization and mining of transcriptomics data, protein modification data, protein interactions, and more. Here, we discuss updated and new features that help biological and biomedical researchers efficiently identify, visualize, analyze, and integrate information and data for Drosophila and other species. Together, these resources facilitate multiple steps in functional genomics workflows, from building gene and reagent lists to management, analysis, and integration of data.}, issn = {1362-4962}, doi = {10.1093/nar/gkaa936}, author = {Hu, Yanhui and Comjean, Aram and Rodiger, Jonathan and Liu, Yifang and Gao, Yue and Chung, Verena and Zirin, Jonathan and Perrimon, Norbert and Mohr, Stephanie E} } @article {1291760, title = {Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies}, journal = {Genetics}, year = {2020}, month = {2020 Feb 18}, abstract = {The Transgenic RNAi Project (TRiP), a functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated \>15,000 RNAi fly stocks. As this covers most genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes. Cutting is triggered by co-expression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.}, issn = {1943-2631}, doi = {10.1534/genetics.119.302964}, author = {Zirin, Jonathan and Hu, Yanhui and Liu, Luping and Yang-Zhou, Donghui and Colbeth, Ryan and Yan, Dong and Ewen-Campen, Ben and Tao, Rong and Vogt, Eric and VanNest, Sara and Cavers, Cooper and Villalta, Christians and Comjean, Aram and Sun, Jin and Wang, Xia and Jia, Yu and Zhu, Ruibao and Peng, Ping and Yu, Jinchao and Shen, Da and Qiu, Yuhao and Ayisi, Limmond and Ragoowansi, Henna and Fenton, Ethan and Efrem, Senait and Parks, Annette and Saito, Kuniaki and Kondo, Shu and Perkins, Liz and Mohr, Stephanie E and Ni, Jianquan and Perrimon, Norbert} } @article {1272329, title = {Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes}, journal = {Curr Protoc Mol Biol}, volume = {130}, year = {2020}, month = {2020 Mar}, pages = {e112}, abstract = {The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or {\textquoteright}knock-in{\textquoteright} of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with \~{}500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. {\textcopyright} 2019 by John Wiley \& Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.}, issn = {1934-3647}, doi = {10.1002/cpmb.112}, author = {Bosch, Justin A and Knight, Shannon and Kanca, Oguz and Zirin, Jonathan and Yang-Zhou, Donghui and Hu, Yanhui and Rodiger, Jonathan and Amador, Gabriel and Bellen, Hugo J and Perrimon, Norbert and Mohr, Stephanie E} } @article {1227208, title = {Drosophila melanogaster: a simple system for understanding complexity.}, journal = {Dis Model Mech}, volume = {12}, year = {2019}, abstract = {

Understanding human gene function is fundamental to understanding and treating diseases. Research using the model organism\ Drosophila melanogaster\ benefits from a wealth of molecular genetic resources and information useful for efficient\ invivo\ experimentation. Moreover,\ Drosophila\ offers a balance as a relatively simple organism that nonetheless exhibits complex multicellular activities. Recent examples demonstrate the power and continued promise of\ Drosophila\ research to further our understanding of conserved gene functions.

}, url = {https://dmm.biologists.org/content/12/10/dmm041871.long}, author = {Mohr, Stephanie E. and Perrimon, Norbert} } @article {1221153, title = {Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids.}, journal = {BioRxiv}, year = {2019}, url = {https://www.biorxiv.org/content/10.1101/639484v1}, author = {Bosch, Justin A. and Colbeth, Ryan and Zirin, Jonathan and Perrimon, Norbert} } @article {1163259, title = {TRiP stocks contain a previously uncharacterized loss-of-function sevenless allele}, journal = {microPublication Biology}, year = {2019}, url = {https://www.micropublication.org/escobedo_etal_2019_tripsev.html}, author = {Escobedo, Spencer E. and Zirin, Jonathan and Weake, Vikki M.} } @article {1427763, title = {The zinc-finger protein CLAMP promotes chromatin insulator function in }, journal = {J Cell Sci}, volume = {132}, year = {2019}, month = {2019 03 08}, abstract = {Chromatin insulators are DNA-protein complexes that establish independent higher-order DNA domains to influence transcription. Insulators are functionally defined by two properties: they can block communication between an enhancer and a promoter, and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is knocked down, gypsy-dependent enhancer-blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap, particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, whereas depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.}, keywords = {Animals, Animals, Genetically Modified, Cells, Cultured, Chromatin, Chromatin Assembly and Disassembly, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Gene Knockdown Techniques, Microtubule-Associated Proteins, Multiprotein Complexes, Nuclear Proteins, Protein Binding, Repressor Proteins, RNA-Binding Proteins, Zinc Fingers}, issn = {1477-9137}, doi = {10.1242/jcs.226092}, author = {Bag, Indira and Dale, Ryan K and Palmer, Cameron and Lei, Elissa P} } @article {1231110, title = {An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Nov 01}, abstract = {We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an - transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely . The approach is fast, cheap, and scalable.}, issn = {2050-084X}, doi = {10.7554/eLife.51539}, author = {Kanca, Oguz and Zirin, Jonathan and Garcia-Marques, Jorge and Knight, Shannon Marie and Yang-Zhou, Donghui and Amador, Gabriel and Chung, Hyunglok and Zuo, Zhongyuan and Ma, Liwen and He, Yuchun and Lin, Wen-Wen and Fang, Ying and Ge, Ming and Yamamoto, Shinya and Schulze, Karen L and Hu, Yanhui and Spradling, Allan C and Mohr, Stephanie E and Perrimon, Norbert and Bellen, Hugo J} } @article {1227742, title = {HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species}, journal = {Sci Signal}, volume = {12}, year = {2019}, month = {2019 Oct 01}, abstract = {Inactivation of the tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and inactivation in two species (human and ) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.}, issn = {1937-9145}, doi = {10.1126/scisignal.aay0482}, author = {Nicholson, Hilary E and Tariq, Zeshan and Housden, Benjamin E and Jennings, Rebecca B and Stransky, Laura A and Perrimon, Norbert and Signoretti, Sabina and Kaelin, William G} } @article {1159605, title = {iProteinDB: An Integrative Database of Post-translational Modifications}, journal = {G3 (Bethesda)}, volume = {9}, year = {2019}, month = {2019 01 09}, pages = {1-11}, abstract = {Post-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing their stability, interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, most commonly serine, threonine and tyrosine in metazoans. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that any given phosphorylation site might be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for At iProteinDB, scientists can view the PTM landscape for any protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related species. Further, iProteinDB enables comparison of PTM data from to that of orthologous proteins from other model organisms, including human, mouse, rat, , , and .}, issn = {2160-1836}, doi = {10.1534/g3.118.200637}, author = {Hu, Yanhui and Sopko, Richelle and Chung, Verena and Foos, Marianna and Studer, Romain A and Landry, Sean D and Liu, Daniel and Rabinow, Leonard and Gnad, Florian and Beltrao, Pedro and Perrimon, Norbert} } @article {1222780, title = {Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species}, journal = {Aging Cell}, year = {2019}, month = {2019 Aug 28}, pages = {e13034}, abstract = {Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S-adenosylmethionine, which, after transferring its methyl group, is converted to S-adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging.}, issn = {1474-9726}, doi = {10.1111/acel.13034}, author = {Parkhitko, Andrey A and Jouandin, Patrick and Mohr, Stephanie E and Perrimon, Norbert} } @article {1233489, title = {Pooled CRISPR Screens in Drosophila Cells}, journal = {Curr Protoc Mol Biol}, volume = {129}, year = {2019}, month = {2019 Dec}, pages = {e111}, abstract = {High-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host-pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array-format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes \~{}3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems. {\textcopyright} 2019 by John Wiley \& Sons, Inc. Basic Protocol: Pooled-format screening with Cas9-expressing Drosophila S2R+ cells in the presence of cytotoxin Support Protocol 1: Optimization of cytotoxin concentration for Drosophila cell screening Support Protocol 2: CRISPR sgRNA library design and production for Drosophila cell screening Support Protocol 3: Barcode deconvolution and analysis of screening data.}, issn = {1934-3647}, doi = {10.1002/cpmb.111}, author = {Viswanatha, Raghuvir and Brathwaite, Roderick and Hu, Yanhui and Li, Zhongchi and Rodiger, Jonathan and Merckaert, Pierre and Chung, Verena and Mohr, Stephanie E and Perrimon, Norbert} } @article {1233488, title = {An RNAi Screen for Genes Required for Growth of Wing Tissue}, journal = {G3 (Bethesda)}, volume = {9}, year = {2019}, month = {2019 10 07}, pages = {3087-3100}, abstract = {Cell division and tissue growth must be coordinated with development. Defects in these processes are the basis for a number of diseases, including developmental malformations and cancer. We have conducted an unbiased RNAi screen for genes that are required for growth in the wing, using GAL4-inducible short hairpin RNA (shRNA) fly strains made by the Drosophila RNAi Screening Center. shRNA expression down the center of the larval wing disc using , and the central region of the adult wing was then scored for tissue growth and wing hair morphology. Out of 4,753 shRNA crosses that survived to adulthood, 18 had impaired wing growth. FlyBase and the new Alliance of Genome Resources knowledgebases were used to determine the known or predicted functions of these genes and the association of their human orthologs with disease. The function of eight of the genes identified has not been previously defined in The genes identified included those with known or predicted functions in cell cycle, chromosome segregation, morphogenesis, metabolism, steroid processing, transcription, and translation. All but one of the genes are similar to those in humans, and many are associated with disease. Knockdown of , a subunit of the Myb-MuvB transcription factor, or β, a gene involved in protein folding and trafficking, resulted in a switch from cell proliferation to an endoreplication growth program through which wing tissue grew by an increase in cell size (hypertrophy). It is anticipated that further analysis of the genes that we have identified will reveal new mechanisms that regulate tissue growth during development.}, issn = {2160-1836}, doi = {10.1534/g3.119.400581}, author = {Rotelli, Michael D and Bolling, Anna M and Killion, Andrew W and Weinberg, Abraham J and Dixon, Michael J and Calvi, Brian R} } @article {1235944, title = {SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing}, journal = {G3 (Bethesda)}, year = {2019}, month = {2019 Dec 10}, abstract = {CRISPR-Cas9 is a powerful genome editing technology in which a short guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and potential applications for design of sgRNAs for disease-associated mutant correction.}, issn = {2160-1836}, doi = {10.1534/g3.119.400904}, author = {Chen, Chiao-Lin and Rodiger, Jonathan and Chung, Verena and Viswanatha, Raghuvir and Mohr, Stephanie E and Hu, Yanhui and Perrimon, Norbert} } @website {1160089, title = {Data portal for "A cell atlas of the adult Drosophila midgut" (BioRxiv)}, year = {2018}, url = {https://www.flyrnai.org/scRNA/}, author = {Hung, R. and Hu, Y. and Kirchner, R. and Li, Fengge and C. Xu and Comjean, A. and Tattikota, S.G. and Song, W.R. and Ho Sui, S. and Perrimon, N.} } @website {1135137, title = {Raw data download access for: Viswanatha et al. 2018 eLife "Pooled genome-wide CRISPR screening ..."}, year = {2018}, url = {https://sharehost.hms.harvard.edu/genetics/?perrimon/CRISPR_fitness_screen_reads}, author = {Viswanatha, Raghuvir and Comjean, Aram and Perrimon, Norbert} } @article {1133711, title = {Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in cells}, journal = {Elife}, volume = {7}, year = {2018}, month = {2018 07 27}, abstract = {Genome-wide screens in cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5\%, representing the highest-resolution fitness gene set yet assembled for , including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells.}, keywords = {Animals, Computational Biology, CRISPR-Cas Systems, Drosophila, Drug Interactions, Gene Expression Regulation, Gene Knockout Techniques, Gene Library, Genes, Essential, Genetic Fitness, Genome-Wide Association Study, Pharmacogenetics, Phenotype, Protein Kinase Inhibitors, Pyridones, Pyrimidinones, Sirolimus}, issn = {2050-084X}, doi = {10.7554/eLife.36333}, author = {Viswanatha, Raghuvir and Li, Zhongchi and Hu, Yanhui and Perrimon, Norbert} } @article {1145832, title = {Identification of insect genes involved in baculovirus AcMNPV entry into insect cells}, journal = {Virology}, volume = {527}, year = {2018}, month = {2018 Nov 13}, pages = {1-11}, abstract = {The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a model enveloped DNA virus that infects and replicates in lepidopteran insect cells, and can efficiently enter a wide variety of non-host cells. Budded virions of AcMNPV enter cells by endocytosis and traffic to the nucleus where the virus initiates gene expression and genome replication. While trafficking of nucleocapsids by actin propulsion has been studied in detail, other important components of trafficking during entry remain poorly understood. We used a recombinant AcMNPV virus expressing an EGFP reporter in combination with an RNAi screen in Drosophila DL1 cells, to identify host proteins involved in AcMNPV entry. The RNAi screen targeted 86 genes involved in vesicular trafficking, including genes coding for VPS and ESCRT proteins, Rab GTPases, Exocyst proteins, and Clathrin adaptor proteins. We identified 24 genes required for efficient virus entry and reporter expression, and 4 genes that appear to restrict virus entry.}, issn = {1096-0341}, doi = {10.1016/j.virol.2018.10.022}, author = {Hodgson, Jeffrey J and Buchon, Nicolas and Blissard, Gary W} } @article {1145831, title = {Whole genome screen reveals a novel relationship between Wolbachia levels and Drosophila host translation}, journal = {PLoS Pathog}, volume = {14}, year = {2018}, month = {2018 Nov 13}, pages = {e1007445}, abstract = {Wolbachia is an intracellular bacterium that infects a remarkable range of insect hosts. Insects such as mosquitos act as vectors for many devastating human viruses such as Dengue, West Nile, and Zika. Remarkably, Wolbachia infection provides insect hosts with resistance to many arboviruses thereby rendering the insects ineffective as vectors. To utilize Wolbachia effectively as a tool against vector-borne viruses a better understanding of the host-Wolbachia relationship is needed. To investigate Wolbachia-insect interactions we used the Wolbachia/Drosophila model that provides a genetically tractable system for studying host-pathogen interactions. We coupled genome-wide RNAi screening with a novel high-throughput fluorescence in situ hybridization (FISH) assay to detect changes in Wolbachia levels in a Wolbachia-infected Drosophila cell line JW18. 1117 genes altered Wolbachia levels when knocked down by RNAi of which 329 genes increased and 788 genes decreased the level of Wolbachia. Validation of hits included in depth secondary screening using in vitro RNAi, Drosophila mutants, and Wolbachia-detection by DNA qPCR. A diverse set of host gene networks was identified to regulate Wolbachia levels and unexpectedly revealed that perturbations of host translation components such as the ribosome and translation initiation factors results in increased Wolbachia levels both in vitro using RNAi and in vivo using mutants and a chemical-based translation inhibition assay. This work provides evidence for Wolbachia-host translation interaction and strengthens our general understanding of the Wolbachia-host intracellular relationship.}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1007445}, author = {Grobler, Yolande and Yun, Chi Y and Kahler, David J and Bergman, Casey M and Lee, Hangnoh and Oliver, Brian and Lehmann, Ruth} } @article {1152056, title = {A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila}, journal = {Dev Cell}, volume = {47}, year = {2018}, month = {2018 11 05}, pages = {294-305.e7}, abstract = {Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In\ vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies.}, issn = {1878-1551}, doi = {10.1016/j.devcel.2018.09.012}, author = {Okamoto, Naoki and Viswanatha, Raghuvir and Bittar, Riyan and Li, Zhongchi and Haga-Yamanaka, Sachiko and Perrimon, Norbert and Yamanaka, Naoki} } @article {1102166, title = {Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress}, journal = {Proc Natl Acad Sci U S A}, year = {2017}, month = {2017 Dec 11}, abstract = {A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.}, issn = {1091-6490}, doi = {10.1073/pnas.1712453115}, author = {Sung, Eui Jae and Ryuda, Masasuke and Matsumoto, Hitoshi and Uryu, Outa and Ochiai, Masanori and Cook, Molly E and Yi, Na Young and Wang, Huanchen and Putney, James W and Bird, Gary S and Shears, Stephen B and Hayakawa, Yoichi} } @article {1102191, title = {Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells}, journal = {G3 (Bethesda)}, year = {2017}, month = {2017 Dec 09}, abstract = {Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wildtype or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and more than 1800 putative zinc-responsive genes. Analysis of the {\textquoteright}omics data points to the relevance of ion transporters, glutathione-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells and that rdog is transcriptionally up-regulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the {\textquoteright}omics datasets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.}, issn = {2160-1836}, doi = {10.1534/g3.117.300447}, author = {Mohr, Stephanie E and Rudd, Kirstin and Hu, Yanhui and Song, Wei R and Gilly, Quentin and Buckner, Michael and Housden, Benjamin E and Kelley, Colleen and Zirin, Jonathan and Tao, Rong and Amador, Gabriel and Sierzputowska, Katarzyna and Comjean, Aram and Perrimon, Norbert} } @article {1096116, title = {Improved detection of synthetic lethal interactions in Drosophila cells using variable dose analysis (VDA)}, journal = {Proc Natl Acad Sci U S A}, year = {2017}, month = {2017 Nov 28}, abstract = {Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells, but this approach generally suffers from low consistency between screens. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called Variable Dose Analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC), and generated a SS/L network for TSC. Using this network, we identified four Food and Drug Administration-approved drugs that selectively affect viability of TSC-deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.}, issn = {1091-6490}, doi = {10.1073/pnas.1713362114}, author = {Housden, Benjamin E and Li, Zhongchi and Kelley, Colleen and Wang, Yuanli and Hu, Yanhui and Valvezan, Alexander J and Manning, Brendan D and Perrimon, Norbert} } @article {1114929, title = {Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data}, journal = {Nucleic Acids Res}, volume = {46}, year = {2017}, month = {2018 Jan 04}, pages = {D567-D574}, abstract = {Model organism and human databases are rich with information about genetic and physical interactions. These data can be used to interpret and guide the analysis of results from new studies and develop new hypotheses. Here, we report the development of the Molecular Interaction Search Tool (MIST; http://fgrtools.hms.harvard.edu/MIST/). The MIST database integrates biological interaction data from yeast, nematode, fly, zebrafish, frog, rat and mouse model systems, as well as human. For individual or short gene lists, the MIST user interface can be used to identify interacting partners based on protein-protein and genetic interaction (GI) data from the species of interest as well as inferred interactions, known as interologs, and to view a corresponding network. The data, interologs and search tools at MIST are also useful for analyzing {\textquoteright}omics datasets. In addition to describing the integrated database, we also demonstrate how MIST can be used to identify an appropriate cut-off value that balances false positive and negative discovery, and present use-cases for additional types of analysis. Altogether, the MIST database and search tools support visualization and navigation of existing protein and GI data, as well as comparison of new and existing data.}, issn = {1362-4962}, doi = {10.1093/nar/gkx1116}, author = {Hu, Yanhui and Vinayagam, Arunachalam and Nand, Ankita and Comjean, Aram and Chung, Verena and Hao, Tong and Mohr, Stephanie E and Perrimon, Norbert} } @article {1070971, title = {Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR}, journal = {Dev Cell}, volume = {43}, year = {2017}, month = {2017 Oct 09}, pages = {6-9}, abstract = {Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research.}, issn = {1878-1551}, doi = {10.1016/j.devcel.2017.09.020}, author = {Ewen-Campen, Ben and Mohr, Stephanie E and Hu, Yanhui and Perrimon, Norbert} } @article {1023846, title = {Gene2Function: An Integrated Online Resource for Gene Function Discovery}, journal = {G3 (Bethesda)}, year = {2017}, month = {2017 Jun 29}, abstract = {One of the most powerful ways to develop hypotheses regarding biological functions of conserved genes in a given species, such as in humans, is to first look at what is known about function in another species. Model organism databases (MODs) and other resources are rich with functional information but difficult to mine. Gene2Function (G2F) addresses a broad need by integrating information about conserved genes in a single online resource.}, issn = {2160-1836}, doi = {10.1534/g3.117.043885}, author = {Hu, Yanhui and Comjean, Aram and Mohr, Stephanie E and The FlyBase Consortium and Perrimon, Norbert} } @article {1036231, title = {MARRVEL: Integration of Human and Model Organism Genetic Resources to Facilitate Functional Annotation of the Human Genome}, journal = {Am J Hum Genet}, volume = {100}, year = {2017}, month = {2017 Jun 01}, pages = {843-853}, abstract = {One major challenge encountered with interpreting human genetic variants is the limited understanding of the functional impact of genetic alterations on biological processes. Furthermore, there remains an unmet demand for an efficient survey of the wealth of information on human homologs in model organisms across numerous databases. To efficiently assess the large volume of publically available information, it is important to provide a concise summary of the most relevant information in a rapid user-friendly format. To this end, we created MARRVEL (model organism aggregated resources for rare variant exploration). MARRVEL is a publicly available website that integrates information from six human genetic databases and seven model organism databases. For any given variant or gene, MARRVEL displays information from OMIM, ExAC, ClinVar, Geno2MP, DGV, and DECIPHER. Importantly, it curates model organism-specific databases to concurrently display a concise summary regarding the human gene homologs in budding and fission yeast, worm, fly, fish, mouse, and rat on a single webpage. Experiment-based information on tissue expression, protein subcellular localization, biological process, and molecular function for the human gene and homologs in the seven model organisms are arranged into a concise output. Hence, rather than visiting multiple separate databases for variant and gene analysis, users can obtain important information by searching once through MARRVEL. Altogether, MARRVEL dramatically improves efficiency and accessibility to data collection and facilitates analysis of human genes and variants by cross-disciplinary integration of 18 million records available in public databases to facilitate clinical diagnosis and basic research.}, keywords = {Databases, Genetic, Genetic Variation, Genome, Human, Humans, Molecular Sequence Annotation, Software}, issn = {1537-6605}, doi = {10.1016/j.ajhg.2017.04.010}, author = {Wang, Julia and Al-Ouran, Rami and Hu, Yanhui and Kim, Seon-Young and Wan, Ying-Wooi and Wangler, Michael F and Yamamoto, Shinya and Chao, Hsiao-Tuan and Comjean, Aram and Mohr, Stephanie E and Diseases Network, Undiagnosed and Perrimon, Norbert and Liu, Zhandong and Bellen, Hugo J} } @website {1006161, title = {Online view of high-content image data generated in the genome-wide screen described in Neum{\"u}ller et al. 2013, "Conserved regulators of nucleolar size revealed by global phenotypic analyses," made possible using OMERO at HMS}, year = {2017}, author = {Copeland, AJ and Comjean, A and N Perrimon and SE Mohr} } @article {964461, title = {The Drosophila Gene Expression Tool (DGET) for expression analyses}, journal = {BMC Bioinformatics}, volume = {18}, year = {2017}, month = {2017 Feb 10}, pages = {98}, abstract = {BACKGROUND: Next-generation sequencing technologies have greatly increased our ability to identify gene expression levels, including at specific developmental stages and in specific tissues. Gene expression data can help researchers understand the diverse functions of genes and gene networks, as well as help in the design of specific and efficient functional studies, such as by helping researchers choose the most appropriate tissue for a study of a group of genes, or conversely, by limiting a long list of gene candidates to the subset that are normally expressed at a given stage or in a given tissue. RESULTS: We report DGET, a Drosophila Gene Expression Tool ( www.flyrnai.org/tools/dget/web/ ), which stores and facilitates search of RNA-Seq based expression profiles available from the modENCODE consortium and other public data sets. Using DGET, researchers are able to look up gene expression profiles, filter results based on threshold expression values, and compare expression data across different developmental stages, tissues and treatments. In addition, at DGET a researcher can analyze tissue or stage-specific enrichment for an inputted list of genes (e.g., {\textquoteright}hits{\textquoteright} from a screen) and search for additional genes with similar expression patterns. We performed a number of analyses to demonstrate the quality and robustness of the resource. In particular, we show that evolutionary conserved genes expressed at high or moderate levels in both fly and human tend to be expressed in similar tissues. Using DGET, we compared whole tissue profile and sub-region/cell-type specific datasets and estimated a potential source of false positives in one dataset. We also demonstrated the usefulness of DGET for synexpression studies by querying genes with expression profile similar to the mesodermal master regulator Twist. CONCLUSION: Altogether, DGET provides a flexible tool for expression data retrieval and analysis with short or long lists of Drosophila genes, which can help scientists to design stage- or tissue-specific in vivo studies and do other subsequent analyses.}, issn = {1471-2105}, doi = {10.1186/s12859-017-1509-z}, author = {Hu, Yanhui and Comjean, Aram and Perrimon, Norbert and Mohr, Stephanie E} } @article {925621, title = {Loss-of-function genetic tools for animal models: cross-species and cross-platform differences}, journal = {Nat Rev Genet}, year = {2016}, month = {31 Oct, 2016}, abstract = {

Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.

}, url = {https://www.ncbi.nlm.nih.gov/pubmed/27795562}, author = {Housden, Benjamin E and Muhar, Matthias and Gemberling, Matthew and Gersbach, Charles A and Stainier, Didier YR and Seydoux, Geraldine and Mohr, Stephanie E and Zuber, Johannes and Perrimon, Norbert} } @article {928061, title = {FlyRNAi.org{\textemdash}the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update}, journal = {Nucleic Acids Research}, year = {2016}, month = {11 Oct 2016}, abstract = {

The FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.

}, url = {http://nar.oxfordjournals.org/content/early/2016/10/20/nar.gkw977.full}, author = {Hu, Yanhui and Comjean, Aram and Roesel, Charles and Vinayagam, Arunachalam and Flockhart, Ian and Zirin, Jonathan and Perkins, Lizabeth and Perrimon, Norbert and Mohr, Stephanie E} } @presentation {910121, title = {Drosophila research resources at the DRSC and TRiP}, year = {2016}, author = {Mohr, Stephanie E} } @article {896531, title = {An Integrative Analysis of the InR/PI3K/Akt Network Identifies the Dynamic Response to Insulin Signaling}, journal = {Cell Reports}, volume = {16}, year = {2016}, month = {2016}, pages = {3062-3074}, abstract = {

Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and meta- bolism. To expand our understanding of the insulin pathway, we combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila InR/PI3K/Akt network. First, we map the dynamic protein-protein interaction network sur- rounding the insulin core pathway using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, we find that 47\% of interacting proteins affect pathway activity, and, using quantitative phospho- proteomics, we demonstrate that $10\% of interact- ing proteins are regulated by insulin stimulation at the level of phosphorylation. Next, we integrate these orthogonal datasets to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate our method by iden- tifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, our study represents a comprehensive resource for the study of the evolutionary conserved insulin network.\ 

}, author = {Vinayagam, Arunachalam and Kulkarni, Meghana M and Sopko, Richelle and Sun, Xiaoyun and Hu, Yanhui and Nand, Ankita and Villalta, Christians and Moghimi, Ahmadali and Yang, Xuemei and Mohr, Stephanie E and Hong, Pengyu and Asara, John M and Perrimon, Norbert} } @article {969486, title = {Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets}, journal = {Proc Natl Acad Sci U S A}, volume = {113}, year = {2016}, month = {2016 May 03}, pages = {4976-81}, abstract = {

The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21\% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network{\textquoteright}s control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets.

}, keywords = {Genetic Predisposition to Disease, Humans, Mutation, Protein Binding, Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.1603992113}, author = {Vinayagam, Arunachalam and Gibson, Travis E and Lee, Ho-Joon and Yilmazel, Bahar and Roesel, Charles and Hu, Yanhui and Kwon, Young and Sharma, Amitabh and Liu, Yang-Yu and Perrimon, Norbert and Barab{\'a}si, Albert-L{\'a}szl{\'o}} } @article {937861, title = {Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway}, journal = {Methods in Molecular Biology}, year = {2016}, abstract = {

The transforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined \>90 \% of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.

}, url = {http://link.springer.com/protocol/10.1007\%2F978-1-4939-2966-5_24}, author = {Chen X and Xu L} } @article {825846, title = {Combined Gene Expression and RNAi Screening to Identify Alkylation Damage Survival Pathways from Fly to Human.}, journal = {PLoS One}, volume = {11}, year = {2016}, month = {2016}, pages = {e0153970}, abstract = {

Alkylating agents are a key component of cancer chemotherapy. Several cellular mechanisms are known to be important for its survival, particularly DNA repair and xenobiotic detoxification, yet genomic screens indicate that additional cellular components may be involved. Elucidating these components has value in either identifying key processes that can be modulated to improve chemotherapeutic efficacy or may be altered in some cancers to confer chemoresistance. We therefore set out to reevaluate our prior Drosophila RNAi screening data by comparison to gene expression arrays in order to determine if we could identify any novel processes in alkylation damage survival. We noted a consistent conservation of alkylation survival pathways across platforms and species when the analysis was conducted on a pathway/process level rather than at an individual gene level. Better results were obtained when combining gene lists from two datasets (RNAi screen plus microarray) prior to analysis. In addition to previously identified DNA damage responses (p53 signaling and Nucleotide Excision Repair), DNA-mRNA-protein metabolism (transcription/translation) and proteasome machinery, we also noted a highly conserved cross-species requirement for NRF2, glutathione (GSH)-mediated drug detoxification and Endoplasmic Reticulum stress (ER stress)/Unfolded Protein Responses (UPR) in cells exposed to alkylation. The requirement for GSH, NRF2 and UPR in alkylation survival was validated by metabolomics, protein studies and functional cell assays. From this we conclude that RNAi/gene expression fusion is a valid strategy to rapidly identify key processes that may be extendable to other contexts beyond damage survival.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0153970}, author = {Zanotto-Filho, Alfeu and Dashnamoorthy, Ravi and Loranc, Eva and de Souza, Luis H T and Moreira, Jos{\'e} C F and Suresh, Uthra and Chen, Yidong and Bishop, Alexander J R} } @article {889076, title = {The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic.}, journal = {Elife}, volume = {5}, year = {2016}, month = {2016}, abstract = {

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83\%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

}, issn = {2050-084X}, doi = {10.7554/eLife.16096}, author = {Swenson, Joel M and Colmenares, Serafin U and Strom, Amy R and Costes, Sylvain V and Karpen, Gary H} } @article {846766, title = {CRISPR guide RNA design for research applications.}, journal = {FEBS J}, year = {2016}, month = {2016 Jun 8}, abstract = {

The rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state-of-the-art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics. This article is protected by copyright. All rights reserved.

}, issn = {1742-4658}, doi = {10.1111/febs.13777}, author = {Mohr, Stephanie E and Hu, Yanhui and Ewen-Campen, Benjamin and Housden, Benjamin E and Viswanatha, Raghuvir and Perrimon, Norbert} } @article {825841, title = {Identification of Drosophila Zfh2 as a Mediator of Hypercapnic Immune Regulation by a Genome-Wide RNA Interference Screen.}, journal = {J Immunol}, volume = {196}, year = {2016}, month = {2016 Jan 15}, pages = {655-67}, abstract = {

Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals. We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes required for host defense in human, mouse, and Drosophila cells, and it increases mortality from bacterial infections in both mice and Drosophila. However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, zinc finger homeodomain 2 (Zfh2; mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNA interference improves survival of flies exposed to elevated CO2 and infected with Staphylococcus aureus. Tissue-specific knockdown of zfh2 in the fat body, the major immune and metabolic organ of the fly, mitigates hypercapnia-induced reductions in Dipt and other antimicrobial peptides and improves resistance of CO2-exposed flies to infection. Zfh2 mutations also partially rescue hypercapnia-induced delays in egg hatching, suggesting that Zfh2{\textquoteright}s role in mediating responses to hypercapnia extends beyond the immune system. Taken together, to our knowledge, these results identify Zfh2 as the first in vivo mediator of hypercapnic immune suppression.

}, issn = {1550-6606}, doi = {10.4049/jimmunol.1501708}, author = {Helenius, Iiro Taneli and Haake, Ryan J and Kwon, Yong-Jae and Hu, Jennifer A and Krupinski, Thomas and Casalino-Matsuda, S Marina and Sporn, Peter H S and Sznajder, Jacob I and Beitel, Greg J} } @article {954476, title = {Seipin is required for converting nascent to mature lipid droplets.}, journal = {Elife}, volume = {5}, year = {2016}, month = {2016 Aug 26}, abstract = {

How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.

}, issn = {2050-084X}, doi = {10.7554/eLife.16582}, author = {Wang, Huajin and Becuwe, Michel and Housden, Benjamin E and Chitraju, Chandramohan and Porras, Ashley J and Graham, Morven M and Liu, Xinran N and Thiam, Abdou Rachid and Savage, David B and Agarwal, Anil K and Garg, Abhimanyu and Olarte, Maria-Jesus and Lin, Qingqing and Fr{\"o}hlich, Florian and Hannibal-Bach, Hans Kristian and Upadhyayula, Srigokul and Perrimon, Norbert and Kirchhausen, Tomas and Ejsing, Christer S and Walther, Tobias C and Farese, Robert V} } @webprojectpage {913271, title = {MitoMax data set and annotations (data portal for Chen et al. 2015 "Proteomic mapping in live Drosophila tissues using an engineeredascorbate peroxidase," Proc Natl Acad Sci U S A. vol. 112(39):12093-8. PMID: 26362788; PMCID: PMC4593093.)}, year = {2015} } @webprojectpage {842181, title = {DRSC: RNA Binding Protein Library S2R+ Baseline Data (data support page for Mohr et al. 2015 "Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells" in G3)}, year = {2015}, url = {http://www.flyrnai.org/DRSC-RBP_data.php} } @article {1137727, title = {Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract}, journal = {Biochem Biophys Res Commun}, volume = {467}, year = {2015}, month = {2015 Nov 13}, pages = {400-6}, abstract = {Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. Here, we demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, we performed ex\ vivo genome-wide RNAi screening in Drosophila cultured cells, and identified several signaling factors that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that our larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.}, keywords = {Adaptor Proteins, Signal Transducing, Animals, Antigens, Differentiation, Antimicrobial Cationic Peptides, Cells, Cultured, drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Genes, Reporter, High-Throughput Screening Assays, Immunity, Innate, Larva, Luciferases, Protein-Serine-Threonine Kinases, Receptors, Immunologic, RNA Interference, Signal Transduction, Staphylococcus saprophyticus, Tissue Extracts, toll-like receptors}, issn = {1090-2104}, doi = {10.1016/j.bbrc.2015.09.138}, author = {Kanoh, Hirotaka and Kuraishi, Takayuki and Tong, Li-Li and Watanabe, Ryo and Nagata, Shinji and Kurata, Shoichiro} } @article {825811, title = {Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.}, journal = {J Cell Sci}, volume = {128}, year = {2015}, month = {2015 Jul 1}, pages = {2388-400}, abstract = {

Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes.

}, keywords = {Actin Depolymerizing Factors, Actins, Animals, Cell Nucleus, Conserved Sequence, drosophila melanogaster, Drosophila Proteins, Evolution, Molecular, Genetic Testing, genome, Mice, Models, Biological, Nerve Tissue Proteins, Phosphorylation, Polymerization, Protein-Serine-Threonine Kinases, RNA Interference}, issn = {1477-9137}, doi = {10.1242/jcs.169441}, author = {Dopie, Joseph and Rajakyl{\"a}, Eeva K and Joensuu, Merja S and Huet, Guillaume and Ferrantelli, Evelina and Xie, Tiao and J{\"a}{\"a}linoja, Harri and Jokitalo, Eija and Vartiainen, Maria K} } @article {1137726, title = {Genome-wide RNAi screening implicates the E3 ubiquitin ligase Sherpa in mediating innate immune signaling by Toll in Drosophila adults}, journal = {Sci Signal}, volume = {8}, year = {2015}, month = {2015 Oct 27}, pages = {ra107}, abstract = {The Drosophila Toll pathway plays important roles in innate immune responses against Gram-positive bacteria and fungi. To identify previously uncharacterized components of this pathway, we performed comparative, ex vivo, genome-wide RNA interference screening. In four screens, we overexpressed the Toll adaptor protein dMyd88, the downstream kinase Pelle, or the nuclear factor κB (NF-κB) homolog Dif, or we knocked down Cactus, the Drosophila homolog of mammalian inhibitor of NF-κB. On the basis of these screens, we identified the E3 ubiquitin ligase Sherpa as being necessary for the activation of Toll signaling. A loss-of-function sherpa mutant fly exhibited compromised production of antimicrobial peptides and enhanced susceptibility to infection by Gram-positive bacteria. In cultured cells, Sherpa mediated ubiquitylation of dMyd88 and Sherpa itself, and Sherpa and Drosophila SUMO (small ubiquitin-like modifier) were required for the proper membrane localization of an adaptor complex containing dMyd88. These findings highlight a role for Sherpa in Drosophila host defense and suggest the SUMOylation-mediated regulation of dMyd88 functions in Toll innate immune signaling.}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Genome-Wide Association Study, Gram-Positive Bacteria, Gram-Positive Bacterial Infections, Protein-Serine-Threonine Kinases, RNA, Small Interfering, SUMO-1 Protein, toll-like receptors, Ubiquitin-Protein Ligases}, issn = {1937-9145}, doi = {10.1126/scisignal.2005971}, author = {Kanoh, Hirotaka and Tong, Li-Li and Kuraishi, Takayuki and Suda, Yamato and Momiuchi, Yoshiki and Shishido, Fumi and Kurata, Shoichiro} } @article {825816, title = {GLAD: an Online Database of Gene List Annotation for Drosophila.}, journal = {J Genomics}, volume = {3}, year = {2015}, month = {2015}, pages = {75-81}, abstract = {

We present a resource of high quality lists of functionally related Drosophila genes, e.g. based on protein domains (kinases, transcription factors, etc.) or cellular function (e.g. autophagy, signal transduction). To establish these lists, we relied on different inputs, including curation from databases or the literature and mapping from other species. Moreover, as an added curation and quality control step, we asked experts in relevant fields to review many of the lists. The resource is available online for scientists to search and view, and is editable based on community input. Annotation of gene groups is an ongoing effort and scientific need will typically drive decisions regarding which gene lists to pursue. We anticipate that the number of lists will increase over time; that the composition of some lists will grow and/or change over time as new information becomes available; and that the lists will benefit the scientific community, e.g. at experimental design and data analysis stages. Based on this, we present an easily updatable online database, available at www.flyrnai.org/glad, at which gene group lists can be viewed, searched and downloaded.

}, issn = {1839-9940}, doi = {10.7150/jgen.12863}, author = {Hu, Yanhui and Comjean, Aram and Perkins, Lizabeth A and Perrimon, Norbert and Mohr, Stephanie E} } @article {825836, title = {Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi.}, journal = {Sci Signal}, volume = {8}, year = {2015}, month = {2015 Sep 8}, pages = {rs9}, abstract = {

The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.

}, issn = {1937-9145}, doi = {10.1126/scisignal.aab3729}, author = {Housden, Benjamin E and Valvezan, Alexander J and Kelley, Colleen and Sopko, Richelle and Hu, Yanhui and Roesel, Charles and Lin, Shuailiang and Buckner, Michael and Tao, Rong and Yilmazel, Bahar and Mohr, Stephanie E and Manning, Brendan D and Perrimon, Norbert} } @article {825826, title = {In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila.}, journal = {Genetics}, volume = {201}, year = {2015}, month = {2015 Oct}, pages = {433-42}, abstract = {

A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.

}, issn = {1943-2631}, doi = {10.1534/genetics.115.181065}, author = {Lin, Shuailiang and Ewen-Campen, Ben and Ni, Xiaochun and Housden, Benjamin E and Perrimon, Norbert} } @article {988736, title = {Pri sORF peptides induce selective proteasome-mediated protein processing}, journal = {Science}, volume = {349}, year = {2015}, month = {2015 Sep 18}, pages = {1356-8}, abstract = {

A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. Here, we show that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. Our data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators.

}, keywords = {Amino Acid Sequence, Animals, DNA-Binding Proteins, drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Molecular Sequence Data, Open Reading Frames, Peptides, Proteasome Endopeptidase Complex, Protein Structure, Tertiary, Proteolysis, RNA Interference, Transcription Factors, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitination}, issn = {1095-9203}, doi = {10.1126/science.aac5677}, author = {Zanet, J and Benrabah, E and Li, T and P{\'e}lissier-Monier, A and Chanut-Delalande, H and Ronsin, B and Bellen, H J and Payre, F and Plaza, S} } @article {825821, title = {Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells.}, journal = {G3 (Bethesda)}, volume = {5}, year = {2015}, month = {2015 Sep}, pages = {1919-24}, abstract = {

RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.

}, issn = {2160-1836}, doi = {10.1534/g3.115.019364}, author = {Mohr, Stephanie E and Hu, Yanhui and Rudd, Kirstin and Buckner, Michael and Gilly, Quentin and Foster, Blake and Sierzputowska, Katarzyna and Comjean, Aram and Ye, Bing and Perrimon, Norbert} } @article {825801, title = {Regulators of autophagosome formation in Drosophila muscles.}, journal = {PLoS Genet}, volume = {11}, year = {2015}, month = {2015 Feb}, pages = {e1005006}, abstract = {

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.

}, keywords = {Animals, Autophagy, Cell Communication, Chloroquine, drosophila melanogaster, Glycogen, GTPase-Activating Proteins, Larva, Muscle Cells, Muscle, Skeletal, Muscular Diseases, Phagosomes, Protein Interaction Maps, rab3 GTP-Binding Proteins, Sirolimus, Ubiquitin}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1005006}, author = {Zirin, Jonathan and Nieuwenhuis, Joppe and Samsonova, Anastasia and Tao, Rong and Perrimon, Norbert} } @article {825806, title = {A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.}, journal = {PLoS Genet}, volume = {11}, year = {2015}, month = {2015 Mar}, pages = {e1005056}, abstract = {

In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.

}, keywords = {Animals, Apoptosis, Cell Line, Cell Survival, drosophila melanogaster, Drosophila Proteins, Genome-Wide Association Study, Hemocytes, Insulin, Receptor Protein-Tyrosine Kinases, Receptors, Steroid, RNA Interference, Signal Transduction}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1005056}, author = {Sopko, Richelle and Lin, You Bin and Makhijani, Kalpana and Alexander, Brandy and Perrimon, Norbert and Br{\"u}ckner, Katja} } @article {825831, title = {The Transgenic RNAi Project at Harvard Medical School: Resources and Validation.}, journal = {Genetics}, volume = {201}, year = {2015}, month = {2015 Nov}, pages = {843-52}, abstract = {

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71\% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

}, issn = {1943-2631}, doi = {10.1534/genetics.115.180208}, author = {Perkins, Lizabeth A and Holderbaum, Laura and Tao, Rong and Hu, Yanhui and Sopko, Richelle and McCall, Kim and Yang-Zhou, Donghui and Flockhart, Ian and Binari, Richard and Shim, Hye-Seok and Miller, Audrey and Housden, Amy and Foos, Marianna and Randkelv, Sakara and Kelley, Colleen and Namgyal, Pema and Villalta, Christians and Liu, Lu-Ping and Jiang, Xia and Huan-Huan, Qiao and Wang, Xia and Fujiyama, Asao and Toyoda, Atsushi and Ayers, Kathleen and Blum, Allison and Czech, Benjamin and Neumuller, Ralph and Yan, Dong and Cavallaro, Amanda and Hibbard, Karen and Hall, Don and Cooley, Lynn and Hannon, Gregory J and Lehmann, Ruth and Parks, Annette and Mohr, Stephanie E and Ueda, Ryu and Kondo, Shu and Ni, Jian-Quan and Perrimon, Norbert} } @article {847501, title = {Cas9-based genome editing in Drosophila.}, journal = {Methods Enzymol}, volume = {546}, year = {2014}, month = {2014}, pages = {415-39}, abstract = {

Our ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.

}, keywords = {Animals, Cloning, Molecular, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Drosophila, Gene Targeting, Genetic Engineering, Genetic Vectors, genome, Homologous Recombination, Mutagenesis, RNA, Guide}, issn = {1557-7988}, doi = {10.1016/B978-0-12-801185-0.00019-2}, author = {Housden, Benjamin E and Lin, Shuailiang and Perrimon, Norbert} } @article {825796, title = {Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos.}, journal = {Dev Cell}, volume = {31}, year = {2014}, month = {2014 Oct 13}, pages = {114-27}, abstract = {

Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. We have generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, we have used multiplexed isobaric labeling for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos to systematically link phosphosites with relevant kinases. We demonstrate how this strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. We validate this approach by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, we show how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E.

}, keywords = {Animals, Drosophila, Drosophila Proteins, Embryo, Nonmammalian, Gene Expression Regulation, Developmental, Gene Knockdown Techniques, Gene Regulatory Networks, Phosphoprotein Phosphatases, Protein Kinases, Proteome}, issn = {1878-1551}, doi = {10.1016/j.devcel.2014.07.027}, author = {Sopko, Richelle and Foos, Marianna and Vinayagam, Arunachalam and Zhai, Bo and Binari, Richard and Hu, Yanhui and Randklev, Sakara and Perkins, Lizabeth A and Gygi, Steven P and Perrimon, Norbert} } @article {825791, title = {A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.}, journal = {PLoS One}, volume = {9}, year = {2014}, month = {2014}, pages = {e107765}, abstract = {

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.

}, keywords = {Animals, Binding Sites, Chromatin, drosophila melanogaster, Drosophila Proteins, Insulator Elements, Mass Spectrometry, Microtubule-Associated Proteins, Nuclear Proteins, Oligonucleotide Array Sequence Analysis, Repressor Proteins}, issn = {1932-6203}, doi = {10.1371/journal.pone.0107765}, author = {Bohla, Dorte and Herold, Martin and Panzer, Imke and Buxa, Melanie K and Ali, Tamer and Demmers, Jeroen and Kr{\"u}ger, Marcus and Scharfe, Maren and Jarek, Michael and Bartkuhn, Marek and Renkawitz, Rainer} } @article {825741, title = {Functional screening in Drosophila identifies Alzheimer{\textquoteright}s disease susceptibility genes and implicates Tau-mediated mechanisms.}, journal = {Hum Mol Genet}, volume = {23}, year = {2014}, month = {2014 Feb 15}, pages = {870-7}, abstract = {

Using a Drosophila model of Alzheimer{\textquoteright}s disease (AD), we systematically evaluated 67 candidate genes based on AD-associated genomic loci (P \< 10(-4)) from published human genome-wide association studies (GWAS). Genetic manipulation of 87 homologous fly genes was tested for modulation of neurotoxicity caused by human Tau, which forms neurofibrillary tangle pathology in AD. RNA interference (RNAi) targeting 9 genes enhanced Tau neurotoxicity, and in most cases reciprocal activation of gene expression suppressed Tau toxicity. Our screen implicates cindr, the fly ortholog of the human CD2AP AD susceptibility gene, as a modulator of Tau-mediated disease mechanisms. Importantly, we also identify the fly orthologs of FERMT2 and CELF1 as Tau modifiers, and these loci have been independently validated as AD susceptibility loci in the latest GWAS meta-analysis. Both CD2AP and FERMT2 have been previously implicated with roles in cell adhesion, and our screen additionally identifies a fly homolog of the human integrin adhesion receptors, ITGAM and ITGA9, as a modifier of Tau neurotoxicity. Our results highlight cell adhesion pathways as important in Tau toxicity and AD susceptibility and demonstrate the power of model organism genetic screens for the functional follow-up of human GWAS.

}, keywords = {Alzheimer Disease, Animals, Animals, Genetically Modified, Antigens, CD11b, Disease Models, Animal, drosophila melanogaster, Drosophila Proteins, Gene Knockdown Techniques, Genetic Association Studies, Genetic Predisposition to Disease, Humans, Integrins, RNA Interference, tau Proteins}, issn = {1460-2083}, doi = {10.1093/hmg/ddt478}, author = {Shulman, Joshua M and Imboywa, Selina and Giagtzoglou, Nikolaos and Powers, Martin P and Hu, Yanhui and Devenport, Danelle and Chipendo, Portia and Chibnik, Lori B and Diamond, Allison and Perrimon, Norbert and Brown, Nicholas H and De Jager, Philip L and Feany, Mel B} } @article {825776, title = {Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen.}, journal = {Mol Cell}, volume = {54}, year = {2014}, month = {2014 May 8}, pages = {485-99}, abstract = {

Polycomb group (PcG) proteins dynamically define cellular identities through epigenetic repression of key developmental genes. PcG target gene repression can be stabilized through the interaction in the nucleus at PcG foci. Here, we report the results of a\ high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. In the absence of SUMO, Pc foci coagulate into larger aggregates. Conversely, loss of function of the SUMO peptidase Velo disperses Pc foci. Moreover, SUMO and Velo colocalize with PcG proteins at PREs, and Pc SUMOylation affects its chromatin targeting, suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci.

}, keywords = {Animals, Cell Line, Cell Nucleus, Chromatin, Cluster Analysis, drosophila melanogaster, Drosophila Proteins, Gene Knockdown Techniques, Gene Ontology, Imaginal Discs, Phenotype, Polycomb-Group Proteins, Protein Binding, Protein Transport, RNA Interference, Sumoylation}, issn = {1097-4164}, doi = {10.1016/j.molcel.2014.03.004}, author = {Gonzalez, Inma and Mateos-Langerak, Julio and Thomas, Aubin and Cheutin, Thierry and Cavalli, Giacomo} } @article {825756, title = {Integrating protein-protein interaction networks with phenotypes reveals signs of interactions.}, journal = {Nat Methods}, volume = {11}, year = {2014}, month = {2014 Jan}, pages = {94-9}, abstract = {

A major objective of systems biology is to organize molecular interactions as networks and to characterize information flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the {\textquoteright}signs{\textquoteright} of interactions (i.e., activation-inhibition relationships). We constructed a Drosophila melanogaster signed PPI network consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes enolase and aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation-inhibition relationships between physically interacting proteins within signaling pathways will affect our understanding of many biological functions, including signal transduction and mechanisms of disease.

}, keywords = {Alcohol Oxidoreductases, Aldehyde Reductase, Animals, Computational Biology, drosophila melanogaster, Gene Expression Regulation, Phenotype, Proteasome Endopeptidase Complex, Protein Interaction Mapping, Protein Interaction Maps, Proteins, RNA Interference, RNA, Double-Stranded, Signal Transduction, Systems Biology}, issn = {1548-7105}, doi = {10.1038/nmeth.2733}, author = {Vinayagam, Arunachalam and Zirin, Jonathan and Roesel, Charles and Hu, Yanhui and Yilmazel, Bahar and Samsonova, Anastasia A and Neum{\"u}ller, Ralph A and Mohr, Stephanie E and Perrimon, Norbert} } @article {825781, title = {Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis.}, journal = {BMC Bioinformatics}, volume = {15}, year = {2014}, month = {2014}, pages = {192}, abstract = {

BACKGROUND: RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial. RESULTS: Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence matches (GESS) was developed to identify potential off-targeted transcripts in large-scale screen data by seed-region analysis. Here, we introduce a user-friendly web application that provides researchers a relatively quick and easy way to perform GESS analysis on data from human or mouse cell-based screens using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), as well as for Drosophila screens using shRNAs. Online GESS relies on up-to-date transcript sequence annotations for human and mouse genes extracted from NCBI Reference Sequence (RefSeq) and Drosophila genes from FlyBase. The tool also accommodates analysis with user-provided reference sequence files. CONCLUSION: Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences.

}, keywords = {Animals, drosophila melanogaster, High-Throughput Nucleotide Sequencing, MicroRNAs, RNA Interference, RNA, Small Interfering, Sequence Analysis, RNA}, issn = {1471-2105}, doi = {10.1186/1471-2105-15-192}, author = {Yilmazel, Bahar and Hu, Yanhui and Sigoillot, Frederic and Smith, Jennifer A and Shamu, Caroline E and Perrimon, Norbert and Mohr, Stephanie E} } @webprojectpage {842316, title = {Protein kinase shRNA phosphoproteomics data from D. melanogaster embryos (data portal for Sopko et al. "Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos" in Dev Cell)}, year = {2014} } @article {825761, title = {A regulatory network of Drosophila germline stem cell self-renewal.}, journal = {Dev Cell}, volume = {28}, year = {2014}, month = {2014 Feb 24}, pages = {459-73}, abstract = {

Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering \~{}25\% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.

}, keywords = {Animals, Cell Differentiation, Cell Division, Cell Lineage, drosophila melanogaster, Drosophila Proteins, Female, Germ Cells, Ovary, RNA Interference, Signal Transduction, Stem Cells}, issn = {1878-1551}, doi = {10.1016/j.devcel.2014.01.020}, author = {Yan, Dong and Neum{\"u}ller, Ralph A and Buckner, Michael and Ayers, Kathleen and Li, Hua and Hu, Yanhui and Yang-Zhou, Donghui and Pan, Lei and Wang, Xiaoxi and Kelley, Colleen and Vinayagam, Arunachalam and Binari, Richard and Randklev, Sakara and Perkins, Lizabeth A and Xie, Ting and Cooley, Lynn and Perrimon, Norbert} } @article {825771, title = {Resources for functional genomics studies in Drosophila melanogaster.}, journal = {Genetics}, volume = {197}, year = {2014}, month = {2014 May}, pages = {1-18}, abstract = {

Drosophila melanogaster has become a system of choice for functional genomic studies. Many resources, including online databases and software tools, are now available to support design or identification of relevant fly stocks and reagents or analysis and mining of existing functional genomic, transcriptomic, proteomic, etc. datasets. These include large community collections of fly stocks and plasmid clones, "meta" information sites like FlyBase and FlyMine, and an increasing number of more specialized reagents, databases, and online tools. Here, we introduce key resources useful to plan large-scale functional genomics studies in Drosophila and to analyze, integrate, and mine the results of those studies in ways that facilitate identification of highest-confidence results and generation of new hypotheses. We also discuss ways in which existing resources can be used and might be improved and suggest a few areas of future development that would further support large- and small-scale studies in Drosophila and facilitate use of Drosophila information by the research community more generally.

}, keywords = {Animals, Databases, Genetic, drosophila melanogaster, Genetic Engineering, Genome, Insect, Genomics, Humans}, issn = {1943-2631}, doi = {10.1534/genetics.113.154344}, author = {Mohr, Stephanie E and Hu, Yanhui and Kim, Kevin and Housden, Benjamin E and Perrimon, Norbert} } @article {825786, title = {RNAi screening comes of age: improved techniques and complementary approaches.}, journal = {Nat Rev Mol Cell Biol}, volume = {15}, year = {2014}, month = {2014 Sep}, pages = {591-600}, abstract = {

Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.

}, keywords = {Animals, Gene Regulatory Networks, Genetic Testing, Humans, Inverted Repeat Sequences, RNA Interference, RNA, Small Interfering}, issn = {1471-0080}, doi = {10.1038/nrm3860}, author = {Mohr, Stephanie E and Smith, Jennifer A and Shamu, Caroline E and Neum{\"u}ller, Ralph A and Perrimon, Norbert} } @article {825766, title = {RNAi screening in Drosophila cells and in vivo.}, journal = {Methods}, volume = {68}, year = {2014}, month = {2014 Jun 15}, pages = {82-8}, abstract = {

Here, I discuss how RNAi screening can be used effectively to uncover gene function. Specifically, I discuss the types of high-throughput assays that can be done in Drosophila cells and in vivo, RNAi reagent design and available reagent collections, automated screen pipelines, analysis of screen results, and approaches to RNAi results verification.

}, keywords = {Animals, developmental biology, Drosophila, Genomics, High-Throughput Screening Assays, RNA Interference}, issn = {1095-9130}, doi = {10.1016/j.ymeth.2014.02.018}, author = {Mohr, Stephanie E} } @article {825736, title = {AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e74296}, abstract = {

Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.

}, keywords = {Animals, Argonaute Proteins, Biosensing Techniques, Cell Line, Drosophila, Drosophila Proteins, Green Fluorescent Proteins, MicroRNAs, Promoter Regions, Genetic, RNA Interference}, issn = {1932-6203}, doi = {10.1371/journal.pone.0074296}, author = {Carr{\'e}, Cl{\'e}ment and Jacquier, Caroline and Boug{\'e}, Anne-Laure and de Chaumont, Fabrice and Besnard-Guerin, Corinne and Thomassin, H{\'e}l{\`e}ne and Pidoux, Josette and Da Silva, Bruno and Chalatsi, Eleftheria and Zahra, Sarah and Olivo-Marin, Jean-Christophe and Munier-Lehmann, H{\'e}l{\`e}ne and Antoniewski, Christophe} } @article {825731, title = {Conserved regulators of nucleolar size revealed by global phenotypic analyses.}, journal = {Sci Signal}, volume = {6}, year = {2013}, month = {2013 Aug 20}, pages = {ra70}, abstract = {

Regulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.

}, keywords = {Cell Nucleolus, DNA, Fungal, DNA, Ribosomal, Genes, Fungal, Genes, rRNA, Histones, RNA Polymerase I, RNA, Fungal, RNA, Ribosomal, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic}, issn = {1937-9145}, doi = {10.1126/scisignal.2004145}, author = {Neum{\"u}ller, Ralph A and Gross, Thomas and Samsonova, Anastasia A and Vinayagam, Arunachalam and Buckner, Michael and Founk, Karen and Hu, Yanhui and Sharifpoor, Sara and Rosebrock, Adam P and Andrews, Brenda and Winston, Fred and Perrimon, Norbert} } @article {825676, title = {Depleting gene activities in early Drosophila embryos with the "maternal-Gal4-shRNA" system.}, journal = {Genetics}, volume = {193}, year = {2013}, month = {2013 Jan}, pages = {51-61}, abstract = {

In a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.

}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Female, Gene Dosage, Gene Expression Regulation, Developmental, Gene Knockdown Techniques, Male, Oogenesis, Phenotype, RNA Interference, RNA, Small Interfering, Transcription Factors, Transcription, Genetic}, issn = {1943-2631}, doi = {10.1534/genetics.112.144915}, author = {Staller, Max V and Yan, Dong and Randklev, Sakara and Bragdon, Meghan D and Wunderlich, Zeba B and Tao, Rong and Perkins, Lizabeth A and Depace, Angela H and Perrimon, Norbert} } @article {825721, title = {An essential and NSF independent role for α-SNAP in store-operated calcium entry.}, journal = {Elife}, volume = {2}, year = {2013}, month = {2013}, pages = {e00802}, abstract = {

Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca(2+) sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE. DOI:http://dx.doi.org/10.7554/eLife.00802.001.

}, keywords = {Animals, calcium, Drosophila, Humans, Ion Transport, NFATC Transcription Factors, Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins}, issn = {2050-084X}, doi = {10.7554/eLife.00802}, author = {Miao, Yong and Miner, Cathrine and Zhang, Lei and Hanson, Phyllis I and Dani, Adish and Vig, Monika} } @article {825701, title = {The evolutionarily conserved protein CG9186 is associated with lipid droplets, required for their positioning and for fat storage.}, journal = {J Cell Sci}, volume = {126}, year = {2013}, month = {2013 May 15}, pages = {2198-212}, abstract = {

Lipid droplets (LDs) are specialized cell organelles for the storage of energy-rich lipids. Although lipid storage is a conserved feature of all cells and organisms, little is known about fundamental aspects of the cell biology of LDs, including their biogenesis, structural assembly and subcellular positioning, and the regulation of organismic energy homeostasis. We identified a novel LD-associated protein family, represented by the Drosophila protein CG9186 and its murine homolog MGI:1916082. In the absence of LDs, both proteins localize at the endoplasmic reticulum (ER). Upon lipid storage induction, they translocate to LDs using an evolutionarily conserved targeting mechanism that acts through a 60-amino-acid targeting motif in the center of the CG9186 protein. Overexpression of CG9186, and MGI:1916082, causes clustering of LDs in both tissue culture and salivary gland cells, whereas RNAi knockdown of CG9186 results in a reduction of LDs. Organismal RNAi knockdown of CG9186 results in a reduction in lipid storage levels of the fly. The results indicate that we identified the first members of a novel and evolutionarily conserved family of lipid storage regulators, which are also required to properly position LDs within cells.

}, keywords = {Amino Acid Sequence, Animals, Cells, Cultured, Conserved Sequence, drosophila melanogaster, Drosophila Proteins, Endoplasmic Reticulum, Evolution, Molecular, Homeostasis, Lipid Metabolism, Lipoprotein Lipase, Mice, Molecular Sequence Data, Phylogeny, Protein Sorting Signals, Proteins, Rats, RNA, Small Interfering, Salivary Glands, Transgenes, Vacuoles}, issn = {1477-9137}, doi = {10.1242/jcs.120493}, author = {Thiel, Katharina and Heier, Christoph and Haberl, Verena and Thul, Peter J and Oberer, Monika and Lass, Achim and J{\"a}ckle, Herbert and Beller, Mathias} } @article {825726, title = {FlyPrimerBank: an online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents.}, journal = {G3 (Bethesda)}, volume = {3}, year = {2013}, month = {2013 Sep}, pages = {1607-16}, abstract = {

The evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.

}, keywords = {Algorithms, Animals, Databases, Genetic, DNA Primers, drosophila melanogaster, Embryo, Nonmammalian, Gene Expression, Internet, Real-Time Polymerase Chain Reaction, RNA Interference, RNA, Small Interfering, User-Computer Interface}, issn = {2160-1836}, doi = {10.1534/g3.113.007021}, author = {Hu, Yanhui and Sopko, Richelle and Foos, Marianna and Kelley, Colleen and Flockhart, Ian and Ammeux, Noemie and Wang, Xiaowei and Perkins, Lizabeth and Perrimon, Norbert and Mohr, Stephanie E} } @article {825696, title = {Genetic determinants of phosphate response in Drosophila.}, journal = {PLoS One}, volume = {8}, year = {2013}, month = {2013}, pages = {e56753}, abstract = {

Phosphate is required for many important cellular processes and having too little phosphate or too much can cause disease and reduce life span in humans. However, the mechanisms underlying homeostatic control of extracellular phosphate levels and cellular effects of phosphate are poorly understood. Here, we establish Drosophila melanogaster as a model system for the study of phosphate effects. We found that Drosophila larval development depends on the availability of phosphate in the medium. Conversely, life span is reduced when adult flies are cultured on high phosphate medium or when hemolymph phosphate is increased in flies with impaired malpighian tubules. In addition, RNAi-mediated inhibition of MAPK-signaling by knockdown of Ras85D, phl/D-Raf or Dsor1/MEK affects larval development, adult life span and hemolymph phosphate, suggesting that some in vivo effects involve activation of this signaling pathway by phosphate. To identify novel genetic determinants of phosphate responses, we used Drosophila hemocyte-like cultured cells (S2R+) to perform a genome-wide RNAi screen using MAPK activation as the readout. We identified a number of candidate genes potentially important for the cellular response to phosphate. Evaluation of 51 genes in live flies revealed some that affect larval development, adult life span and hemolymph phosphate levels.

}, keywords = {Animals, Cell Line, drosophila melanogaster, Drosophila Proteins, Hemocytes, Hemolymph, Longevity, Malpighian Tubules, MAP Kinase Signaling System, Phosphates, RNA Interference}, issn = {1932-6203}, doi = {10.1371/journal.pone.0056753}, author = {Bergwitz, Clemens and Wee, Mark J and Sinha, Sumi and Huang, Joanne and DeRobertis, Charles and Mensah, Lawrence B and Cohen, Jonathan and Friedman, Adam and Kulkarni, Meghana and Hu, Yanhui and Vinayagam, Arunachalam and Schnall-Levin, Michael and Berger, Bonnie and Perkins, Lizabeth A and Mohr, Stephanie E and Perrimon, Norbert} } @article {825686, title = {A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells.}, journal = {Genome Res}, volume = {23}, year = {2013}, month = {2013 Apr}, pages = {581-91}, abstract = {

The androgen receptor (AR) is a mediator of both androgen-dependent and castration-resistant prostate cancers. Identification of cellular factors affecting AR transcriptional activity could in principle yield new targets that reduce AR activity and combat prostate cancer, yet a comprehensive analysis of the genes required for AR-dependent transcriptional activity has not been determined. Using an unbiased genetic approach that takes advantage of the evolutionary conservation of AR signaling, we have conducted a genome-wide RNAi screen in Drosophila cells for genes required for AR transcriptional activity and applied the results to human prostate cancer cells. We identified 45 AR-regulators, which include known pathway components and genes with functions not previously linked to AR regulation, such as HIPK2 (a protein kinase) and MED19 (a subunit of the Mediator complex). Depletion of HIPK2 and MED19 in human prostate cancer cells decreased AR target gene expression and, importantly, reduced the proliferation of androgen-dependent and castration-resistant prostate cancer cells. We also systematically analyzed additional Mediator subunits and uncovered a small subset of Mediator subunits that interpret AR signaling and affect AR-dependent transcription and prostate cancer cell proliferation. Importantly, targeting of HIPK2 by an FDA-approved kinase inhibitor phenocopied the effect of depletion by RNAi and reduced the growth of AR-positive, but not AR-negative, treatment-resistant prostate cancer cells. Thus, our screen has yielded new AR regulators including drugable targets that reduce the proliferation of castration-resistant prostate cancer cells.

}, keywords = {Animals, Carrier Proteins, Cell Line, Tumor, Cell Proliferation, Cluster Analysis, Drosophila, Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Genome-Wide Association Study, Humans, Male, Mediator Complex, Prostatic Neoplasms, Protein Binding, Protein Kinase Inhibitors, Protein-Serine-Threonine Kinases, Receptors, Androgen, RNA Interference, Transcription, Genetic}, issn = {1549-5469}, doi = {10.1101/gr.144774.112}, author = {Imberg-Kazdan, Keren and Ha, Susan and Greenfield, Alex and Poultney, Christopher S and Bonneau, Richard and Logan, Susan K and Garabedian, Michael J} } @article {825706, title = {A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.}, journal = {Mol Cell}, volume = {50}, year = {2013}, month = {2013 Jun 6}, pages = {736-48}, abstract = {

A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and components of the piRNA pathway, we\ carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

}, keywords = {Animals, DNA Transposable Elements, drosophila melanogaster, Drosophila Proteins, Female, Gene Knockdown Techniques, Gene Silencing, Genome, Insect, Ovary, Reproducibility of Results, RNA Interference, RNA, Small Interfering, SUMO-1 Protein}, issn = {1097-4164}, doi = {10.1016/j.molcel.2013.04.006}, author = {Muerdter, Felix and Guzzardo, Paloma M and Gillis, Jesse and Luo, Yicheng and Yu, Yang and Chen, Caifu and Fekete, Richard and Hannon, Gregory J} } @article {825746, title = {The Hippo signaling pathway interactome.}, journal = {Science}, volume = {342}, year = {2013}, month = {2013 Nov 8}, pages = {737-40}, abstract = {

The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67\% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer.

}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Intracellular Signaling Peptides and Proteins, Nuclear Proteins, Protein Interaction Maps, Protein-Serine-Threonine Kinases, Proteome, RNA Interference, Trans-Activators}, issn = {1095-9203}, doi = {10.1126/science.1243971}, author = {Kwon, Young and Vinayagam, Arunachalam and Sun, Xiaoyun and Dephoure, Noah and Gygi, Steven P and Hong, Pengyu and Perrimon, Norbert} } @article {825751, title = {Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9.}, journal = {Proc Natl Acad Sci U S A}, volume = {110}, year = {2013}, month = {2013 Nov 19}, pages = {19012-7}, abstract = {

The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.

}, keywords = {Animals, Animals, Genetically Modified, CRISPR-Cas Systems, Databases, Genetic, drosophila melanogaster, Drosophila Proteins, Genetic Engineering, Genomics, Germ Cells, Mutagenesis, Promoter Regions, Genetic, RNA-Binding Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.1318481110}, author = {Ren, Xingjie and Sun, Jin and Housden, Benjamin E and Hu, Yanhui and Roesel, Charles and Lin, Shuailiang and Liu, Lu-Ping and Yang, Zhihao and Mao, Decai and Sun, Lingzhu and Wu, Qujie and Ji, Jun-Yuan and Xi, Jianzhong and Mohr, Stephanie E and Xu, Jiang and Perrimon, Norbert and Ni, Jian-Quan} } @article {825691, title = {Protein complex-based analysis framework for high-throughput data sets.}, journal = {Sci Signal}, volume = {6}, year = {2013}, month = {2013 Feb 26}, pages = {rs5}, abstract = {

Analysis of high-throughput data increasingly relies on pathway annotation and functional information derived from Gene Ontology. This approach has limitations, in particular for the analysis of network dynamics over time or under different experimental conditions, in which modules within a network rather than complete pathways might respond and change. We report an analysis framework based on protein complexes, which are at the core of network reorganization. We generated a protein complex resource for human, Drosophila, and yeast from the literature and databases of protein-protein interaction networks, with each species having thousands of complexes. We developed COMPLEAT (http://www.flyrnai.org/compleat), a tool for data mining and visualization for complex-based analysis of high-throughput data sets, as well as analysis and integration of heterogeneous proteomics and gene expression data sets. With COMPLEAT, we identified dynamically regulated protein complexes among genome-wide RNA interference data sets that used the abundance of phosphorylated extracellular signal-regulated kinase in cells stimulated with either insulin or epidermal growth factor as the output. The analysis predicted that the Brahma complex participated in the insulin response.

}, keywords = {Animals, Cell Cycle Proteins, Data Mining, Databases, Genetic, drosophila melanogaster, Drosophila Proteins, Gene Expression Profiling, High Mobility Group Proteins, High-Throughput Screening Assays, Humans, Insulin, Internet, Molecular Sequence Annotation, Multiprotein Complexes, Protein Interaction Maps, proteomics, RNA Interference, Saccharomyces cerevisiae, Software, Species Specificity, Systems Biology, Trans-Activators}, issn = {1937-9145}, doi = {10.1126/scisignal.2003629}, author = {Vinayagam, Arunachalam and Hu, Yanhui and Kulkarni, Meghana and Roesel, Charles and Sopko, Richelle and Mohr, Stephanie E and Perrimon, Norbert} } @article {825711, title = {A screen for morphological complexity identifies regulators of switch-like transitions between discrete cell shapes.}, journal = {Nat Cell Biol}, volume = {15}, year = {2013}, month = {2013 Jul}, pages = {860-71}, abstract = {

The way in which cells adopt different morphologies is not fully understood. Cell shape could be a continuous variable or restricted to a set of discrete forms. We developed quantitative methods to describe cell shape and show that Drosophila haemocytes in culture are a heterogeneous mixture of five discrete morphologies. In an RNAi screen of genes affecting the morphological complexity of heterogeneous cell populations, we found that most genes regulate the transition between discrete shapes rather than generating new morphologies. In particular, we identified a subset of genes, including the tumour suppressor PTEN, that decrease the heterogeneity of the population, leading to populations enriched in rounded or elongated forms. We show that these genes have a highly conserved function as regulators of cell shape in both mouse and human metastatic melanoma cells.

}, keywords = {Animals, Cell Shape, drosophila melanogaster, Genes, Tumor Suppressor, Genetic Testing, Humans, Melanoma, Mice, Phenotype, PTEN Phosphohydrolase, RNA Interference, Tumor Cells, Cultured}, issn = {1476-4679}, doi = {10.1038/ncb2764}, author = {Yin, Zheng and Sadok, Amine and Sailem, Heba and McCarthy, Afshan and Xia, Xiaofeng and Li, Fuhai and Garcia, Mar Arias and Evans, Louise and Barr, Alexis R and Perrimon, Norbert and Marshall, Christopher J and Wong, Stephen T C and Bakal, Chris} } @article {825716, title = {UP-TORR: online tool for accurate and Up-to-Date annotation of RNAi Reagents.}, journal = {Genetics}, volume = {195}, year = {2013}, month = {2013 Sep}, pages = {37-45}, abstract = {

RNA interference (RNAi) is a widely adopted tool for loss-of-function studies but RNAi results only have biological relevance if the reagents are appropriately mapped to genes. Several groups have designed and generated RNAi reagent libraries for studies in cells or in vivo for Drosophila and other species. At first glance, matching RNAi reagents to genes appears to be a simple problem, as each reagent is typically designed to target a single gene. In practice, however, the reagent-gene relationship is complex. Although the sequences of oligonucleotides used to generate most types of RNAi reagents are static, the reference genome and gene annotations are regularly updated. Thus, at the time a researcher chooses an RNAi reagent or analyzes RNAi data, the most current interpretation of the RNAi reagent-gene relationship, as well as related information regarding specificity (e.g., predicted off-target effects), can be different from the original interpretation. Here, we describe a set of strategies and an accompanying online tool, UP-TORR (for Updated Targets of RNAi Reagents; www.flyrnai.org/up-torr), useful for accurate and up-to-date annotation of cell-based and in vivo RNAi reagents. Importantly, UP-TORR automatically synchronizes with gene annotations daily, retrieving the most current information available, and for Drosophila, also synchronizes with the major reagent collections. Thus, UP-TORR allows users to choose the most appropriate RNAi reagents at the onset of a study, as well as to perform the most appropriate analyses of results of RNAi-based studies.

}, keywords = {Animals, Drosophila, Indicators and Reagents, Internet, Molecular Sequence Annotation, RNA Interference, RNA, Small Interfering, Software}, issn = {1943-2631}, doi = {10.1534/genetics.113.151340}, author = {Hu, Yanhui and Roesel, Charles and Flockhart, Ian and Perkins, Lizabeth and Perrimon, Norbert and Mohr, Stephanie E} } @article {825636, title = {Active maintenance of nuclear actin by importin 9 supports transcription.}, journal = {Proc Natl Acad Sci U S A}, volume = {109}, year = {2012}, month = {2012 Feb 28}, pages = {E544-52}, abstract = {

Besides its essential and well established role as a component of the cytoskeleton, actin is also present in the cell nucleus, where it has been linked to many processes that control gene expression. For example, nuclear actin regulates the activity of specific transcription factors, associates with all three RNA polymerases, and is a component of many chromatin remodelling complexes. Despite the fact that two export receptors, Crm1 and exportin 6, have been linked to nuclear export of actin, the mechanism by which actin enters the nucleus to elicit these essential functions has not been determined. It is also unclear whether actin is actively exchanged between the nucleus and the cytoplasm, and whether this connection has any functional significance for the cell. By applying a variety of live-cell imaging techniques we revealed that actin constantly shuttles in and out of the nucleus. The fast transport rates, which depend on the availability of actin monomers, suggest an active transport mechanism in both directions. Importantly, we identified importin 9 as the nuclear import factor for actin. Furthermore, our RNAi experiments showed that the active maintenance of nuclear actin levels by importin 9 is required for maximal transcriptional activity. Measurements of nuclear export rates and depletion studies also clarified that nuclear export of actin is mediated by exportin 6, and not by Crm1. These results demonstrate that cytoplasmic and nuclear actin pools are dynamically connected and identify the nuclear import and export mechanisms of actin.

}, keywords = {Actin Depolymerizing Factors, Actins, Active Transport, Cell Nucleus, Animals, beta Karyopherins, Cell Line, Cytoplasm, drosophila melanogaster, Drosophila Proteins, Genes, Reporter, Genetic Complementation Test, Green Fluorescent Proteins, Humans, Karyopherins, Mice, Microscopy, Confocal, NIH 3T3 Cells, Photobleaching, ran GTP-Binding Protein, Receptors, Cytoplasmic and Nuclear, Recombinant Fusion Proteins, RNA Interference, RNA, Small Interfering, Transcription, Genetic}, issn = {1091-6490}, doi = {10.1073/pnas.1118880109}, author = {Dopie, Joseph and Skarp, Kari-Pekka and Rajakyl{\"a}, Eeva Kaisa and Tanhuanp{\"a}{\"a}, Kimmo and Vartiainen, Maria K} } @article {825671, title = {Anion-sensitive fluorophore identifies the Drosophila swell-activated chloride channel in a genome-wide RNA interference screen.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e46865}, abstract = {

When cells swell in hypo-osmotic solutions, chloride-selective ion channels (Cl(swell)) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian Cl(swell) channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila Cl(swell) channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the Cl(swell) current (I(Clswell)). We further demonstrate the essential contribution of dBest1 to Drosophila I(Clswell) with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous I(Clswell). We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila-like I(Clswell). In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the Cl(swell) channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.

}, keywords = {Amino Acid Sequence, Animals, Chloride Channels, drosophila melanogaster, Drosophila Proteins, Genomics, Humans, Luminescent Proteins, Mice, Molecular Sequence Data, Mutation, RNA Interference}, issn = {1932-6203}, doi = {10.1371/journal.pone.0046865}, author = {Stotz, Stephanie C and Clapham, David E} } @article {825681, title = {BUHO: a MATLAB script for the study of stress granules and processing bodies by high-throughput image analysis.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e51495}, abstract = {

The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5\% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny.

}, keywords = {Algorithms, Animals, drosophila melanogaster, Image Processing, Computer-Assisted, Molecular Imaging, Organelles, oxidative stress, RNA Interference, RNA, Messenger, Software, Synapses, Time Factors}, issn = {1932-6203}, doi = {10.1371/journal.pone.0051495}, author = {Perez-Pepe, Marcelo and Slomiansky, Victoria and Loschi, Mariela and Luchelli, Luciana and Neme, Maximiliano and Thomas, Mar{\'\i}a Gabriela and Boccaccio, Graciela Lidia} } @article {825666, title = {Control of proinflammatory gene programs by regulated trimethylation and demethylation of histone H4K20.}, journal = {Mol Cell}, volume = {48}, year = {2012}, month = {2012 Oct 12}, pages = {28-38}, abstract = {

Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of coregulator complexes that function to read, write, and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll-like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 trimethylation/demethylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.

}, keywords = {Animals, Cell Line, Co-Repressor Proteins, Drosophila, Gene Expression Regulation, HEK293 Cells, Histone-Lysine N-Methyltransferase, Histones, Humans, Inflammation, Macrophages, Methylation, Mice, Models, Biological, NF-kappa B, Promoter Regions, Genetic, Signal Transduction, Toll-Like Receptor 4}, issn = {1097-4164}, doi = {10.1016/j.molcel.2012.07.020}, author = {Stender, Joshua D and Pascual, Gabriel and Liu, Wen and Kaikkonen, Minna U and Do, Kevin and Spann, Nathanael J and Boutros, Michael and Perrimon, Norbert and Rosenfeld, Michael G and Glass, Christopher K} } @article {825656, title = {Differential RNAi screening provides insights into the rewiring of signalling networks during oxidative stress.}, journal = {Mol Biosyst}, volume = {8}, year = {2012}, month = {2012 Oct}, pages = {2605-13}, abstract = {

Reactive Oxygen Species (ROS) are a natural by-product of cellular growth and proliferation, and are required for fundamental processes such as protein-folding and signal transduction. However, ROS accumulation, and the onset of oxidative stress, can negatively impact cellular and genomic integrity. Signalling networks have evolved to respond to oxidative stress by engaging diverse enzymatic and non-enzymatic antioxidant mechanisms to restore redox homeostasis. The architecture of oxidative stress response networks during periods of normal growth, and how increased ROS levels dynamically reconfigure these networks are largely unknown. In order to gain insight into the structure of signalling networks that promote redox homeostasis we first performed genome-scale RNAi screens to identify novel suppressors of superoxide accumulation. We then infer relationships between redox regulators by hierarchical clustering of phenotypic signatures describing how gene inhibition affects superoxide levels, cellular viability, and morphology across different genetic backgrounds. Genes that cluster together are likely to act in the same signalling pathway/complex and thus make "functional interactions". Moreover we also calculate differential phenotypic signatures describing the difference in cellular phenotypes following RNAi between untreated cells and cells submitted to oxidative stress. Using both phenotypic signatures and differential signatures we construct a network model of functional interactions that occur between components of the redox homeostasis network, and how such interactions become rewired in the presence of oxidative stress. This network model predicts a functional interaction between the transcription factor Jun and the IRE1 kinase, which we validate in an orthogonal assay. We thus demonstrate the ability of systems-biology approaches to identify novel signalling events.

}, keywords = {Animals, Antioxidants, Cell Survival, Cells, Cultured, drosophila melanogaster, Drosophila Proteins, Endoribonucleases, Gene Expression, Gene Regulatory Networks, Multigene Family, Oxidation-Reduction, oxidative stress, Paraquat, Protein Interaction Mapping, Proto-Oncogene Proteins c-jun, RNA Interference, RNA, Small Interfering, Signal Transduction, Stress, Physiological, Superoxides}, issn = {1742-2051}, doi = {10.1039/c2mb25092f}, author = {Garcia, Mar Arias and Alvarez, Miguel Sanchez and Sailem, Heba and Bousgouni, Vicky and Sero, Julia and Bakal, Chris} } @article {844236, title = {FlyRNAi.org--the database of the Drosophila RNAi screening center: 2012 update.}, journal = {Nucleic Acids Res}, volume = {40}, year = {2012}, month = {2012 Jan}, pages = {D715-9}, abstract = {

FlyRNAi (http://www.flyrnai.org), the database and website of the Drosophila RNAi Screening Center (DRSC) at Harvard Medical School, serves a dual role, tracking both production of reagents for RNA interference (RNAi) screening in Drosophila cells and RNAi screen results. The database and website is used as a platform for community availability of protocols, tools, and other resources useful to researchers planning, conducting, analyzing or interpreting the results of Drosophila RNAi screens. Based on our own experience and user feedback, we have made several changes. Specifically, we have restructured the database to accommodate new types of reagents; added information about new RNAi libraries and other reagents; updated the user interface and website; and added new tools of use to the Drosophila community and others. Overall, the result is a more useful, flexible and comprehensive website and database.

}, keywords = {Animals, Databases, Genetic, Drosophila, Genes, Insect, Genome, Insect, Indicators and Reagents, Internet, RNA Interference, Software}, issn = {1362-4962}, doi = {10.1093/nar/gkr953}, author = {Flockhart, Ian T and Booker, Matthew and Hu, Yanhui and McElvany, Benjamin and Gilly, Quentin and Mathey-Prevot, Bernard and Perrimon, Norbert and Mohr, Stephanie E} } @article {825641, title = {A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.}, journal = {PLoS One}, volume = {7}, year = {2012}, month = {2012}, pages = {e33665}, abstract = {

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

}, keywords = {Animals, Animals, Genetically Modified, Cholesterol, drosophila melanogaster, Drosophila Proteins, Genes, Insect, Genetic Testing, Genome, Insect, Golgi Apparatus, Hedgehog Proteins, Intracellular Space, Luciferases, Renilla, Protein Processing, Post-Translational, Protein Transport, Recombinant Fusion Proteins, Reproducibility of Results, RNA Interference, RNA, Double-Stranded, Subcellular Fractions, Wnt1 Protein}, issn = {1932-6203}, doi = {10.1371/journal.pone.0033665}, author = {Aikin, Reid and Cervantes, Alexandra and D{\textquoteright}Angelo, Gisela and Ruel, Laurent and Lacas-Gervais, Sandra and Schaub, S{\'e}bastien and Th{\'e}rond, Pascal} } @article {825661, title = {Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.}, journal = {PLoS Genet}, volume = {8}, year = {2012}, month = {2012}, pages = {e1002830}, abstract = {

Sex chromosome dosage compensation in Drosophila provides a model for understanding how chromatin organization can modulate coordinate gene regulation. Male Drosophila increase the transcript levels of genes on the single male X approximately two-fold to equal the gene expression in females, which have two X-chromosomes. Dosage compensation is mediated by the Male-Specific Lethal (MSL) histone acetyltransferase complex. Five core components of the MSL complex were identified by genetic screens for genes that are specifically required for male viability and are dispensable for females. However, because dosage compensation must interface with the general transcriptional machinery, it is likely that identifying additional regulators that are not strictly male-specific will be key to understanding the process at a mechanistic level. Such regulators would not have been recovered from previous male-specific lethal screening strategies. Therefore, we have performed a cell culture-based, genome-wide RNAi screen to search for factors required for MSL targeting or function. Here we focus on the discovery of proteins that function to promote MSL complex recruitment to "chromatin entry sites," which are proposed to be the initial sites of MSL targeting. We find that components of the NSL (Non-specific lethal) complex, and a previously unstudied zinc-finger protein, facilitate MSL targeting and display a striking enrichment at MSL entry sites. Identification of these factors provides new insight into how MSL complex establishes the specialized hyperactive chromatin required for dosage compensation in Drosophila.

}, keywords = {Animals, Cell Line, Chromatin, DNA-Binding Proteins, Dosage Compensation, Genetic, drosophila melanogaster, Drosophila Proteins, Female, Gene Expression Regulation, Male, Nuclear Proteins, RNA Interference, Transcription Factors, X Chromosome}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1002830}, author = {Larschan, Erica and Soruco, Marcela M L and Lee, Ok-Kyung and Peng, Shouyong and Bishop, Eric and Chery, Jessica and Goebel, Karen and Feng, Jessica and Park, Peter J and Kuroda, Mitzi I} } @article {825651, title = {Identification of genes that promote or antagonize somatic homolog pairing using a high-throughput FISH-based screen.}, journal = {PLoS Genet}, volume = {8}, year = {2012}, month = {2012}, pages = {e1002667}, abstract = {

The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate "pairing promoting genes" and candidate "anti-pairing genes," providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing.

}, keywords = {Anaphase-Promoting Complex-Cyclosome, Aneuploidy, Animals, Cell Culture Techniques, Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, Chromosome Pairing, DNA Breaks, Double-Stranded, DNA-Binding Proteins, drosophila melanogaster, Drosophila Proteins, Heterochromatin, In Situ Hybridization, Fluorescence, Meiosis, Microtubule-Associated Proteins, Mitosis, Recombination, Genetic, RNA Interference, Ubiquitin-Protein Ligase Complexes}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1002667}, author = {Joyce, Eric F and Williams, Benjamin R and Xie, Tiao and Wu, C-Ting} } @article {825626, title = {RNAi screening: new approaches, understandings, and organisms.}, journal = {Wiley Interdiscip Rev RNA}, volume = {3}, year = {2012}, month = {2012 Mar-Apr}, pages = {145-58}, abstract = {

RNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity, and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.

}, keywords = {Animals, Gene Knockdown Techniques, Genetic Testing, Genomics, High-Throughput Screening Assays, Humans, Phenotype, RNA Interference, RNA, Small Interfering}, issn = {1757-7012}, doi = {10.1002/wrna.110}, author = {Mohr, Stephanie E and Perrimon, Norbert} } @article {825646, title = {Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context.}, journal = {PLoS Genet}, volume = {8}, year = {2012}, month = {2012}, pages = {e1002646}, abstract = {

The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at "entry sites" that contain a consensus sequence motif ("MSL recognition element" or MRE). However, this motif is only \~{}2 fold enriched on X, and only a fraction of the motifs on X are initially targeted. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells (which contain MSL complex) and female Kc cells (which lack the complex), we find that the presence of active chromatin modifications, together with an elevated local GC content in the surrounding sequences, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our analysis can serve as a model for identifying chromatin and local sequence features that may contribute to selection of functional protein binding sites in the genome.

}, keywords = {Acetylation, Animals, Base Composition, Binding Sites, Chromatin, Dosage Compensation, Genetic, drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Genes, X-Linked, Histones, Male, Nuclear Proteins, Nucleosomes, Nucleotide Motifs, Protein-Serine-Threonine Kinases, RNA Interference, RNA-Binding Proteins, Transcription Factors, Transcription, Genetic, X Chromosome}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1002646}, author = {Alekseyenko, Artyom A and Ho, Joshua W K and Peng, Shouyong and Gelbart, Marnie and Tolstorukov, Michael Y and Plachetka, Annette and Kharchenko, Peter V and Jung, Youngsook L and Gorchakov, Andrey A and Larschan, Erica and Gu, Tingting and Minoda, Aki and Riddle, Nicole C and Schwartz, Yuri B and Elgin, Sarah C R and Karpen, Gary H and Pirrotta, Vincenzo and Kuroda, Mitzi I and Park, Peter J} } @article {991911, title = {Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes}, journal = {Genetics}, volume = {190}, year = {2012}, month = {2012 Mar}, pages = {931-40}, abstract = {

In Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.

}, keywords = {Animals, Cell Line, Cell Survival, Drosophila, Drosophila Proteins, Embryo, Nonmammalian, Female, Fluorescent Dyes, Gene Order, Gene Silencing, Green Fluorescent Proteins, Multiprotein Complexes, Peptide Elongation Factors, Protein Binding, Protein Interaction Mapping, Recombinant Fusion Proteins, RNA, Small Interfering, Stem Cells}, issn = {1943-2631}, doi = {10.1534/genetics.111.136465}, author = {Neum{\"u}ller, Ralph A and Wirtz-Peitz, Frederik and Lee, Stella and Kwon, Young and Buckner, Michael and Hoskins, Roger A and Venken, Koen J T and Bellen, Hugo J and Mohr, Stephanie E and Perrimon, Norbert} } @article {825581, title = {Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.}, journal = {Mol Cell Biol}, volume = {31}, year = {2011}, month = {2011 Feb}, pages = {897-909}, abstract = {

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap {\textquoteright}n{\textquoteright} collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

}, keywords = {Amino Acid Sequence, Animals, Base Sequence, Carrier Proteins, Cell Line, drosophila melanogaster, Drosophila Proteins, Evolution, Molecular, Gene Knockdown Techniques, Genes, Insect, Intracellular Signaling Peptides and Proteins, Mammals, Models, Biological, Molecular Sequence Data, oxidative stress, Phylogeny, Proteasome Endopeptidase Complex, Repressor Proteins, RNA Interference, Sequence Homology, Amino Acid}, issn = {1098-5549}, doi = {10.1128/MCB.00799-10}, author = {Grimberg, Kristian Bj{\"o}rk and Beskow, Anne and Lundin, Daniel and Davis, Monica M and Young, Patrick} } @article {825616, title = {Comparative RNAi screening identifies a conserved core metazoan actinome by phenotype.}, journal = {J Cell Biol}, volume = {194}, year = {2011}, month = {2011 Sep 5}, pages = {789-805}, abstract = {

Although a large number of actin-binding proteins and their regulators have been identified through classical approaches, gaps in our knowledge remain. Here, we used genome-wide RNA interference as a systematic method to define metazoan actin regulators based on visual phenotype. Using comparative screens in cultured Drosophila and human cells, we generated phenotypic profiles for annotated actin regulators together with proteins bearing predicted actin-binding domains. These phenotypic clusters for the known metazoan "actinome" were used to identify putative new core actin regulators, together with a number of genes with conserved but poorly studied roles in the regulation of the actin cytoskeleton, several of which we studied in detail. This work suggests that although our search for new components of the core actin machinery is nearing saturation, regulation at the level of nuclear actin export, RNA splicing, ubiquitination, and other upstream processes remains an important but unexplored frontier of actin biology.

}, keywords = {Actin Cytoskeleton, Actins, Animals, Carboxy-Lyases, Carrier Proteins, Cell Line, Cell Nucleus, Cell Shape, Cluster Analysis, DNA, drosophila melanogaster, Drosophila Proteins, HeLa Cells, Hemocytes, High-Throughput Screening Assays, Humans, Microfilament Proteins, Phenotype, rho GTP-Binding Proteins, RNA Interference, RNA Splicing, RNA, Double-Stranded, RNA, Small Interfering, SKP Cullin F-Box Protein Ligases, Tubulin}, issn = {1540-8140}, doi = {10.1083/jcb.201103168}, author = {Rohn, Jennifer L and Sims, David and Liu, Tao and Fedorova, Marina and Sch{\"o}ck, Frieder and Dopie, Joseph and Vartiainen, Maria K and Kiger, Amy A and Perrimon, Norbert and Baum, Buzz} } @article {825596, title = {False negative rates in Drosophila cell-based RNAi screens: a case study.}, journal = {BMC Genomics}, volume = {12}, year = {2011}, month = {2011}, pages = {50}, abstract = {

BACKGROUND: High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention. RESULTS: We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8\%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene. CONCLUSIONS: RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.

}, keywords = {Animals, Cluster Analysis, Drosophila, Gene Expression Profiling, Proteasome Endopeptidase Complex, Ribosomes, RNA Interference, RNA, Double-Stranded}, issn = {1471-2164}, doi = {10.1186/1471-2164-12-50}, author = {Booker, Matthew and Samsonova, Anastasia A and Kwon, Young and Flockhart, Ian and Mohr, Stephanie E and Perrimon, Norbert} } @article {858436, title = {A genome-scale shRNA resource for transgenic RNAi in Drosophila.}, journal = {Nat Methods}, volume = {8}, year = {2011}, month = {2011 May}, pages = {405-7}, abstract = {

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

}, keywords = {Animals, Animals, Genetically Modified, Base Sequence, DNA Primers, drosophila melanogaster, Female, Gene Knockdown Techniques, Genetic techniques, Genetic Vectors, Genome, Insect, MicroRNAs, Oogenesis, RNA Interference, RNA, Small Interfering}, issn = {1548-7105}, doi = {10.1038/nmeth.1592}, author = {Ni, Jian-Quan and Zhou, Rui and Czech, Benjamin and Liu, Lu-Ping and Holderbaum, Laura and Yang-Zhou, Donghui and Shim, Hye-Seok and Tao, Rong and Handler, Dominik and Karpowicz, Phillip and Binari, Richard and Booker, Matthew and Brennecke, Julius and Perkins, Lizabeth A and Hannon, Gregory J and Perrimon, Norbert} } @article {825611, title = {A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.}, journal = {EMBO Rep}, volume = {12}, year = {2011}, month = {2011 Nov}, pages = {1144-52}, abstract = {

Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.

}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Endoplasmic Reticulum, Genes, Insect, Genetic Association Studies, Genetic Testing, Genome, Insect, Membrane Transport Proteins, RNA Interference, Vesicular Transport Proteins, Wnt1 Protein}, issn = {1469-3178}, doi = {10.1038/embor.2011.165}, author = {Port, Fillip and Hausmann, George and Basler, Konrad} } @article {825591, title = {A genome-wide RNAi screen identifies core components of the G$_{2}$-M DNA damage checkpoint.}, journal = {Sci Signal}, volume = {4}, year = {2011}, month = {2011}, pages = {rs1}, abstract = {

The DNA damage checkpoint, the first pathway known to be activated in response to DNA damage, is a mechanism by which the cell cycle is temporarily arrested to allow DNA repair. The checkpoint pathway transmits signals from the sites of DNA damage to the cell cycle machinery through the evolutionarily conserved ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinase cascades. We conducted a genome-wide RNAi (RNA interference) screen in Drosophila cells to identify previously unknown genes and pathways required for the G$_{2}$-M checkpoint induced by DNA double-strand breaks (DSBs). Our large-scale analysis provided a systems-level view of the G$_{2}$-M checkpoint and revealed the coordinated actions of particular classes of proteins, which include those involved in DNA repair, DNA replication, cell cycle control, chromatin regulation, and RNA processing. Further, from the screen and in vivo analysis, we identified previously unrecognized roles of two DNA damage response genes, mus101 and mus312. Our results suggest that the DNA replication preinitiation complex, which includes MUS101, and the MUS312-containing nuclease complexes, which are important for DSB repair, also function in the G$_{2}$-M checkpoint. Our results provide insight into the diverse mechanisms that link DNA damage and the checkpoint signaling pathway.

}, keywords = {Animals, Cell Cycle Proteins, Cell Division, DNA Damage, Drosophila, G1 Phase, genome, RNA, Small Interfering}, issn = {1937-9145}, doi = {10.1126/scisignal.2001350}, author = {Kondo, Shu and Perrimon, Norbert} } @article {825621, title = {High-content chemical and RNAi screens for suppressors of neurotoxicity in a Huntington{\textquoteright}s disease model.}, journal = {PLoS One}, volume = {6}, year = {2011}, month = {2011}, pages = {e23841}, abstract = {

To identify Huntington{\textquoteright}s Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington{\textquoteright}s Disease model, making them attractive candidates for further therapeutic evaluation.

}, keywords = {Algorithms, Animals, Cells, Cultured, Disease Models, Animal, drosophila melanogaster, Genes, Suppressor, High-Throughput Screening Assays, Humans, Huntington Disease, Mutant Proteins, Nerve Tissue Proteins, Neurites, Neurons, Neurotoxins, Nuclear Proteins, Protein Structure, Quaternary, Reproducibility of Results, RNA Interference}, issn = {1932-6203}, doi = {10.1371/journal.pone.0023841}, author = {Schulte, Joost and Sepp, Katharine J and Wu, Chaohong and Hong, Pengyu and Littleton, J Troy} } @article {825606, title = {An integrative approach to ortholog prediction for disease-focused and other functional studies.}, journal = {BMC Bioinformatics}, volume = {12}, year = {2011}, month = {2011}, pages = {357}, abstract = {

BACKGROUND: Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. RESULTS: We report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately {\textquoteright}cast a wide net{\textquoteright} or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist). CONCLUSIONS: DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.

}, keywords = {Animals, Databases, Genetic, disease, Disease Models, Animal, Evolution, Molecular, Genetic Predisposition to Disease, Genome-Wide Association Study, Humans}, issn = {1471-2105}, doi = {10.1186/1471-2105-12-357}, author = {Hu, Yanhui and Flockhart, Ian and Vinayagam, Arunachalam and Bergwitz, Clemens and Berger, Bonnie and Perrimon, Norbert and Mohr, Stephanie E} } @article {825601, title = {Primary cell cultures from Drosophila gastrula embryos.}, journal = {J Vis Exp}, year = {2011}, month = {2011}, abstract = {

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

}, keywords = {Animals, Cell Culture Techniques, Culture Media, Drosophila, RNA Interference, RNA, Double-Stranded}, issn = {1940-087X}, doi = {10.3791/2215}, author = {Perrimon, Norbert and Zirin, Jonathan and Bai, Jianwu} } @article {825631, title = {Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.}, journal = {Sci Signal}, volume = {4}, year = {2011}, month = {2011}, pages = {rs10}, abstract = {

Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.

}, keywords = {Algorithms, Animals, Blotting, Western, Cell Line, Drosophila, Drosophila Proteins, Extracellular Signal-Regulated MAP Kinases, Gene Regulatory Networks, Genomics, Immunoprecipitation, MAP Kinase Signaling System, Models, Genetic, Protein Binding, Protein Interaction Mapping, proteomics, ras Proteins, Receptor Protein-Tyrosine Kinases, RNA Interference, Wings, Animal}, issn = {1937-9145}, doi = {10.1126/scisignal.2002029}, author = {Friedman, Adam A and Tucker, George and Singh, Rohit and Yan, Dong and Vinayagam, Arunachalam and Hu, Yanhui and Binari, Richard and Hong, Pengyu and Sun, Xiaoyun and Porto, Maura and Pacifico, Svetlana and Murali, Thilakam and Finley, Russell L and Asara, John M and Berger, Bonnie and Perrimon, Norbert} } @article {825586, title = {Where gene discovery turns into systems biology: genome-scale RNAi screens in Drosophila.}, journal = {Wiley Interdiscip Rev Syst Biol Med}, volume = {3}, year = {2011}, month = {2011 Jul-Aug}, pages = {471-8}, abstract = {

Systems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline. WIREs Syst Biol Med 2011 3 471-478 DOI: 10.1002/wsbm.127

}, keywords = {Animals, Cells, Cultured, Databases, Genetic, Drosophila, Genetic Association Studies, Genome, Insect, RNA Interference, Systems Biology}, issn = {1939-005X}, doi = {10.1002/wsbm.127}, author = {Neum{\"u}ller, Ralph A and Perrimon, Norbert} } @article {825546, title = {Automatic robust neurite detection and morphological analysis of neuronal cell cultures in high-content screening.}, journal = {Neuroinformatics}, volume = {8}, year = {2010}, month = {2010 Jun}, pages = {83-100}, abstract = {

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutamine-mediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington{\textquoteright}s Disease (HD) model.

}, keywords = {Algorithms, Animals, Animals, Genetically Modified, Automation, Cells, Cultured, Databases as Topic, Disease Models, Animal, Drosophila, Fluorescence, Humans, Huntington Disease, Image Processing, Computer-Assisted, Neurites, Neurons, Peptides, Software, Time Factors}, issn = {1559-0089}, doi = {10.1007/s12021-010-9067-9}, author = {Wu, Chaohong and Schulte, Joost and Sepp, Katharine J and Littleton, J Troy and Hong, Pengyu} } @article {825536, title = {Building and analyzing protein interactome networks by cross-species comparisons.}, journal = {BMC Syst Biol}, volume = {4}, year = {2010}, month = {2010}, pages = {36}, abstract = {

BACKGROUND: A genomic catalogue of protein-protein interactions is a rich source of information, particularly for exploring the relationships between proteins. Numerous systems-wide and small-scale experiments have been conducted to identify interactions; however, our knowledge of all interactions for any one species is incomplete, and alternative means to expand these network maps is needed. We therefore took a comparative biology approach to predict protein-protein interactions across five species (human, mouse, fly, worm, and yeast) and developed InterologFinder for research biologists to easily navigate this data. We also developed a confidence score for interactions based on available experimental evidence and conservation across species. RESULTS: The connectivity of the resultant networks was determined to have scale-free distribution, small-world properties, and increased local modularity, indicating that the added interactions do not disrupt our current understanding of protein network structures. We show examples of how these improved interactomes can be used to analyze a genome-scale dataset (RNAi screen) and to assign new function to proteins. Predicted interactions within this dataset were tested by co-immunoprecipitation, resulting in a high rate of validation, suggesting the high quality of networks produced. CONCLUSIONS: Protein-protein interactions were predicted in five species, based on orthology. An InteroScore, a score accounting for homology, number of orthologues with evidence of interactions, and number of unique observations of interactions, is given to each known and predicted interaction. Our website http://www.interologfinder.org provides research biologists intuitive access to this data.

}, keywords = {Animals, Caenorhabditis elegans, Computational Biology, Drosophila, Humans, Mice, Models, Statistical, Proteasome Endopeptidase Complex, Protein Interaction Mapping, Proteins, RNA Interference, Saccharomyces cerevisiae, Software, Species Specificity, Transfection}, issn = {1752-0509}, doi = {10.1186/1752-0509-4-36}, author = {Wiles, Amy M and Doderer, Mark and Ruan, Jianhua and Gu, Ting-Ting and Ravi, Dashnamoorthy and Blackman, Barron and Bishop, Alexander J R} } @article {855856, title = {Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3{\textquoteright}UTRs.}, journal = {Proc Natl Acad Sci U S A}, volume = {107}, year = {2010}, month = {2010 Sep 7}, pages = {15751-6}, abstract = {

MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate protein-coding genes posttranscriptionally. In animals, most known miRNA targeting occurs within the 3{\textquoteright}UTR of mRNAs, but the extent of biologically relevant targeting in the ORF or 5{\textquoteright}UTR of mRNAs remains unknown. Here, we develop an algorithm (MinoTar-miRNA ORF Targets) to identify conserved regulatory motifs within protein-coding regions and use it to estimate the number of preferentially conserved miRNA-target sites in ORFs. We show that, in Drosophila, preferentially conserved miRNA targeting in ORFs is as widespread as it is in 3{\textquoteright}UTRs and that, while far less abundant, conserved targets in Drosophila 5{\textquoteright}UTRs number in the hundreds. Using our algorithm, we predicted a set of high-confidence ORF targets and selected seven miRNA-target pairs from among these for experimental validation. We observed down-regulation by the miRNA in five out of seven cases, indicating our approach can recover functional sites with high confidence. Additionally, we observed additive targeting by multiple sites within a single ORF. Altogether, our results demonstrate that the scale of biologically important miRNA targeting in ORFs is extensive and that computational tools such as ours can aid in the identification of such targets. Further evidence suggests that our results extend to mammals, but that the extent of ORF and 5{\textquoteright}UTR targeting relative to 3{\textquoteright}UTR targeting may be greater in Drosophila.

}, keywords = {3{\textquoteright} Untranslated Regions, Algorithms, Animals, Drosophila, MicroRNAs, Open Reading Frames}, issn = {1091-6490}, doi = {10.1073/pnas.1006172107}, author = {Schnall-Levin, Michael and Zhao, Yong and Perrimon, Norbert and Berger, Bonnie} } @article {825571, title = {Drosophila genome-wide RNAi screen identifies multiple regulators of HIF-dependent transcription in hypoxia.}, journal = {PLoS Genet}, volume = {6}, year = {2010}, month = {2010 Jun}, pages = {e1000994}, abstract = {

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

}, keywords = {Animals, Anoxia, Argonaute Proteins, Basic Helix-Loop-Helix Transcription Factors, Cell Line, drosophila melanogaster, Drosophila Proteins, Eukaryotic Initiation Factors, Genome-Wide Association Study, RNA Interference, Transcription, Genetic}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1000994}, author = {Dekanty, Andr{\'e}s and Romero, Nuria M and Bertolin, Agustina P and Thomas, Mar{\'\i}a G and Leishman, Claudia C and Perez-Perri, Joel I and Boccaccio, Graciela L and Wappner, Pablo} } @article {825561, title = {Dynamic switch of negative feedback regulation in Drosophila Akt-TOR signaling.}, journal = {PLoS Genet}, volume = {6}, year = {2010}, month = {2010 Jun}, pages = {e1000990}, abstract = {

Akt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)-dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K-independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt-TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo.

}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Enzyme Activation, Epistasis, Genetic, Genome-Wide Association Study, Phosphorylation, Protein Kinases, Proto-Oncogene Proteins c-akt, Ribosomal Protein S6 Kinases, 70-kDa, Signal Transduction, TOR Serine-Threonine Kinases}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1000990}, author = {Kockel, Lutz and Kerr, Kimberly S and Melnick, Michael and Br{\"u}ckner, Katja and Hebrok, Matthias and Perrimon, Norbert} } @article {825526, title = {A genomewide RNA interference screen for modifiers of aggregates formation by mutant Huntingtin in Drosophila.}, journal = {Genetics}, volume = {184}, year = {2010}, month = {2010 Apr}, pages = {1165-79}, abstract = {

Protein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntington{\textquoteright}s disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.

}, keywords = {Age Factors, Animals, drosophila melanogaster, Drosophila Proteins, Genome, Insect, Genomics, High-Throughput Screening Assays, HSP110 Heat-Shock Proteins, Humans, Microtubule-Associated Proteins, Mutant Proteins, Mutation, Peptides, Protein Multimerization, Protein Structure, Quaternary, RNA Interference, Transcription Factors}, issn = {1943-2631}, doi = {10.1534/genetics.109.112516}, author = {Zhang, Sheng and Binari, Richard and Zhou, Rui and Perrimon, Norbert} } @article {825521, title = {A genome-wide RNA interference screen identifies two novel components of the metazoan secretory pathway.}, journal = {EMBO J}, volume = {29}, year = {2010}, month = {2010 Jan 20}, pages = {304-14}, abstract = {

Genetic screens in the yeast Saccharomyces cerevisiae have identified many proteins involved in the secretory pathway, most of which have orthologues in higher eukaryotes. To investigate whether there are additional proteins that are required for secretion in metazoans but are absent from yeast, we used genome-wide RNA interference (RNAi) to look for genes required for secretion of recombinant luciferase from Drosophila S2 cells. This identified two novel components of the secretory pathway that are conserved from humans to plants. Gryzun is distantly related to, but distinct from, the Trs130 subunit of the TRAPP complex but is absent from S. cerevisiae. RNAi of human Gryzun (C4orf41) blocks Golgi exit. Kish is a small membrane protein with a previously uncharacterised orthologue in yeast. The screen also identified Drosophila orthologues of almost 60\% of the yeast genes essential for secretion. Given this coverage, the small number of novel components suggests that contrary to previous indications the number of essential core components of the secretory pathway is not much greater in metazoans than in yeasts.

}, keywords = {Animals, Cell Line, Drosophila, Drosophila Proteins, Eukaryota, Genes, Insect, Golgi Apparatus, Humans, RNA Interference, Saccharomyces cerevisiae, Secretory Pathway}, issn = {1460-2075}, doi = {10.1038/emboj.2009.350}, author = {Wendler, Franz and Gillingham, Alison K and Sinka, Rita and Rosa-Ferreira, Cl{\'a}udia and Gordon, David E and Franch-Marro, Xavier and Peden, Andrew A and Vincent, Jean-Paul and Munro, Sean} } @article {825541, title = {Genomic screening with RNAi: results and challenges.}, journal = {Annu Rev Biochem}, volume = {79}, year = {2010}, month = {2010}, pages = {37-64}, abstract = {

RNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.

}, keywords = {Animals, Genes, Genetic techniques, genome, Humans, RNA Interference}, issn = {1545-4509}, doi = {10.1146/annurev-biochem-060408-092949}, author = {Mohr, Stephanie and Bakal, Chris and Perrimon, Norbert} } @article {825556, title = {Host factors required for modulation of phagosome biogenesis and proliferation of Francisella tularensis within the cytosol.}, journal = {PLoS One}, volume = {5}, year = {2010}, month = {2010}, pages = {e11025}, abstract = {

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of approximately 21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.

}, keywords = {Animals, Cell Line, Cytosol, drosophila melanogaster, Francisella tularensis, Humans, Integration Host Factors, Phagosomes, Phosphotransferases (Alcohol Group Acceptor), RNA Interference, Thiolester Hydrolases}, issn = {1932-6203}, doi = {10.1371/journal.pone.0011025}, author = {Akimana, Christine and Al-Khodor, Souhaila and Abu Kwaik, Yousef} } @article {825551, title = {In vivo RNAi: today and tomorrow.}, journal = {Cold Spring Harb Perspect Biol}, volume = {2}, year = {2010}, month = {2010 Aug}, pages = {a003640}, abstract = {

RNA interference (RNAi) provides a powerful reverse genetics approach to analyze gene functions both in tissue culture and in vivo. Because of its widespread applicability and effectiveness it has become an essential part of the tool box kits of model organisms such as Caenorhabditis elegans, Drosophila, and the mouse. In addition, the use of RNAi in animals in which genetic tools are either poorly developed or nonexistent enables a myriad of fundamental questions to be asked. Here, we review the methods and applications of in vivo RNAi to characterize gene functions in model organisms and discuss their impact to the study of developmental as well as evolutionary questions. Further, we discuss the applications of RNAi technologies to crop improvement, pest control and RNAi therapeutics, thus providing an appreciation of the potential for phenomenal applications of RNAi to agriculture and medicine.

}, keywords = {Animals, Caenorhabditis elegans, Drosophila, Evolution, Molecular, Genetic techniques, Humans, Mice, Models, Animal, Models, Genetic, Molecular Biology, Plants, Genetically Modified, RNA Interference}, issn = {1943-0264}, doi = {10.1101/cshperspect.a003640}, author = {Perrimon, Norbert and Ni, Jian-Quan and Perkins, Lizabeth} } @article {825531, title = {Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen.}, journal = {Genome Res}, volume = {20}, year = {2010}, month = {2010 Mar}, pages = {372-80}, abstract = {

Biological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.

}, keywords = {Animals, Drosophila, GTP Phosphohydrolases, GTPase-Activating Proteins, Protein Processing, Post-Translational, RNA Interference, Signal Transduction}, issn = {1549-5469}, doi = {10.1101/gr.100248.109}, author = {Nir, Oaz and Bakal, Chris and Perrimon, Norbert and Berger, Bonnie} } @article {825566, title = {A kinome RNAi screen identified AMPK as promoting poxvirus entry through the control of actin dynamics.}, journal = {PLoS Pathog}, volume = {6}, year = {2010}, month = {2010}, pages = {e1000954}, abstract = {

Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator.

}, keywords = {Actins, Animals, Blotting, Northern, Cell Movement, Cells, Cultured, drosophila melanogaster, Embryo, Mammalian, Fluorescent Antibody Technique, Genome, Insect, Immunoblotting, Mice, Mice, Knockout, Phosphorylation, Pinocytosis, Protein Kinases, Protein-Serine-Threonine Kinases, Pseudopodia, Reverse Transcriptase Polymerase Chain Reaction, RNA Interference, RNA, Messenger, Vaccinia, Vaccinia virus, Virus Internalization, Virus Replication, Wound Healing}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1000954}, author = {Moser, Theresa S and Jones, Russell G and Thompson, Craig B and Coyne, Carolyn B and Cherry, Sara} } @article {825576, title = {Modifiers of notch transcriptional activity identified by genome-wide RNAi.}, journal = {BMC Dev Biol}, volume = {10}, year = {2010}, month = {2010}, pages = {107}, abstract = {

BACKGROUND: The Notch signaling pathway regulates a diverse array of developmental processes, and aberrant Notch signaling can lead to diseases, including cancer. To obtain a more comprehensive understanding of the genetic network that integrates into Notch signaling, we performed a genome-wide RNAi screen in Drosophila cell culture to identify genes that modify Notch-dependent transcription. RESULTS: Employing complementary data analyses, we found 399 putative modifiers: 189 promoting and 210 antagonizing Notch activated transcription. These modifiers included several known Notch interactors, validating the robustness of the assay. Many novel modifiers were also identified, covering a range of cellular localizations from the extracellular matrix to the nucleus, as well as a large number of proteins with unknown function. Chromatin-modifying proteins represent a major class of genes identified, including histone deacetylase and demethylase complex components and other chromatin modifying, remodeling and replacement factors. A protein-protein interaction map of the Notch-dependent transcription modifiers revealed that a large number of the identified proteins interact physically with these core chromatin components. CONCLUSIONS: The genome-wide RNAi screen identified many genes that can modulate Notch transcriptional output. A protein interaction map of the identified genes highlighted a network of chromatin-modifying enzymes and remodelers that regulate Notch transcription. Our results open new avenues to explore the mechanisms of Notch signal regulation and the integration of this pathway into diverse cellular processes.

}, keywords = {Animals, Cell Line, Chromatin, drosophila melanogaster, Drosophila Proteins, Epistasis, Genetic, genome, Mutation, Protein Interaction Mapping, Receptors, Notch, Ribosomes, RNA Interference, Signal Transduction, Transcription Factors, Transcription, Genetic}, issn = {1471-213X}, doi = {10.1186/1471-213X-10-107}, author = {Mourikis, Philippos and Lake, Robert J and Firnhaber, Christopher B and DeDecker, Brian S} } @article {825506, title = {Bili inhibits Wnt/beta-catenin signaling by regulating the recruitment of axin to LRP6.}, journal = {PLoS One}, volume = {4}, year = {2009}, month = {2009}, pages = {e6129}, abstract = {

BACKGROUND: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway. METHODOLOGY/PRINCIPAL FINDINGS: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a beta-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/beta-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/beta-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation. CONCLUSIONS: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/beta-catenin pathway.

}, keywords = {Animals, Axin Protein, Base Sequence, beta Catenin, Cells, Cultured, Cytoskeletal Proteins, DNA Primers, Humans, Immunoprecipitation, In Situ Hybridization, Low Density Lipoprotein Receptor-Related Protein-6, Membrane Proteins, Protein Binding, Receptors, LDL, Repressor Proteins, Reverse Transcriptase Polymerase Chain Reaction, RNA Interference, Signal Transduction, Wnt Proteins}, issn = {1932-6203}, doi = {10.1371/journal.pone.0006129}, author = {Kategaya, Lorna S and Changkakoty, Binita and Biechele, Travis and Conrad, William H and Kaykas, Ajamete and DasGupta, Ramanuj and Moon, Randall T} } @article {855071, title = {Cross-species RNAi rescue platform in Drosophila melanogaster.}, journal = {Genetics}, volume = {183}, year = {2009}, month = {2009 Nov}, pages = {1165-73}, abstract = {

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

}, keywords = {Animals, Animals, Genetically Modified, Aurora Kinases, Base Sequence, Caspases, Drosophila, drosophila melanogaster, Drosophila Proteins, Genetic Vectors, Molecular Sequence Data, Mutation, Phenotype, Phylogeny, Protein-Serine-Threonine Kinases, RNA Interference, RNA, Double-Stranded, Sequence Homology, Nucleic Acid, Species Specificity, Transfection}, issn = {1943-2631}, doi = {10.1534/genetics.109.106567}, author = {Kondo, Shu and Booker, Matthew and Perrimon, Norbert} } @article {825516, title = {Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening.}, journal = {Nat Protoc}, volume = {4}, year = {2009}, month = {2009}, pages = {1502-12}, abstract = {

We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.

}, keywords = {Animals, Cell Culture Techniques, Cells, Cultured, Culture Media, Drosophila, Gastrula, Genomics, RNA Interference, RNA, Double-Stranded}, issn = {1750-2799}, doi = {10.1038/nprot.2009.147}, author = {Bai, Jianwu and Sepp, Katharine J and Perrimon, Norbert} } @article {825491, title = {Discovery of insect and human dengue virus host factors.}, journal = {Nature}, volume = {458}, year = {2009}, month = {2009 Apr 23}, pages = {1047-50}, abstract = {

Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world{\textquoteright}s population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1-4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and alpha-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

}, keywords = {Aedes, Animals, Cell Line, Conserved Sequence, Dengue Virus, drosophila melanogaster, Gene Knockdown Techniques, Genome, Insect, Host-Pathogen Interactions, Humans, Insect Vectors, RNA Interference, RNA, Double-Stranded, Virus Replication}, issn = {1476-4687}, doi = {10.1038/nature07967}, author = {Sessions, October M and Barrows, Nicholas J and Souza-Neto, Jayme A and Robinson, Timothy J and Hershey, Christine L and Rodgers, Mary A and Ramirez, Jose L and Dimopoulos, George and Yang, Priscilla L and Pearson, James L and Garcia-Blanco, Mariano A} } @article {858431, title = {A Drosophila resource of transgenic RNAi lines for neurogenetics.}, journal = {Genetics}, volume = {182}, year = {2009}, month = {2009 Aug}, pages = {1089-100}, abstract = {

Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the "VALIUM" series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.

}, keywords = {Animals, Carrier Proteins, Drosophila, Gene Knockdown Techniques, ion channels, Methods, Nervous System, RNA Interference, RNA, Small Interfering, Transcription Factors}, issn = {1943-2631}, doi = {10.1534/genetics.109.103630}, author = {Ni, Jian-Quan and Liu, Lu-Ping and Binari, Richard and Hardy, Robert and Shim, Hye-Seok and Cavallaro, Amanda and Booker, Matthew and Pfeiffer, Barret D and Markstein, Michele and Wang, Hui and Villalta, Christians and Laverty, Todd R and Perkins, Lizabeth A and Perrimon, Norbert} } @article {825511, title = {Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.}, journal = {Science}, volume = {326}, year = {2009}, month = {2009 Oct 2}, pages = {144-7}, abstract = {

Mitochondria are integral components of cellular calcium (Ca2+) signaling. Calcium stimulates mitochondrial adenosine 5{\textquoteright}-triphosphate production, but can also initiate apoptosis. In turn, cytoplasmic Ca2+ concentrations are regulated by mitochondria. Although several transporter and ion-channel mechanisms have been measured in mitochondria, the molecules that govern Ca2+ movement across the inner mitochondrial membrane are unknown. We searched for genes that regulate mitochondrial Ca2+ and H+ concentrations using a genome-wide Drosophila RNA interference (RNAi) screen. The mammalian homolog of one Drosophila gene identified in the screen, Letm1, was found to specifically mediate coupled Ca2+/H+ exchange. RNAi knockdown, overexpression, and liposome reconstitution of the purified Letm1 protein demonstrate that Letm1 is a mitochondrial Ca2+/H+ antiporter.

}, keywords = {Animals, Antiporters, calcium, Calcium-Binding Proteins, Cation Transport Proteins, Cell Line, drosophila melanogaster, Drosophila Proteins, Genome, Human, Genome, Insect, HeLa Cells, Humans, Hydrogen, Hydrogen-Ion Concentration, Ion Transport, Membrane Potential, Mitochondrial, Membrane Proteins, mitochondria, Mitochondrial Membranes, Mitochondrial Proteins, Proteolipids, RNA Interference}, issn = {1095-9203}, doi = {10.1126/science.1175145}, author = {Jiang, Dawei and Zhao, Linlin and Clapham, David E} } @article {825496, title = {A network of conserved damage survival pathways revealed by a genomic RNAi screen.}, journal = {PLoS Genet}, volume = {5}, year = {2009}, month = {2009 Jun}, pages = {e1000527}, abstract = {

Damage initiates a pleiotropic cellular response aimed at cellular survival when appropriate. To identify genes required for damage survival, we used a cell-based RNAi screen against the Drosophila genome and the alkylating agent methyl methanesulphonate (MMS). Similar studies performed in other model organisms report that damage response may involve pleiotropic cellular processes other than the central DNA repair components, yet an intuitive systems level view of the cellular components required for damage survival, their interrelationship, and contextual importance has been lacking. Further, by comparing data from different model organisms, identification of conserved and presumably core survival components should be forthcoming. We identified 307 genes, representing 13 signaling, metabolic, or enzymatic pathways, affecting cellular survival of MMS-induced damage. As expected, the majority of these pathways are involved in DNA repair; however, several pathways with more diverse biological functions were also identified, including the TOR pathway, transcription, translation, proteasome, glutathione synthesis, ATP synthesis, and Notch signaling, and these were equally important in damage survival. Comparison with genomic screen data from Saccharomyces cerevisiae revealed no overlap enrichment of individual genes between the species, but a conservation of the pathways. To demonstrate the functional conservation of pathways, five were tested in Drosophila and mouse cells, with each pathway responding to alkylation damage in both species. Using the protein interactome, a significant level of connectivity was observed between Drosophila MMS survival proteins, suggesting a higher order relationship. This connectivity was dramatically improved by incorporating the components of the 13 identified pathways within the network. Grouping proteins into "pathway nodes" qualitatively improved the interactome organization, revealing a highly organized "MMS survival network." We conclude that identification of pathways can facilitate comparative biology analysis when direct gene/orthologue comparisons fail. A biologically intuitive, highly interconnected MMS survival network was revealed after we incorporated pathway data in our interactome analysis.

}, keywords = {Animals, Cell Line, Cells, Cultured, DNA Damage, Drosophila, Drosophila Proteins, Gene Regulatory Networks, Genome, Insect, Methyl Methanesulfonate, Mice, Mice, Inbred C57BL, Mutagens, RNA Interference, Saccharomyces cerevisiae, Signal Transduction}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1000527}, author = {Ravi, Dashnamoorthy and Wiles, Amy M and Bhavani, Selvaraj and Ruan, Jianhua and Leder, Philip and Bishop, Alexander J R} } @article {825486, title = {PDGF/VEGF signaling controls cell size in Drosophila.}, journal = {Genome Biol}, volume = {10}, year = {2009}, month = {2009}, pages = {R20}, abstract = {

BACKGROUND: In multicellular animals, cell size is controlled by a limited set of conserved intracellular signaling pathways, which when deregulated contribute to tumorigenesis by enabling cells to grow outside their usual niche. To delineate the pathways controlling this process, we screened a genome-scale, image-based Drosophila RNA interference dataset for double-stranded RNAs that reduce the average size of adherent S2R+ cells. RESULTS: Automated analysis of images from this RNA interference screen identified the receptor tyrosine kinase Pvr, Ras pathway components and several novel genes as regulators of cell size. Significantly, Pvr/Ras signaling also affected the size of other Drosophila cell lines and of larval hemocytes. A detailed genetic analysis of this growth signaling pathway revealed a role for redundant secreted ligands, Pvf2 and Pvf3, in the establishment of an autocrine growth signaling loop. Downstream of Ras1, growth signaling was found to depend on parallel mitogen-activated protein kinase (MAPK) and phospho-inositide-3-kinase (PI3K) signaling modules, as well as the Tor pathway. CONCLUSIONS: This automated genome-wide screen identifies autocrine Pvf/Pvr signaling, upstream of Ras, MAPK and PI3K, as rate-limiting for the growth of immortalized fly cells in culture. Since, Pvf2/3 and Pvr show mutually exclusive in vivo patterns of gene expression, these data suggest that co-expression of this receptor-ligand pair plays a key role in driving cell autonomous growth during the establishment of Drosophila cell lines, as has been suggested to occur during tumor development.

}, keywords = {Animals, Autocrine Communication, Cell Line, Cell Size, Drosophila, Drosophila Proteins, Egg Proteins, Genomics, Neoplasms, Platelet-Derived Growth Factor, Receptor Protein-Tyrosine Kinases, RNA Interference, RNA, Double-Stranded, Signal Transduction, Vascular Endothelial Growth Factors}, issn = {1474-760X}, doi = {10.1186/gb-2009-10-2-r20}, author = {Sims, David and Duchek, Peter and Baum, Buzz} } @article {825501, title = {RNAiCut: automated detection of significant genes from functional genomic screens.}, journal = {Nat Methods}, volume = {6}, year = {2009}, month = {2009 Jul}, pages = {476-7}, keywords = {Animals, Databases, Genetic, Genome, Insect, Genomics, Insulin, MAP Kinase Signaling System, Protein Interaction Mapping, RNA Interference, Software}, issn = {1548-7105}, doi = {10.1038/nmeth0709-476}, author = {Kaplow, Irene M and Singh, Rohit and Friedman, Adam and Bakal, Chris and Perrimon, Norbert and Berger, Bonnie} } @article {825456, title = {An analysis of normalization methods for Drosophila RNAi genomic screens and development of a robust validation scheme.}, journal = {J Biomol Screen}, volume = {13}, year = {2008}, month = {2008 Sep}, pages = {777-84}, abstract = {

Genome-wide RNA interference (RNAi) screening allows investigation of the role of individual genes in a process of choice. Most RNAi screens identify a large number of genes with a continuous gradient in the assessed phenotype. Screeners must decide whether to examine genes with the most robust phenotype or the full gradient of genes that cause an effect and how to identify candidate genes. The authors have used RNAi in Drosophila cells to examine viability in a 384-well plate format and compare 2 screens, untreated control and treatment. They compare multiple normalization methods, which take advantage of different features within the data, including quantile normalization, background subtraction, scaling, cellHTS2 (Boutros et al. 2006), and interquartile range measurement. Considering the false-positive potential that arises from RNAi technology, a robust validation method was designed for the purpose of gene selection for future investigations. In a retrospective analysis, the authors describe the use of validation data to evaluate each normalization method. Although no method worked ideally, a combination of 2 methods, background subtraction followed by quantile normalization and cellHTS2, at different thresholds, captures the most dependable and diverse candidate genes. Thresholds are suggested depending on whether a few candidate genes are desired or a more extensive systems-level analysis is sought. The normalization approaches and experimental design to perform validation experiments are likely to apply to those high-throughput screening systems attempting to identify genes for systems-level analysis.

}, keywords = {Animals, Cell Line, Drosophila, Reproducibility of Results, RNA Interference, RNA, Double-Stranded}, issn = {1087-0571}, doi = {10.1177/1087057108323125}, author = {Wiles, Amy M and Ravi, Dashnamoorthy and Bhavani, Selvaraj and Bishop, Alexander J R} } @article {825421, title = {Cellular phenotype recognition for high-content RNA interference genome-wide screening.}, journal = {J Biomol Screen}, volume = {13}, year = {2008}, month = {2008 Jan}, pages = {29-39}, abstract = {

Genome-wide, cell-based screens using high-content screening (HCS) techniques and automated fluorescence microscopy generate thousands of high-content images that contain an enormous wealth of cell biological information. Such screens are key to the analysis of basic cell biological principles, such as control of cell cycle and cell morphology. However, these screens will ultimately only shed light on human disease mechanisms and potential cures if the analysis can keep up with the generation of data. A fundamental step toward automated analysis of high-content screening is to construct a robust platform for automatic cellular phenotype identification. The authors present a framework, consisting of microscopic image segmentation and analysis components, for automatic recognition of cellular phenotypes in the context of the Rho family of small GTPases. To implicate genes involved in Rac signaling, RNA interference (RNAi) was used to perturb gene functions, and the corresponding cellular phenotypes were analyzed for changes. The data used in the experiments are high-content, 3-channel, fluorescence microscopy images of Drosophila Kc167 cultured cells stained with markers that allow visualization of DNA, polymerized actin filaments, and the constitutively activated Rho protein Rac(V12). The performance of this approach was tested using a cellular database that contained more than 1000 samples of 3 predefined cellular phenotypes, and the generalization error was estimated using a cross-validation technique. Moreover, the authors applied this approach to analyze the whole high-content fluorescence images of Drosophila cells for further HCS-based gene function analysis.

}, keywords = {Algorithms, Animals, Cell Line, Cell Shape, Cytoskeleton, Drosophila, Drosophila Proteins, Genomics, Microscopy, Fluorescence, Phenotype, rac GTP-Binding Proteins, RNA Interference, Signal Transduction}, issn = {1087-0571}, doi = {10.1177/1087057107311223}, author = {Wang, Jun and Zhou, Xiaobo and Bradley, Pamela L and Chang, Shih-Fu and Perrimon, Norbert and Wong, Stephen T C} } @article {825471, title = {Comparative analysis of argonaute-dependent small RNA pathways in Drosophila.}, journal = {Mol Cell}, volume = {32}, year = {2008}, month = {2008 Nov 21}, pages = {592-9}, abstract = {

The specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.

}, keywords = {Animals, Argonaute Proteins, Cell Line, Drosophila, drosophila melanogaster, Drosophila Proteins, Eukaryotic Initiation Factors, Gene Silencing, Genes, Insect, MicroRNAs, Models, Biological, RNA Interference, RNA, Small Interfering, RNA, Untranslated, RNA-Induced Silencing Complex}, issn = {1097-4164}, doi = {10.1016/j.molcel.2008.10.018}, author = {Zhou, Rui and Hotta, Ikuko and Denli, Ahmet M and Hong, Pengyu and Perrimon, Norbert and Hannon, Gregory J} } @article {825481, title = {COPI complex is a regulator of lipid homeostasis.}, journal = {PLoS Biol}, volume = {6}, year = {2008}, month = {2008 Nov 25}, pages = {e292}, abstract = {

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that these regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. We found that COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets.

}, keywords = {Adipocytes, Animals, Carrier Proteins, Coat Protein Complex I, Drosophila, Drosophila Proteins, Fat Body, Fatty Acids, Nonesterified, Gene Expression Regulation, Homeostasis, Lipid Metabolism, Lipolysis, Mice, Phenotype, Phosphoproteins, Proteome, RNA Interference}, issn = {1545-7885}, doi = {10.1371/journal.pbio.0060292}, author = {Beller, Mathias and Sztalryd, Carole and Southall, Noel and Bell, Ming and J{\"a}ckle, Herbert and Auld, Douglas S and Oliver, Brian} } @article {825416, title = {Definition of global and transcript-specific mRNA export pathways in metazoans.}, journal = {Genes Dev}, volume = {22}, year = {2008}, month = {2008 Jan 1}, pages = {66-78}, abstract = {

Eukaryotic gene expression requires export of messenger RNAs (mRNAs) from their site of transcription in the nucleus to the cytoplasm where they are translated. While mRNA export has been studied in yeast, the complexity of gene structure and cellular function in metazoan cells has likely led to increased diversification of these organisms{\textquoteright} export pathways. Here we report the results of a genome-wide RNAi screen in which we identify 72 factors required for polyadenylated [poly-(A(+))] mRNA export from the nucleus in Drosophila cells. Using structural and functional conservation analysis of yeast and Drosophila mRNA export factors, we expose the evolutionary divergence of eukaryotic mRNA export pathways. Additionally, we demonstrate the differential export requirements of two endogenous heat-inducible transcripts--intronless heat-shock protein 70 (HSP70) and intron-containing HSP83--and identify novel export factors that participate in HSP83 mRNA splicing. We characterize several novel factors and demonstrate their participation in interactions with known components of the Drosophila export machinery. One of these factors, Drosophila melanogaster PCI domain-containing protein 2 (dmPCID2), associates with polysomes and may bridge the transition between exported messenger ribonucleoprotein particles (mRNPs) and polysomes. Our results define the global network of factors involved in Drosophila mRNA export, reveal specificity in the export requirements of different transcripts, and expose new avenues for future work in mRNA export.

}, keywords = {Animals, Cell Cycle, Drosophila, Drosophila Proteins, genome, Models, Biological, Molecular Sequence Data, Nucleocytoplasmic Transport Proteins, Protein Biosynthesis, RNA Interference, RNA Splicing, RNA Transport, RNA, Messenger, Saccharomyces cerevisiae, Sequence Alignment}, issn = {0890-9369}, doi = {10.1101/gad.1616008}, author = {Farny, Natalie G and Hurt, Jessica A and Silver, Pamela A} } @article {825426, title = {ESCRT factors restrict mycobacterial growth.}, journal = {Proc Natl Acad Sci U S A}, volume = {105}, year = {2008}, month = {2008 Feb 26}, pages = {3070-5}, abstract = {

Nearly 1.7 billion people are infected with Mycobacterium tuberculosis. Its ability to survive intracellularly is thought to be central to its success as a pathogen, but how it does this is poorly understood. Using a Drosophila model of infection, we identify three host cell activities, Rab7, CG8743, and the ESCRT machinery, that modulate the mycobacterial phagosome. In the absence of these factors the cell no longer restricts growth of the non-pathogen Mycobacterium smegmatis. Hence, we identify factors that represent unique vulnerabilities of the host cell, because manipulation of any one of them alone is sufficient to allow a nonpathogenic mycobacterial species to proliferate. Furthermore, we demonstrate that, in mammalian cells, the ESCRT machinery plays a conserved role in restricting bacterial growth.

}, keywords = {Animals, Cell Line, DNA-Binding Proteins, Drosophila, Endosomal Sorting Complexes Required for Transport, Endosomes, Microscopy, Fluorescence, Multiprotein Complexes, Mycobacterium, Mycobacterium Infections, rab GTP-Binding Proteins, RNA Interference, Species Specificity, Transcription Factors, Vesicular Transport Proteins, Virulence}, issn = {1091-6490}, doi = {10.1073/pnas.0707206105}, author = {Philips, Jennifer A and Porto, Maura C and Wang, Hui and Rubin, Eric J and Perrimon, Norbert} } @article {825476, title = {Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation.}, journal = {J Cell Biol}, volume = {183}, year = {2008}, month = {2008 Dec 1}, pages = {805-18}, abstract = {

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, we isolated factors required for centromere propagation using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila melanogaster. We identified the proteins CAL1 and CENP-C as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. We also identified the mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 as regulators of centromere propagation. We show that CYCA is centromere localized and that CYCA and RCA1/Emi1 couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. Our findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division.

}, keywords = {Anaphase-Promoting Complex-Cyclosome, Animals, Annexin A2, Cell Cycle, Cell Cycle Proteins, Cell Line, Centromere, Chromosomal Proteins, Non-Histone, Chromosome Segregation, Cyclin A, DNA-Binding Proteins, drosophila melanogaster, Drosophila Proteins, Epigenesis, Genetic, Genome-Wide Association Study, Histones, Mitosis, Mutation, RNA Interference, S100 Proteins, Ubiquitin-Protein Ligase Complexes}, issn = {1540-8140}, doi = {10.1083/jcb.200806038}, author = {Erhardt, Sylvia and Mellone, Barbara G and Betts, Craig M and Zhang, Weiguo and Karpen, Gary H and Straight, Aaron F} } @article {825446, title = {Identification of neural outgrowth genes using genome-wide RNAi.}, journal = {PLoS Genet}, volume = {4}, year = {2008}, month = {2008 Jul}, pages = {e1000111}, abstract = {

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system.

}, keywords = {Animals, Cells, Cultured, Drosophila, genome, Genomics, Mice, Nervous System, Neurons, Phenotype, ran GTP-Binding Protein, RNA Interference, RNA, Small Interfering}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1000111}, author = {Sepp, Katharine J and Hong, Pengyu and Lizarraga, Sofia B and Liu, Judy S and Mejia, Luis A and Walsh, Christopher A and Perrimon, Norbert} } @article {825441, title = {Identification of novel genes involved in light-dependent CRY degradation through a genome-wide RNAi screen.}, journal = {Genes Dev}, volume = {22}, year = {2008}, month = {2008 Jun 1}, pages = {1522-33}, abstract = {

Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor CRYPTOCHROME (CRY). In response to light, CRY promotes the degradation of the circadian clock protein TIMELESS (TIM) and then is itself degraded. To identify novel genes involved in circadian entrainment, we performed an unbiased genome-wide screen in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of CRY. We systematically knocked down the expression of approximately 21,000 genes and identified those that regulate CRY stability. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for CRY degradation and do not affect degradation of the TIM protein in response to light, suggesting that, for the most part, these two pathways are distinct. We further validated the effect of three candidate genes on CRY stability in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent CRY degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology.

}, keywords = {Animals, Cells, Cultured, Circadian Rhythm, CLOCK Proteins, Drosophila, Light, Photoreceptor Cells, Invertebrate, RNA Interference, Trans-Activators}, issn = {0890-9369}, doi = {10.1101/gad.1652308}, author = {Sathyanarayanan, Sriram and Zheng, Xiangzhong and Kumar, Shailesh and Chen, Chun-Hong and Chen, Dechun and Hay, Bruce and Sehgal, Amita} } @article {825451, title = {Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes.}, journal = {Genes Dev}, volume = {22}, year = {2008}, month = {2008 Aug 15}, pages = {2189-203}, abstract = {

Multiple centrosomes in tumor cells create the potential for multipolar divisions that can lead to aneuploidy and cell death. Nevertheless, many cancer cells successfully divide because of mechanisms that suppress multipolar mitoses. A genome-wide RNAi screen in Drosophila S2 cells and a secondary analysis in cancer cells defined mechanisms that suppress multipolar mitoses. In addition to proteins that organize microtubules at the spindle poles, we identified novel roles for the spindle assembly checkpoint, cortical actin cytoskeleton, and cell adhesion. Using live cell imaging and fibronectin micropatterns, we found that interphase cell shape and adhesion pattern can determine the success of the subsequent mitosis in cells with extra centrosomes. These findings may identify cancer-selective therapeutic targets: HSET, a normally nonessential kinesin motor, was essential for the viability of certain extra centrosome-containing cancer cells. Thus, morphological features of cancer cells can be linked to unique genetic requirements for survival.

}, keywords = {Actins, Animals, Cell Adhesion, Cell Division, Cell Line, Cell Polarity, Cell Shape, Centrosome, Cytoskeleton, drosophila melanogaster, Drosophila Proteins, genome, Interphase, Kinesin, Microscopy, Fluorescence, microtubules, Mitosis, Neoplasms, Phenotype, RNA, Small Interfering, Spindle Apparatus}, issn = {0890-9369}, doi = {10.1101/gad.1700908}, author = {Kwon, Mijung and Godinho, Susana A and Chandhok, Namrata S and Ganem, Neil J and Azioune, Ammar and Thery, Manuel and Pellman, David} } @article {825431, title = {The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells.}, journal = {Mol Biol Cell}, volume = {19}, year = {2008}, month = {2008 Jun}, pages = {2402-12}, abstract = {

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.

}, keywords = {Amino Acid Sequence, Animals, Apoptosis, Carbonyl Cyanide m-Chlorophenyl Hydrazone, Drosophila, Genes, Dominant, GTP Phosphohydrolases, HeLa Cells, Humans, Membrane Potential, Mitochondrial, Membrane Proteins, Membrane Transport Proteins, mitochondria, Mitochondrial Membrane Transport Proteins, Mitochondrial Proteins, Molecular Sequence Data, Multiprotein Complexes, Mutation, Organelle Shape, Peroxisomes, Protein Transport, RNA Interference, RNA, Small Interfering, Sequence Homology, Amino Acid}, issn = {1939-4586}, doi = {10.1091/mbc.E07-12-1287}, author = {Gandre-Babbe, Shilpa and van der Bliek, Alexander M} } @article {825461, title = {Phosphorylation networks regulating JNK activity in diverse genetic backgrounds.}, journal = {Science}, volume = {322}, year = {2008}, month = {2008 Oct 17}, pages = {453-6}, abstract = {

Cellular signaling networks have evolved to enable swift and accurate responses, even in the face of genetic or environmental perturbation. Thus, genetic screens may not identify all the genes that regulate different biological processes. Moreover, although classical screening approaches have succeeded in providing parts lists of the essential components of signaling networks, they typically do not provide much insight into the hierarchical and functional relations that exist among these components. We describe a high-throughput screen in which we used RNA interference to systematically inhibit two genes simultaneously in 17,724 combinations to identify regulators of Drosophila JUN NH(2)-terminal kinase (JNK). Using both genetic and phosphoproteomics data, we then implemented an integrative network algorithm to construct a JNK phosphorylation network, which provides structural and mechanistic insights into the systems architecture of JNK signaling.

}, keywords = {Algorithms, Animals, Cell Line, Computational Biology, Drosophila, Drosophila Proteins, Fluorescence Resonance Energy Transfer, Genes, Insect, JNK Mitogen-Activated Protein Kinases, MAP Kinase Signaling System, Metabolic Networks and Pathways, Phosphorylation, proteomics, RNA Interference, Signal Transduction}, issn = {1095-9203}, doi = {10.1126/science.1158739}, author = {Bakal, Chris and Linding, Rune and Llense, Flora and Heffern, Elleard and Martin-Blanco, Enrique and Pawson, Tony and Perrimon, Norbert} } @article {825436, title = {RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance.}, journal = {Development}, volume = {135}, year = {2008}, month = {2008 Apr}, pages = {1439-49}, abstract = {

To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.

}, keywords = {Animals, Animals, Genetically Modified, Base Sequence, Cells, Cultured, DNA Primers, Drosophila, Drosophila Proteins, Gene Expression Regulation, Developmental, Genes, Insect, Humans, Muscle Development, Muscular Diseases, Phenotype, Plasma Membrane Calcium-Transporting ATPases, RNA Interference}, issn = {0950-1991}, doi = {10.1242/dev.012849}, author = {Bai, Jianwu and Binari, Richard and Ni, Jian-Quan and Vijayakanthan, Marina and Li, Hong-Sheng and Perrimon, Norbert} } @article {825466, title = {Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis.}, journal = {Cell}, volume = {135}, year = {2008}, month = {2008 Oct 31}, pages = {524-34}, abstract = {

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In Caenorhabditis elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. We report that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. We place Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM, and the Ca2+-release-activated Ca2+ channel dOrai in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, our results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway.

}, keywords = {Animals, Animals, Genetically Modified, Apoptosis, calcium, drosophila melanogaster, Drosophila Proteins, Embryo, Nonmammalian, Membrane Proteins, Phagocytosis}, issn = {1097-4172}, doi = {10.1016/j.cell.2008.08.033}, author = {Cuttell, Leigh and Vaughan, Andrew and Silva, Elizabeth and Escaron, Claire J and Lavine, Mark and Van Goethem, Emeline and Eid, Jean-Pierre and Quirin, Magali and Franc, Nathalie C} } @article {825411, title = {Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster.}, journal = {Nat Methods}, volume = {5}, year = {2008}, month = {2008 Jan}, pages = {49-51}, abstract = {

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.

}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Gene Targeting, Genetic Vectors, RNA Interference}, issn = {1548-7105}, doi = {10.1038/nmeth1146}, author = {Ni, Jian-Quan and Markstein, Michele and Binari, Richard and Pfeiffer, Barret and Liu, Lu-Ping and Villalta, Christians and Booker, Matthew and Perkins, Lizabeth and Perrimon, Norbert} } @article {825361, title = {Applications of high-throughput RNA interference screens to problems in cell and developmental biology.}, journal = {Genetics}, volume = {175}, year = {2007}, month = {2007 Jan}, pages = {7-16}, abstract = {

RNA interference (RNAi) in tissue culture cells has emerged as an excellent methodology for identifying gene functions systematically and in an unbiased manner. Here, we describe how RNAi high-throughput screening (HTS) in Drosophila cells are currently being performed and emphasize the strengths and weaknesses of the approach. Further, to demonstrate the versatility of the technology, we provide examples of the various applications of the method to problems in signal transduction and cell and developmental biology. Finally, we discuss emerging technological advances that will extend RNAi-based screening methods.

}, keywords = {Animals, developmental biology, genome, Humans, Phenotype, RNA Interference}, issn = {0016-6731}, doi = {10.1534/genetics.106.069963}, author = {Perrimon, Norbert and Mathey-Prevot, Bernard} } @article {825366, title = {Biochemical and functional characterization of Orai proteins.}, journal = {J Biol Chem}, volume = {282}, year = {2007}, month = {2007 Jun 1}, pages = {16232-43}, abstract = {

Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

}, keywords = {Amino Acid Substitution, Animals, Calcium Channels, Calcium Signaling, Cell Line, Cell Membrane, Cell Proliferation, Cytokines, Drosophila, Drosophila Proteins, Fibroblasts, Glycosylation, Humans, Membrane Proteins, Mutation, Missense, Neoplasm Proteins, Protein Processing, Post-Translational, Protein Structure, Secondary, Sequence Homology, Amino Acid, T-Lymphocytes}, issn = {0021-9258}, doi = {10.1074/jbc.M609630200}, author = {Gwack, Yousang and Srikanth, Sonal and Feske, Stefan and Cruz-Guilloty, Fernando and Oh-hora, Masatsugu and Neems, Daniel S and Hogan, Patrick G and Rao, Anjana} } @article {825391, title = {A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila.}, journal = {Genome Biol}, volume = {8}, year = {2007}, month = {2007}, pages = {R203}, abstract = {

Off-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.

}, keywords = {Animals, Drosophila, Drosophila Proteins, Gene Expression Regulation, Gene Library, Genes, Insect, Genetic techniques, genome, Models, Genetic, RNA, RNA Interference, RNA, Double-Stranded, Sensitivity and Specificity, Transcription, Genetic}, issn = {1474-760X}, doi = {10.1186/gb-2007-8-9-r203}, author = {DasGupta, Ramanuj and Nybakken, Kent and Booker, Matthew and Mathey-Prevot, Bernard and Gonsalves, Foster and Changkakoty, Binita and Perrimon, Norbert} } @article {825386, title = {Design and implementation of high-throughput RNAi screens in cultured Drosophila cells.}, journal = {Nat Protoc}, volume = {2}, year = {2007}, month = {2007}, pages = {2245-64}, abstract = {

This protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.

}, keywords = {Animals, Artifacts, Cells, Cultured, Drosophila, Gene Library, Genomics, Polymerase Chain Reaction, RNA Interference, RNA, Double-Stranded}, issn = {1750-2799}, doi = {10.1038/nprot.2007.250}, author = {Ramadan, Nadire and Flockhart, Ian and Booker, Matthew and Perrimon, Norbert and Mathey-Prevot, Bernard} } @article {825371, title = {A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression.}, journal = {Proc Natl Acad Sci U S A}, volume = {104}, year = {2007}, month = {2007 May 29}, pages = {9381-6}, abstract = {

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, we have conducted a genome-wide RNAi screen in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related CG16975/dSfmbt. These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth.

}, keywords = {Animals, Cells, Cultured, Chromatin, Cyclin H, Cyclins, Down-Regulation, drosophila melanogaster, Drosophila Proteins, E2F Transcription Factors, Genetic Testing, Genome, Insect, Mutation, Phenotype, Promoter Regions, Genetic, Protein Binding, Protein Kinases, RNA Interference, Transcription Factors}, issn = {0027-8424}, doi = {10.1073/pnas.0610279104}, author = {Lu, Jianrong and Ruhf, Marie-Laure and Perrimon, Norbert and Leder, Philip} } @article {825406, title = {A genome-wide RNA interference screen reveals that variant histones are necessary for replication-dependent histone pre-mRNA processing.}, journal = {Mol Cell}, volume = {28}, year = {2007}, month = {2007 Nov 30}, pages = {692-9}, abstract = {

Metazoan replication-dependent histone mRNAs are not polyadenylated and instead end in a conserved stem loop that is the cis element responsible for coordinate posttranscriptional regulation of these mRNAs. Using biochemical approaches, only a limited number of factors required for cleavage of histone pre-mRNA have been identified. We therefore performed a genome-wide RNA interference screen in Drosophila cells using a GFP reporter that is expressed only when histone pre-mRNA processing is disrupted. Four of the 24 genes identified encode proteins also necessary for cleavage/polyadenylation, indicating mechanistic conservation in formation of different mRNA 3{\textquoteright} ends. We also unexpectedly identified the histone variants H2Av and H3.3A/B. In H2Av mutant cells, U7 snRNP remains active but fails to accumulate at the histone locus, suggesting there is a regulatory pathway that coordinates the production of variant and canonical histones that acts via localization of essential histone pre-mRNA processing factors.

}, keywords = {Animals, Base Sequence, DNA Replication, drosophila melanogaster, Drosophila Proteins, Genes, Reporter, Genome, Insect, Histones, Molecular Sequence Data, Mutant Proteins, Polyadenylation, Protein Transport, Ribonucleoprotein, U7 Small Nuclear, RNA Interference, RNA Precursors, RNA Processing, Post-Transcriptional}, issn = {1097-2765}, doi = {10.1016/j.molcel.2007.10.009}, author = {Wagner, Eric J and Burch, Brandon D and Godfrey, Ashley C and Salzler, Harmony R and Duronio, Robert J and Marzluff, William F} } @article {825401, title = {A genome-wide RNAi screen reveals multiple regulators of caspase activation.}, journal = {J Cell Biol}, volume = {179}, year = {2007}, month = {2007 Nov 19}, pages = {619-26}, abstract = {

Apoptosis is an evolutionally conserved cellular suicide mechanism that can be activated in response to a variety of stressful stimuli. Increasing evidence suggests that apoptotic regulation relies on specialized cell death signaling pathways and also integrates diverse signals from additional regulatory circuits, including those of cellular homeostasis. We present a genome-wide RNA interference screen to systematically identify regulators of apoptosis induced by DNA damage in Drosophila melanogaster cells. We identify 47 double- stranded RNA that target a functionally diverse set of genes, including several with a known function in promoting cell death. Further characterization uncovers 10 genes that influence caspase activation upon the removal of Drosophila inhibitor of apoptosis 1. This set includes the Drosophila initiator caspase Dronc and, surprisingly, several metabolic regulators, a candidate tumor suppressor, Charlatan, and an N-acetyltransferase, ARD1. Importantly, several of these genes show functional conservation in regulating apoptosis in mammalian cells. Our data suggest a previously unappreciated fundamental connection between various cellular processes and caspase-dependent cell death.

}, keywords = {Acetyltransferases, Animals, Apoptosis, bcl-2 Homologous Antagonist-Killer Protein, bcl-2-Associated X Protein, Caspases, Cell Death, Cell Survival, Cells, Cultured, DNA Damage, Doxorubicin, drosophila melanogaster, Drosophila Proteins, Embryo, Nonmammalian, Enzyme Activation, Epistasis, Genetic, Gene Silencing, genome, HeLa Cells, Hemocytes, Humans, Inhibitor of Apoptosis Proteins, N-Terminal Acetyltransferase A, N-Terminal Acetyltransferase E, Protein-Serine-Threonine Kinases, RNA Interference, RNA, Small Interfering, Transcription Factors, Transfection}, issn = {1540-8140}, doi = {10.1083/jcb.200708090}, author = {Yi, Caroline H and Sogah, Dodzie K and Boyce, Michael and Degterev, Alexei and Christofferson, Dana E and Yuan, Junying} } @article {825381, title = {Msk is required for nuclear import of TGF-{beta}/BMP-activated Smads.}, journal = {J Cell Biol}, volume = {178}, year = {2007}, month = {2007 Sep 10}, pages = {981-94}, abstract = {

Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.

}, keywords = {Active Transport, Cell Nucleus, Animals, Bone Morphogenetic Protein 2, Bone Morphogenetic Proteins, Cell Nucleus, DNA-Binding Proteins, Drosophila, Drosophila Proteins, Genome, Insect, Humans, Karyopherins, Receptors, Cytoplasmic and Nuclear, RNA Interference, Smad Proteins, Transcription Factors, Transforming Growth Factor beta}, issn = {0021-9525}, doi = {10.1083/jcb.200703106}, author = {Xu, Lan and Yao, Xiaohao and Chen, Xiaochu and Lu, Peiyuan and Zhang, Biliang and Ip, Y Tony} } @article {825376, title = {Quantitative morphological signatures define local signaling networks regulating cell morphology.}, journal = {Science}, volume = {316}, year = {2007}, month = {2007 Jun 22}, pages = {1753-6}, abstract = {

Although classical genetic and biochemical approaches have identified hundreds of proteins that function in the dynamic remodeling of cell shape in response to upstream signals, there is currently little systems-level understanding of the organization and composition of signaling networks that regulate cell morphology. We have developed quantitative morphological profiling methods to systematically investigate the role of individual genes in the regulation of cell morphology in a fast, robust, and cost-efficient manner. We analyzed a compendium of quantitative morphological signatures and described the existence of local signaling networks that act to regulate cell protrusion, adhesion, and tension.

}, keywords = {Animals, Cell Line, Cell Movement, Cell Shape, Drosophila, Green Fluorescent Proteins, Metabolic Networks and Pathways, Phenotype, RNA Interference, Signal Transduction}, issn = {1095-9203}, doi = {10.1126/science.1140324}, author = {Bakal, Chris and Aach, John and Church, George and Perrimon, Norbert} } @article {825396, title = {RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection.}, journal = {PLoS Pathog}, volume = {3}, year = {2007}, month = {2007 Oct 26}, pages = {1446-58}, abstract = {

Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate{\textquoteright}s knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.

}, keywords = {Animals, Carrier Proteins, Chlamydia, Chlamydia Infections, Drosophila, Fluorescent Antibody Technique, Guinea Pigs, HeLa Cells, Host-Parasite Interactions, Humans, Image Processing, Computer-Assisted, Inclusion Bodies, Microscopy, Electron, Transmission, mitochondria, RNA Interference}, issn = {1553-7374}, doi = {10.1371/journal.ppat.0030155}, author = {Derr{\'e}, Isabelle and Pypaert, Marc and Dautry-Varsat, Alice and Agaisse, Herv{\'e}} } @article {825351, title = {COPI activity coupled with fatty acid biosynthesis is required for viral replication.}, journal = {PLoS Pathog}, volume = {2}, year = {2006}, month = {2006 Oct}, pages = {e102}, abstract = {

During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.

}, keywords = {Animals, Cell Line, Coat Protein Complex I, Drosophila, Fatty Acids, Golgi Apparatus, Humans, Nodaviridae, Poliovirus, RNA Interference, RNA, Viral, Virus Replication}, issn = {1553-7374}, doi = {10.1371/journal.ppat.0020102}, author = {Cherry, Sara and Kunte, Amit and Wang, Hui and Coyne, Carolyn and Rawson, Robert B and Perrimon, Norbert} } @article {825336, title = {CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry.}, journal = {Science}, volume = {312}, year = {2006}, month = {2006 May 26}, pages = {1220-3}, abstract = {

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.

}, keywords = {Animals, calcium, Calcium Channels, Cell Line, Cell Membrane, drosophila melanogaster, Drosophila Proteins, Endoplasmic Reticulum, Humans, Ion Transport, Jurkat Cells, Membrane Proteins, Patch-Clamp Techniques, Reverse Transcriptase Polymerase Chain Reaction, RNA Interference, RNA, Small Interfering}, issn = {1095-9203}, doi = {10.1126/science.1127883}, author = {Vig, M and Peinelt, C and Beck, A and Koomoa, D L and Rabah, D and Koblan-Huberson, M and Kraft, S and Turner, H and Fleig, A and Penner, R and Kinet, J-P} } @article {825346, title = {Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays.}, journal = {Nat Methods}, volume = {3}, year = {2006}, month = {2006 Oct}, pages = {833-8}, abstract = {

To evaluate the specificity of long dsRNAs used in high-throughput RNA interference (RNAi) screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Notably, our analysis predicts that dsRNAs containing \> or = 19-nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to efficient knockdown of a cross-hybridizing transcript, raising a cautionary note about interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high-quality information from RNAi high-throughput screens.

}, keywords = {Animals, drosophila melanogaster, False Positive Reactions, Gene Library, Genetic Testing, RNA Interference, RNA, Double-Stranded, Sensitivity and Specificity}, issn = {1548-7091}, doi = {10.1038/nmeth935}, author = {Kulkarni, Meghana M and Booker, Matthew and Silver, Serena J and Friedman, Adam and Hong, Pengyu and Perrimon, Norbert and Mathey-Prevot, Bernard} } @article {825311, title = {FlyRNAi: the Drosophila RNAi screening center database.}, journal = {Nucleic Acids Res}, volume = {34}, year = {2006}, month = {2006 Jan 1}, pages = {D489-94}, abstract = {

RNA interference (RNAi) has become a powerful tool for genetic screening in Drosophila. At the Drosophila RNAi Screening Center (DRSC), we are using a library of over 21,000 double-stranded RNAs targeting known and predicted genes in Drosophila. This library is available for the use of visiting scientists wishing to perform full-genome RNAi screens. The data generated from these screens are collected in the DRSC database (http://flyRNAi.org/cgi-bin/RNAi_screens.pl) in a flexible format for the convenience of the scientist and for archiving data. The long-term goal of this database is to provide annotations for as many of the uncharacterized genes in Drosophila as possible. Data from published screens are available to the public through a highly configurable interface that allows detailed examination of the data and provides access to a number of other databases and bioinformatics tools.

}, keywords = {Animals, Computational Biology, Databases, Genetic, Drosophila, Gene Library, Genome, Insect, Internet, RNA Interference, RNA, Double-Stranded, Software, User-Computer Interface}, issn = {1362-4962}, doi = {10.1093/nar/gkj114}, author = {Flockhart, Ian and Booker, Matthew and Kiger, Amy and Boutros, Michael and Armknecht, Susan and Ramadan, Nadire and Richardson, Kris and Xu, Andrew and Perrimon, Norbert and Mathey-Prevot, Bernard} } @article {825316, title = {Functional genomics reveals genes involved in protein secretion and Golgi organization.}, journal = {Nature}, volume = {439}, year = {2006}, month = {2006 Feb 2}, pages = {604-7}, abstract = {

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.

}, keywords = {Animals, Cell Line, Drosophila, Drosophila Proteins, Endoplasmic Reticulum, Genes, Insect, Genes, Reporter, Genomics, Golgi Apparatus, Horseradish Peroxidase, Intracellular Membranes, Protein Sorting Signals, RNA Interference}, issn = {1476-4687}, doi = {10.1038/nature04377}, author = {Bard, Frederic and Casano, Laetitia and Mallabiabarrena, Arrate and Wallace, Erin and Saito, Kota and Kitayama, Hitoshi and Guizzunti, Gianni and Hu, Yue and Wendler, Franz and DasGupta, Ramanuj and Perrimon, Norbert and Malhotra, Vivek} } @article {825356, title = {A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling.}, journal = {Nature}, volume = {444}, year = {2006}, month = {2006 Nov 9}, pages = {230-4}, abstract = {

Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.

}, keywords = {Animals, Drosophila, Drug Evaluation, Preclinical, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases, Genome, Insect, Genomics, Ligands, MAP Kinase Signaling System, Receptor Protein-Tyrosine Kinases, RNA Interference}, issn = {1476-4687}, doi = {10.1038/nature05280}, author = {Friedman, Adam and Perrimon, Norbert} } @article {825321, title = {A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.}, journal = {Nature}, volume = {441}, year = {2006}, month = {2006 Jun 1}, pages = {646-50}, abstract = {

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

}, keywords = {Animals, Casein Kinase I, Drosophila, Genome, Insect, Genomics, Glycogen Synthase Kinase 3, Interleukin-2, NFATC Transcription Factors, Phosphorylation, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Protein-Tyrosine Kinases, RNA Interference, Transcription, Genetic}, issn = {1476-4687}, doi = {10.1038/nature04631}, author = {Gwack, Yousang and Sharma, Sonia and Nardone, Julie and Tanasa, Bogdan and Iuga, Alina and Srikanth, Sonal and Okamura, Heidi and Bolton, Diana and Feske, Stefan and Hogan, Patrick G and Rao, Anjana} } @article {825341, title = {Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity.}, journal = {Proc Natl Acad Sci U S A}, volume = {103}, year = {2006}, month = {2006 Jun 13}, pages = {9357-62}, abstract = {

Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca(2+) influx and Ca(2+) release-activated Ca(2+) (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca(2+) influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca(2+) entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca(2+) pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca(2+) depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

}, keywords = {Animals, calcium, Calcium Channels, drosophila melanogaster, Drosophila Proteins, Enzyme Inhibitors, Genome, Insect, Humans, Patch-Clamp Techniques, Recombinant Fusion Proteins, RNA Interference, RNA, Double-Stranded, Signal Transduction, Thapsigargin}, issn = {0027-8424}, doi = {10.1073/pnas.0603161103}, author = {Zhang, Shenyuan L and Yeromin, Andriy V and Zhang, Xiang H-F and Yu, Ying and Safrina, Olga and Penna, Aubin and Roos, Jack and Stauderman, Kenneth A and Cahalan, Michael D} } @article {825331, title = {High-throughput RNAi screening in cultured cells: a user{\textquoteright}s guide.}, journal = {Nat Rev Genet}, volume = {7}, year = {2006}, month = {2006 May}, pages = {373-84}, abstract = {

RNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user{\textquoteright}s guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.

}, keywords = {Animals, Automation, Cells, Cultured, drosophila melanogaster, Gene Expression Profiling, Gene Transfer Techniques, Humans, Models, Biological, Phenotype, Quality Control, RNA Interference, RNA, Small Interfering}, issn = {1471-0056}, doi = {10.1038/nrg1836}, author = {Echeverri, Christophe J and Perrimon, Norbert} } @article {825326, title = {A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function.}, journal = {Nature}, volume = {441}, year = {2006}, month = {2006 May 11}, pages = {179-85}, abstract = {

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

}, keywords = {Animals, Biological Transport, calcium, Calcium Channels, Carrier State, Chromosomes, Human, Pair 12, drosophila melanogaster, Drosophila Proteins, Electric Conductivity, Gene Dosage, Genome, Human, Heterozygote, Humans, Lod Score, Membrane Proteins, Mutation, NFATC Transcription Factors, Phenotype, Polymorphism, Single Nucleotide, RNA Interference, Severe Combined Immunodeficiency, T-Lymphocytes}, issn = {1476-4687}, doi = {10.1038/nature04702}, author = {Feske, Stefan and Gwack, Yousang and Prakriya, Murali and Srikanth, Sonal and Puppel, Sven-Holger and Tanasa, Bogdan and Hogan, Patrick G and Lewis, Richard S and Daly, Mark and Rao, Anjana} } @article {825296, title = {Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection.}, journal = {Science}, volume = {309}, year = {2005}, month = {2005 Aug 19}, pages = {1251-3}, abstract = {

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.

}, keywords = {Animals, Antigens, CD36, Cell Line, Cytoskeleton, drosophila melanogaster, Escherichia coli, Humans, Immunity, Innate, Lysosome-Associated Membrane Glycoproteins, Macrophages, Membrane Proteins, Mice, Mycobacterium fortuitum, Phagocytosis, Receptors, Immunologic, Receptors, Scavenger, RNA Interference, RNA, Double-Stranded, Scavenger Receptors, Class B, Sialoglycoproteins, Staphylococcus aureus, Transfection, Transport Vesicles}, issn = {1095-9203}, doi = {10.1126/science.1116006}, author = {Philips, Jennifer A and Rubin, Eric J and Perrimon, Norbert} } @article {825286, title = {Functional genomic analysis of the Wnt-wingless signaling pathway.}, journal = {Science}, volume = {308}, year = {2005}, month = {2005 May 6}, pages = {826-33}, abstract = {

The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50\% of the genes identified in the screen have human orthologs, of which approximately 18\% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.

}, keywords = {Animals, beta Catenin, Binding Sites, Cell Line, Cloning, Molecular, Computational Biology, Cytoskeletal Proteins, drosophila melanogaster, Drosophila Proteins, Embryo, Nonmammalian, Embryonic Development, Epistasis, Genetic, Gene Expression Regulation, Genes, Insect, Genes, Reporter, Genomics, Mutation, Phenotype, Phosphorylation, Protein Kinases, Proteins, Proto-Oncogene Proteins, rab5 GTP-Binding Proteins, RNA Interference, Signal Transduction, Trans-Activators, Transcription Factors, Transfection, Wnt Proteins, Wnt1 Protein, Wnt3 Protein, zebrafish, Zebrafish Proteins}, issn = {1095-9203}, doi = {10.1126/science.1109374}, author = {DasGupta, Ramanuj and Kaykas, Ajamete and Moon, Randall T and Perrimon, Norbert} } @article {825306, title = {A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway.}, journal = {Nat Genet}, volume = {37}, year = {2005}, month = {2005 Dec}, pages = {1323-32}, abstract = {

Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. Here we report the results of a genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI) complex, the ribosome and the proteasome. We identified the multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, as Hh regulators. We also identified a large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, we showed that several of these genes have conserved roles in mammalian Hh signaling.

}, keywords = {Animals, drosophila melanogaster, Drosophila Proteins, Gene Expression Regulation, Genes, Insect, Genome, Insect, Genomics, Hedgehog Proteins, Insect Proteins, Phosphoprotein Phosphatases, Phosphotransferases, Protein Phosphatase 2, RNA Interference, RNA Splicing, RNA, Messenger, Signal Transduction}, issn = {1061-4036}, doi = {10.1038/ng1682}, author = {Nybakken, Kent and Vokes, Steven A and Lin, Ting-Yi and McMahon, Andrew P and Perrimon, Norbert} } @article {825301, title = {Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila.}, journal = {Genes Dev}, volume = {19}, year = {2005}, month = {2005 Aug 15}, pages = {1861-70}, abstract = {

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

}, keywords = {Active Transport, Cell Nucleus, Animals, Cell Line, Cell Nucleus, DNA-Binding Proteins, Drosophila Proteins, Janus Kinase 1, Phosphorylation, Protein Tyrosine Phosphatases, Protein Tyrosine Phosphatases, Non-Receptor, Protein-Tyrosine Kinases, Repressor Proteins, RNA Interference, Signal Transduction, STAT Transcription Factors, Suppressor of Cytokine Signaling Proteins, Trans-Activators, Tyrosine}, issn = {0890-9369}, doi = {10.1101/gad.1320705}, author = {Baeg, Gyeong-Hun and Zhou, Rui and Perrimon, Norbert} } @article {825291, title = {Genome-wide RNAi screen for host factors required for intracellular bacterial infection.}, journal = {Science}, volume = {309}, year = {2005}, month = {2005 Aug 19}, pages = {1248-51}, abstract = {

Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

}, keywords = {Animals, Cell Cycle, Cell Line, Cytoskeleton, Cytosol, drosophila melanogaster, Drosophila Proteins, Genes, Insect, genome, Green Fluorescent Proteins, Listeria monocytogenes, Macrophages, Mycobacterium fortuitum, Phenotype, RNA Interference, RNA Processing, Post-Transcriptional, RNA, Double-Stranded, Signal Transduction, Vacuoles, Vesicular Transport Proteins}, issn = {1095-9203}, doi = {10.1126/science.1116008}, author = {Agaisse, Herv{\'e} and Burrack, Laura S and Philips, Jennifer A and Rubin, Eric J and Perrimon, Norbert and Higgins, Darren E} } @article {825281, title = {Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.}, journal = {Genes Dev}, volume = {19}, year = {2005}, month = {2005 Feb 15}, pages = {445-52}, abstract = {

The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.

}, keywords = {Animals, Base Sequence, DNA Primers, Drosophila, Genome, Viral, HeLa Cells, Humans, Protein Biosynthesis, RNA Interference, RNA Viruses, Virus Replication}, issn = {0890-9369}, doi = {10.1101/gad.1267905}, author = {Cherry, Sara and Doukas, Tammy and Armknecht, Susan and Whelan, Sean and Wang, Hui and Sarnow, Peter and Perrimon, Norbert} } @article {825276, title = {High-throughput RNA interference screens in Drosophila tissue culture cells.}, journal = {Methods Enzymol}, volume = {392}, year = {2005}, month = {2005}, pages = {55-73}, abstract = {

This chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.

}, keywords = {Animals, Cell Line, Drosophila, Genes, Reporter, RNA Interference, Tissue Culture Techniques, Transfection}, issn = {0076-6879}, doi = {10.1016/S0076-6879(04)92004-6}, author = {Armknecht, Susan and Boutros, Michael and Kiger, Amy and Nybakken, Kent and Mathey-Prevot, Bernard and Perrimon, Norbert} } @article {825266, title = {Genome-wide RNAi analysis of growth and viability in Drosophila cells.}, journal = {Science}, volume = {303}, year = {2004}, month = {2004 Feb 6}, pages = {832-5}, abstract = {

A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91\%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80\% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.

}, keywords = {Animals, Apoptosis, Cell Cycle, Cell Survival, Cells, Cultured, Computational Biology, Core Binding Factor Alpha 2 Subunit, DNA-Binding Proteins, drosophila melanogaster, Drosophila Proteins, Genes, Essential, Genes, Insect, genome, Humans, Inhibitor of Apoptosis Proteins, Phenotype, Proteome, Proto-Oncogene Proteins, Reproducibility of Results, RNA Interference, RNA, Double-Stranded, Sequence Homology, Transcription Factors}, issn = {1095-9203}, doi = {10.1126/science.1091266}, author = {Boutros, Michael and Kiger, Amy A and Armknecht, Susan and Kerr, Kim and Hild, Marc and Koch, Britta and Haas, Stefan A and Paro, Renato and Perrimon, Norbert and Heidelberg Fly Array Consortium} } @article {825271, title = {Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets.}, journal = {PLoS Biol}, volume = {2}, year = {2004}, month = {2004 Dec}, pages = {e379}, abstract = {

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.

}, keywords = {Animals, Aurora Kinases, Caenorhabditis elegans, Cell Line, Cytokinesis, Drosophila, Formamides, Genetic techniques, genome, Genomics, Microscopy, Fluorescence, Phenotype, Protein-Serine-Threonine Kinases, Pyrazoles, RNA Interference, Saccharomyces cerevisiae}, issn = {1545-7885}, doi = {10.1371/journal.pbio.0020379}, author = {Eggert, Ulrike S and Kiger, Amy A and Richter, Constance and Perlman, Zachary E and Perrimon, Norbert and Mitchison, Timothy J and Field, Christine M} } @article {825261, title = {A functional genomic analysis of cell morphology using RNA interference.}, journal = {J Biol}, volume = {2}, year = {2003}, month = {2003}, pages = {27}, abstract = {

BACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

}, keywords = {Animals, Cell Line, Cell Shape, Cytoskeleton, Drosophila, Drosophila Proteins, Genes, Insect, genome, Genomics, Microscopy, Fluorescence, Mutation, Phenotype, Phosphoric Monoester Hydrolases, PTEN Phosphohydrolase, RNA Interference, RNA, Double-Stranded}, issn = {1475-4924}, doi = {10.1186/1475-4924-2-27}, author = {Kiger, A A and Baum, B and Jones, S and Jones, M R and Coulson, A and Echeverri, C and N Perrimon} } @article {1467181, title = {Task delegation to physician extenders--some comparisons}, journal = {Am J Public Health}, volume = {66}, year = {1976}, month = {1976 Jan}, pages = {64-6}, abstract = {

This study uses a task delegation questionnaire to compare 1973 physician extender practices in seven primary care-oriented sites with a physician attitude survey made in 1969. One additional site using no physician extenders was included as a control. The study involves both major types of physician extenders (physician assistants and nurse practitioners) in ambulatory practices with at least one year of experience in using such personnel. With minor exceptions, actual task delegation patterns conform with the 1969 attitudes of physicians as to which tasks "could and should" be delegated to physician extenders.

}, keywords = {Humans, Nurse Practitioners, Physician Assistants, Primary Health Care, Professional Practice, Task Performance and Analysis, United States}, issn = {0090-0036}, doi = {10.2105/ajph.66.1.64}, author = {Glenn, J K and Goldman, J} } @booklet {1488881, title = {An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination}, howpublished = {Elife}, volume = {11}, year = {Published}, note = {2050-084xKanca, Oguz Orcid: 0000-0001-5438-0879 Zirin, Jonathan Hu, Yanhui Tepe, Burak Dutta, Debdeep Lin, Wen-Wen Ma, Liwen Ge, Ming Zuo, Zhongyuan Liu, Lu-Ping Levis, Robert W Orcid: 0000-0003-3453-2390 Perrimon, Norbert Orcid: 0000-0001-7542-472x Bellen, Hugo J Orcid: 0000-0001-5992-5989 U54 HD083092/HD/NICHD NIH HHS/United States R01 GM067761/GM/NIGMS NIH HHS/United States R01 GM067858/GM/NIGMS NIH HHS/United States R01 GM084947/GM/NIGMS NIH HHS/United States HHMI/Howard Hughes Medical Institute/United States R24 OD031447/OD/NIH HHS/United States U54 NS093793/NS/NINDS NIH HHS/United States R24 OD022005/OD/NIH HHS/United States Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov{\textquoteright}t England 2022/06/21 Elife. 2022 Jun 20;11:e76077. doi: 10.7554/eLife.76077. }, month = {Jun 20}, abstract = {Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58\% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80\% success rate.}, keywords = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics, *Drosophila/genetics, Animals, Crispr, CRISPR-Cas Systems/genetics, D. melanogaster, Exons/genetics, gene trap, Genetics, Genomics, Homologous Recombination, knock-in, knock-out, Plasmids, protein trap}, isbn = {2050-084x}, author = {Kanca, O. and Zirin, J. and Hu, Y. and Tepe, B. and Dutta, D. and Lin, W. W. and Ma, L. and Ge, M. and Zuo, Z. and Liu, L. P. and Levis, R. W. and Perrimon, N. and Bellen, H. J.} } @article {1488886, title = {Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11}, journal = {Genet Med}, volume = {23}, year = {Published}, note = {1530-0366Ravenscroft, Thomas A Phillips, Jennifer B Fieg, Elizabeth Bajikar, Sameer S Peirce, Judy Wegner, Jeremy Luna, Alia A Fox, Eric J Yan, Yi-Lin Rosenfeld, Jill A Zirin, Jonathan Kanca, Oguz Undiagnosed Diseases Network Benke, Paul J Cameron, Eric S Strehlow, Vincent Platzer, Konrad Jamra, Rami Abou Kl{\"o}ckner, Chiara Osmond, Matthew Licata, Thomas Rojas, Samantha Dyment, David Chong, Josephine S C Lincoln, Sharyn Stoler, Joan M Postlethwait, John H Wangler, Michael F Yamamoto, Shinya Krier, Joel Westerfield, Monte Bellen, Hugo J U01 HG007690/HG/NHGRI NIH HHS/United States P41 GM132087/GM/NIGMS NIH HHS/United States R24 OD031447/OD/NIH HHS/United States F32 HD100048/HD/NICHD NIH HHS/United States U01 HG007942/HG/NHGRI NIH HHS/United States R24 OD026591/OD/NIH HHS/United States U54 NS093793/NS/NINDS NIH HHS/United States R24 OD022005/OD/NIH HHS/United States Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov{\textquoteright}t United States 2021/06/12 Genet Med. 2021 Oct;23(10):1889-1900. doi: 10.1038/s41436-021-01216-8. Epub 2021 Jun 10. }, month = {Oct}, pages = {1889-1900}, abstract = {PURPOSE: Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. METHODS: We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. RESULTS: Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients{\textquoteright} variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants. CONCLUSION: GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.}, keywords = {*Bone Morphogenetic Proteins/genetics, *Growth Differentiation Factors/genetics, Animals, Craniofacial Abnormalities/*genetics, Humans, Mutation, Missense, Phenotype, Spine, Zebrafish/genetics}, isbn = {1098-3600 (Print)1098-3600 }, author = {Ravenscroft, T. A. and Phillips, J. B. and Fieg, E. and Bajikar, S. S. and Peirce, J. and Wegner, J. and Luna, A. A. and Fox, E. J. and Yan, Y. L. and Rosenfeld, J. A. and Zirin, J. and Kanca, O. and Benke, P. J. and Cameron, E. S. and Strehlow, V. and Platzer, K. and Jamra, R. A. and Kl{\"o}ckner, C. and Osmond, M. and Licata, T. and Rojas, S. and Dyment, D. and Chong, J. S. C. and Lincoln, S. and Stoler, J. M. and Postlethwait, J. H. and Wangler, M. F. and Yamamoto, S. and Krier, J. and Westerfield, M. and Bellen, H. J.} }