Person: Zirin, Jonathan
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Zirin
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Jonathan
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Zirin, Jonathan
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Publication Ecdysone Signaling at Metamorphosis Triggers Apoptosis of Drosophila Abdominal Muscles(Elsevier BV, 2013-11-15) Zirin, Jonathan; Cheng, Daojun; Dhanyasi, Nagaraju; Cho, Julio; Dura, Jean-Maurice; VijayRaghavan, Krishnaswamy; Perrimon, NorbertOne of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24 hrs of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.Publication Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy(Public Library of Science, 2013) Zirin, Jonathan; Nieuwenhuis, Joppe; Perrimon, NorbertSeveral myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8.Publication Integrating protein-protein interaction networks with phenotypes reveals signs of interactions(2013) Vinayagam, Arunachalam; Zirin, Jonathan; Roesel, Charles; Hu, Yanhui; Yilmazel, Bahar; Samsonova, Anastasia A.; Neumüller, Ralph A.; Mohr, Stephanie; Perrimon, NorbertA 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 “signs” 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 impact our understanding of many biological functions, including signal transduction and mechanisms of disease.Publication Regulators of Autophagosome Formation in Drosophila Muscles(Public Library of Science, 2015) Zirin, Jonathan; Nieuwenhuis, Joppe; Samsonova, Anastasia; Tao, Rong; Perrimon, NorbertGiven 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.Publication FlyRNAi.org—the database of the Drosophila RNAi screening center and transgenic RNAi project: 2017 update(Oxford University Press, 2017) Hu, Yanhui; Comjean, Aram; Roesel, Charles; Vinayagam, Arunachalam; Flockhart, Ian; Zirin, Jonathan; Perkins, Lizabeth A.; Perrimon, Norbert; Mohr, StephanieThe 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.Publication Optimized Strategy for in Vivo Cas9-Activation in Drosophila(National Academy of Sciences, 2017-08-29) Ewen-Campen, Benjamin; Yang-Zhou, Donghui; Fernandes, Vitória R.; González, Delfina P.; Liu, Lu-Ping; Tao, Rong; Ren, Xingjie; Sun, Jin; Hu, Yanhui; Zirin, Jonathan; Mohr, Stephanie; Ni, Jian-Quan; Perrimon, NorbertWhile several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.Publication Activin Signaling Mediates Muscle-to-Adipose Communication in a Mitochondria Dysfunction-Associated Obesity Model(National Academy of Sciences, 2017-08-08) Song, Wei; Owusu-Ansah, Edward; Hu, Yanhui; Cheng, Daojun; Ni, Xiaochun; Zirin, Jonathan; Perrimon, NorbertMitochondrial perturbation-associated dysregulation of one organ has been shown to nonautonomously affect the functions of other organs in both vertebrates and invertebrates. Using Drosophila as a genetic model organism, we characterized mitochondrial synchrony dysregulation across organs and uncovered that mitochondrial perturbation caused by complex I disruption in muscles remotely impairs mitochondrial function and lipid mobilization in the fat body, leading to obesity. We further identified that the TGF-β ligand Actβ, which is autonomously increased by muscular mitochondrial perturbation, mediates muscle-to-fat-body communication and synchronized mitochondrial dysregulation.Publication The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming(Elsevier BV, 2018-05-01) Tang, Hong-Wen; Hu, Yanhui; Chen, Chiao-Lin; Xia, Baolong; Zirin, Jonathan; Yuan, Min; Asara, John; Rabinow, Leonard; Perrimon, NorbertNutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.Publication Primary Cell Cultures from Drosophila Gastrula Embryos(MyJove Corporation, 2011-02-28) Perrimon, Norbert; Zirin, Jonathan; Bai, JianwuHere 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.Publication A gene-specific T2A-GAL4 library for Drosophila(eLife Sciences Publications, Ltd, 2018) Lee, Pei-Tseng; Zirin, Jonathan; Kanca, Oguz; Lin, Wen-Wen; Schulze, Karen L; Li-Kroeger, David; Tao, Rong; Devereaux, Colby; Hu, Yanhui; Chung, Verena; Fang, Ying; He, Yuchun; Pan, Hongling; Ge, Ming; Zuo, Zhongyuan; Housden, Benjamin E; Mohr, Stephanie; Yamamoto, Shinya; Levis, Robert W; Spradling, Allan C; Perrimon, Norbert; Bellen, Hugo JWe generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3’ of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.