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Tao, Rong

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Tao, Rong
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    Regulators of Autophagosome Formation in Drosophila Muscles
    (Public Library of Science, 2015) Zirin, Jonathan; Nieuwenhuis, Joppe; Samsonova, Anastasia; Tao, Rong; Perrimon, Norbert
    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.
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    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, Norbert
    While 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.
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    A Genome-Scale shRNA Resource for Transgenic RNAi in Drosophila
    (Springer Science and Business Media LLC, 2011-05) Ni, Jian-Quan; Zhou, Rui; Czech, Benjamin; Liu, Lu-Ping; Holderbaum, Laura; Yang-Zhou, Donghui; Shim, Hye-Seok; Tao, Rong; Handler, Dominik; Karpowicz, Phillip; Binari, Richard; Booker, Matthew; Brennecke, Julius; Perkins, Lizabeth A.; Hannon, Gregory J.; Perrimon, Norbert
    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.
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    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 J
    We 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.
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    Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells
    (Genetics Society of America, 2018) Mohr, Stephanie; Rudd, Kirstin; Hu, Yanhui; Song, Wei; Gilly, Quentin; Buckner, Michael; Housden, Benjamin E.; Kelley, Colleen; Zirin, Jonathan; Tao, Rong; Amador, Gabriel; Sierzputowska, Katarzyna; Comjean, Aram; Perrimon, Norbert
    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 wild-type 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 >1800 putative zinc-responsive genes. Analysis of the ‘omics data points to the relevance of ion transporters, glutathione (GSH)-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 upregulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the ‘omics data sets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.