Cell-based assays

Stephanie E Mohr, Yanhui Hu, Kirstin Rudd, Michael Buckner, Quentin Gilly, Blake Foster, Katarzyna Sierzputowska, Aram Comjean, Bing Ye, and Norbert Perrimon. 2015. “Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells.” G3 (Bethesda), 5, 9, Pp. 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.

2015_G3_Mohr.pdf Reagent Table S1.xlsx
Jonathan Zirin, Joppe Nieuwenhuis, Anastasia Samsonova, Rong Tao, and Norbert Perrimon. 2015. “Regulators of autophagosome formation in Drosophila muscles.” PLoS Genet, 11, 2, Pp. 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.

2015_PLOS Genet_Zirin.pdf Supplemental Files.zip
Richelle Sopko, You Bin Lin, Kalpana Makhijani, Brandy Alexander, Norbert Perrimon, and Katja Brückner. 2015. “A systems-level interrogation identifies regulators of Drosophila blood cell number and survival.” PLoS Genet, 11, 3, Pp. 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.

2015_PLOS Gen_Sopko.pdf Supplemental Files.zip
Dorte Bohla, Martin Herold, Imke Panzer, Melanie K Buxa, Tamer Ali, Jeroen Demmers, Marcus Krüger, Maren Scharfe, Michael Jarek, Marek Bartkuhn, and Rainer Renkawitz. 2014. “A functional insulator screen identifies NURF and dREAM components to be required for enhancer-blocking.” PLoS One, 9, 9, Pp. 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.

2014_PLOS One_Bohla.pdf Supplemental Files.zip
Inma Gonzalez, Julio Mateos-Langerak, Aubin Thomas, Thierry Cheutin, and Giacomo Cavalli. 2014. “Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen.” Mol Cell, 54, 3, Pp. 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.

2014_Mol Cell_Gonzalez.pdf Supplement.pdf
Clément Carré, Caroline Jacquier, Anne-Laure Bougé, Fabrice de Chaumont, Corinne Besnard-Guerin, Hélène Thomassin, Josette Pidoux, Bruno Da Silva, Eleftheria Chalatsi, Sarah Zahra, Jean-Christophe Olivo-Marin, Hélène Munier-Lehmann, and Christophe Antoniewski. 2013. “AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.” PLoS One, 8, 9, Pp. 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.

2013_PLOS One_Carre.pdf Supplemental Files.zip
Ralph A Neumüller, Thomas Gross, Anastasia A Samsonova, Arunachalam Vinayagam, Michael Buckner, Karen Founk, Yanhui Hu, Sara Sharifpoor, Adam P Rosebrock, Brenda Andrews, Fred Winston, and Norbert Perrimon. 2013. “Conserved regulators of nucleolar size revealed by global phenotypic analyses.” Sci Signal, 6, 289, Pp. 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.

2013_Sci Sig_Neumuller.pdf Supplemental Files.zip
Clemens Bergwitz, Mark J Wee, Sumi Sinha, Joanne Huang, Charles DeRobertis, Lawrence B Mensah, Jonathan Cohen, Adam Friedman, Meghana Kulkarni, Yanhui Hu, Arunachalam Vinayagam, Michael Schnall-Levin, Bonnie Berger, Lizabeth A Perkins, Stephanie E Mohr, and Norbert Perrimon. 2013. “Genetic determinants of phosphate response in Drosophila.” PLoS One, 8, 3, Pp. 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.

2013_PLOS One_Bergwitz.pdf Supplemental Files.zip
Keren Imberg-Kazdan, Susan Ha, Alex Greenfield, Christopher S Poultney, Richard Bonneau, Susan K Logan, and Michael J Garabedian. 2013. “A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells.” Genome Res, 23, 4, Pp. 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.

2013_Genome Res_Imberg-Kazdan.pdf Supplement.pdf
Felix Muerdter, Paloma M Guzzardo, Jesse Gillis, Yicheng Luo, Yang Yu, Caifu Chen, Richard Fekete, and Gregory J Hannon. 2013. “A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.” Mol Cell, 50, 5, Pp. 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.

2013_Mol Cell_Muerdter.pdf Supplemental Files.zip
Young Kwon, Arunachalam Vinayagam, Xiaoyun Sun, Noah Dephoure, Steven P Gygi, Pengyu Hong, and Norbert Perrimon. 2013. “The Hippo signaling pathway interactome.” Science, 342, 6159, Pp. 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.

2013_Science_Kwon.pdf Supplemental Files.zip
Zheng Yin, Amine Sadok, Heba Sailem, Afshan McCarthy, Xiaofeng Xia, Fuhai Li, Mar Arias Garcia, Louise Evans, Alexis R Barr, Norbert Perrimon, Christopher J Marshall, Stephen TC Wong, and Chris Bakal. 2013. “A screen for morphological complexity identifies regulators of switch-like transitions between discrete cell shapes.” Nat Cell Biol, 15, 7, Pp. 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.

2013_Nat Cell Bio_Yin.pdf Supplemental Files.zip
Joseph Dopie, Kari-Pekka Skarp, Eeva Kaisa Rajakylä, Kimmo Tanhuanpää, and Maria K Vartiainen. 2012. “Active maintenance of nuclear actin by importin 9 supports transcription.” Proc Natl Acad Sci U S A, 109, 9, Pp. 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.

2012_PNAS_Dopie.pdf Supplement.pdf
Stephanie C Stotz and David E Clapham. 2012. “Anion-sensitive fluorophore identifies the Drosophila swell-activated chloride channel in a genome-wide RNA interference screen.” PLoS One, 7, 10, Pp. 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.

2012_PLOS One_Stotz.pdf Supplemental Files.zip
Marcelo Perez-Pepe, Victoria Slomiansky, Mariela Loschi, Luciana Luchelli, Maximiliano Neme, María Gabriela Thomas, and Graciela Lidia Boccaccio. 2012. “BUHO: a MATLAB script for the study of stress granules and processing bodies by high-throughput image analysis.” PLoS One, 7, 12, Pp. 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.

2012_PLOS One_Perez-Pepe.pdf Supplemental Files.zip
Mar Arias Garcia, Miguel Sanchez Alvarez, Heba Sailem, Vicky Bousgouni, Julia Sero, and Chris Bakal. 2012. “Differential RNAi screening provides insights into the rewiring of signalling networks during oxidative stress.” Mol Biosyst, 8, 10, Pp. 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.

2012_Mol BioSys_Garcia.pdf Supplemental Files.zip
Ian T Flockhart, Matthew Booker, Yanhui Hu, Benjamin McElvany, Quentin Gilly, Bernard Mathey-Prevot, Norbert Perrimon, and Stephanie E Mohr. 2012. “FlyRNAi.org--the database of the Drosophila RNAi screening center: 2012 update.” Nucleic Acids Res, 40, Database issue, Pp. 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.

2012_Nuc Acids Res_Flockhart.pdf
Reid Aikin, Alexandra Cervantes, Gisela D'Angelo, Laurent Ruel, Sandra Lacas-Gervais, Sébastien Schaub, and Pascal Thérond. 2012. “A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila.” PLoS One, 7, 3, Pp. 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.

2012_PLOS One_Aikin.pdf Supplemental Files.zip
Erica Larschan, Marcela ML Soruco, Ok-Kyung Lee, Shouyong Peng, Eric Bishop, Jessica Chery, Karen Goebel, Jessica Feng, Peter J Park, and Mitzi I Kuroda. 2012. “Identification of chromatin-associated regulators of MSL complex targeting in Drosophila dosage compensation.” PLoS Genet, 8, 7, Pp. 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.

2012_PLOS Gene_Larschan.pdf Supplemental Files.zip
Eric F Joyce, Benjamin R Williams, Tiao Xie, and C-Ting Wu. 2012. “Identification of genes that promote or antagonize somatic homolog pairing using a high-throughput FISH-based screen.” PLoS Genet, 8, 5, Pp. 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.

2012_PLOS Gene_Joyce.pdf Supplemental Files.zip