Research Article

2019
Zirin J, Ni X, Sack LM, Yang-Zhou D, Hu Y, Brathwaite R, et al. Interspecies analysis of MYC targets identifies tRNA synthetases as mediators of growth and survival in MYC-overexpressing cells. Proc Natl Acad Sci U S A. 2019;Abstract
Aberrant MYC oncogene activation is one of the most prevalent characteristics of cancer. By overlapping datasets of genes that are insulin-responsive and also regulate nucleolus size, we enriched for Myc target genes required for cellular biosynthesis. Among these, we identified the aminoacyl tRNA synthetases (aaRSs) as essential mediators of Myc growth control in and found that their pharmacologic inhibition is sufficient to kill MYC-overexpressing human cells, indicating that aaRS inhibitors might be used to selectively target MYC-driven cancers. We suggest a general principle in which oncogenic increases in cellular biosynthesis sensitize cells to disruption of protein homeostasis.
2019_PNAS_Zirin.pdf Supplement.pdf Supp. Datasets.zip
Dumesic PA, Egan DF, Gut P, Tran MT, Parisi A, Chatterjee N, et al. An Evolutionarily Conserved uORF Regulates PGC1α and Oxidative Metabolism in Mice, Flies, and Bluefin Tuna. Cell Metab. 2019;Abstract
Mitochondrial abundance and function are tightly controlled during metabolic adaptation but dysregulated in pathological states such as diabetes, neurodegeneration, cancer, and kidney disease. We show here that translation of PGC1α, a key governor of mitochondrial biogenesis and oxidative metabolism, is negatively regulated by an upstream open reading frame (uORF) in the 5' untranslated region of its gene (PPARGC1A). We find that uORF-mediated translational repression is a feature of PPARGC1A orthologs from human to fly. Strikingly, whereas multiple inhibitory uORFs are broadly present in fish PPARGC1A orthologs, they are completely absent in the Atlantic bluefin tuna, an animal with exceptionally high mitochondrial content. In mice, an engineered mutation disrupting the PPARGC1A uORF increases PGC1α protein levels and oxidative metabolism and confers protection from acute kidney injury. These studies identify a translational regulatory element governing oxidative metabolism and highlight its potential contribution to the evolution of organismal mitochondrial function.
2019_Cell Metab_Dumesic.pdf Supplement.zip
He L, Binari R, Huang J, Falo-Sanjuan J, Perrimon N. study of gene expression with an enhanced dual-color fluorescent transcriptional timer. Elife. 2019;8. Abstract
Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in . In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.
2019_eLife_He.pdf Supplement.zip
Bosch JA, Colbeth R, Zirin J, Perrimon N. Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids. BioRxiv [Internet]. 2019; Publisher's Version 2019_BioRxiv_Bosch.pdf
Strumillo MJ, Oplová M, Viéitez C, Ochoa D, Shahraz M, Busby BP, et al. Conserved phosphorylation hotspots in eukaryotic protein domain families. Nat Commun. 2019;10 (1) :1977. Abstract
Protein phosphorylation is the best characterized post-translational modification that regulates almost all cellular processes through diverse mechanisms such as changing protein conformations, interactions, and localization. While the inventory for phosphorylation sites across different species has rapidly expanded, their functional role remains poorly investigated. Here, we combine 537,321 phosphosites from 40 eukaryotic species to identify highly conserved phosphorylation hotspot regions within domain families. Mapping these regions onto structural data reveals that they are often found at interfaces, near catalytic residues and tend to harbor functionally important phosphosites. Notably, functional studies of a phospho-deficient mutant in the C-terminal hotspot region within the ribosomal S11 domain in the yeast ribosomal protein uS11 shows impaired growth and defective cytoplasmic 20S pre-rRNA processing at 16 °C and 20 °C. Altogether, our study identifies phosphorylation hotspots for 162 protein domains suggestive of an ancient role for the control of diverse eukaryotic domain families.
2019_Nat Comm_Strumillo.pdf Supplement.zip
Hunter GL, He L, Perrimon N, Charras G, Giniger E, Baum B. A role for actomyosin contractility in Notch signaling. BMC Biol. 2019;17 (1) :12. Abstract
BACKGROUND: Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling. RESULTS: To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems-as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture. CONCLUSIONS: Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation.
2019_BMC Bio_Hunter.pdf Supplemental Files.zip
Xu C, Tang H-W, Hung R-J, Hu Y, Ni X, Housden BE, et al. The Septate Junction Protein Tsp2A Restricts Intestinal Stem Cell Activity via Endocytic Regulation of aPKC and Hippo Signaling. Cell Rep. 2019;26 (3) :670-688.e6. Abstract
Hippo signaling and the activity of its transcriptional coactivator, Yorkie (Yki), are conserved and crucial regulators of tissue homeostasis. In the Drosophila midgut, after tissue damage, Yki activity increases to stimulate stem cell proliferation, but how Yki activity is turned off once the tissue is repaired is unknown. From an RNAi screen, we identified the septate junction (SJ) protein tetraspanin 2A (Tsp2A) as a tumor suppressor. Tsp2A undergoes internalization to facilitate the endocytic degradation of atypical protein kinase C (aPKC), a negative regulator of Hippo signaling. In the Drosophila midgut epithelium, adherens junctions (AJs) and SJs are prominent in intestinal stem cells or enteroblasts (ISCs or EBs) and enterocytes (ECs), respectively. We show that when ISCs differentiate toward ECs, Tsp2A is produced, participates in SJ assembly, and turns off aPKC and Yki-JAK-Stat activity. Altogether, our study uncovers a mechanism allowing the midgut to restore Hippo signaling and restrict proliferation once tissue repair is accomplished.
2019_Cell Rep_Xu.pdf Supplement.pdf Supplemental Tables.zip
Song W, Kir S, Hong S, Hu Y, Wang X, Binari R, et al. Tumor-Derived Ligands Trigger Tumor Growth and Host Wasting via Differential MEK Activation. Dev Cell. 2019;48 (2) :277-286.e6. Abstract
Interactions between tumors and host tissues play essential roles in tumor-induced systemic wasting and cancer cachexia, including muscle wasting and lipid loss. However, the pathogenic molecular mechanisms of wasting are still poorly understood. Using a fly model of tumor-induced organ wasting, we observed aberrant MEK activation in both tumors and host tissues of flies bearing gut-yki tumors. We found that host MEK activation results in muscle wasting and lipid loss, while tumor MEK activation is required for tumor growth. Strikingly, host MEK suppression alone is sufficient to abolish the wasting phenotypes without affecting tumor growth. We further uncovered that yki tumors produce the vein (vn) ligand to trigger autonomous Egfr/MEK-induced tumor growth and produce the PDGF- and VEGF-related factor 1 (Pvf1) ligand to non-autonomously activate host Pvr/MEK signaling and wasting. Altogether, our results demonstrate the essential roles and molecular mechanisms of differential MEK activation in tumor-induced host wasting.
2019_Dev Cell_Song.pdf Supplement.pdf Supplemental Tables.zip
2018
Doupé DP, Marshall OJ, Dayton H, Brand AH, Perrimon N. Drosophila intestinal stem and progenitor cells are major sources and regulators of homeostatic niche signals. Proc Natl Acad Sci U S A. 2018;115 (48) :12218-12223. Abstract
Epithelial homeostasis requires the precise balance of epithelial stem/progenitor proliferation and differentiation. While many signaling pathways that regulate epithelial stem cells have been identified, it is probable that other regulators remain unidentified. Here, we use gene-expression profiling by targeted DamID to identify the stem/progenitor-specific transcription and signaling factors in the midgut. Many signaling pathway components, including ligands of most major pathways, exhibit stem/progenitor-specific expression and have regulatory regions bound by both intrinsic and extrinsic transcription factors. In addition to previously identified stem/progenitor-derived ligands, we show that both the insulin-like factor Ilp6 and TNF ligand eiger are specifically expressed in the stem/progenitors and regulate normal tissue homeostasis. We propose that intestinal stem cells not only integrate multiple signals but also contribute to and regulate the homeostatic signaling microenvironmental niche through the expression of autocrine and paracrine factors.
2018_PNAS_Doupe.pdf Supplement.pdf Supp. Tables.zip
Parasram K, Bernardon N, Hammoud M, Chang H, He L, Perrimon N, et al. Intestinal Stem Cells Exhibit Conditional Circadian Clock Function. Stem Cell Reports. 2018;11 (5) :1287-1301. Abstract
The circadian clock is a molecular pacemaker that produces 24-hr physiological cycles known as circadian rhythms. How the clock regulates stem cells is an emerging area of research with many outstanding questions. We tested clock function in vivo at the single cell resolution in the Drosophila intestine, a tissue that is exquisitely sensitive to environmental cues and has circadian rhythms in regeneration. Our results indicate that circadian clocks function in intestinal stem cells and enterocytes but are downregulated during enteroendocrine cell differentiation. Drosophila intestinal cells are principally synchronized by the photoperiod, but intestinal stem cell clocks are highly responsive to signaling pathways that comprise their niche, and we find that the Wnt and Hippo signaling pathways positively regulate stem cell circadian clock function. These data reveal that intestinal stem cell circadian rhythms are regulated by cellular signaling and provide insight as to how clocks may be altered during physiological changes such as regeneration and aging.
2018_Stem Cell Rep_Parasram.pdf Supplement.pdf
Thurmond J, Goodman JL, Strelets VB, Attrill H, Gramates SL, Marygold SJ, et al. FlyBase 2.0: the next generation. Nucleic Acids Res. 2018;Abstract
FlyBase (flybase.org) is a knowledge base that supports the community of researchers that use the fruit fly, Drosophila melanogaster, as a model organism. The FlyBase team curates and organizes a diverse array of genetic, molecular, genomic, and developmental information about Drosophila. At the beginning of 2018, 'FlyBase 2.0' was released with a significantly improved user interface and new tools. Among these important changes are a new organization of search results into interactive lists or tables (hitlists), enhanced reference lists, and new protein domain graphics. An important new data class called 'experimental tools' consolidates information on useful fly strains and other resources related to a specific gene, which significantly enhances the ability of the Drosophila researcher to design and carry out experiments. With the release of FlyBase 2.0, there has also been a restructuring of backend architecture and a continued development of application programming interfaces (APIs) for programmatic access to FlyBase data. In this review, we describe these major new features and functionalities of the FlyBase 2.0 site and how they support the use of Drosophila as a model organism for biological discovery and translational research.
2018_Nuc Acids Res_Thurmond.pdf
Hu Y, Sopko R, Chung V, Foos M, Studer RA, Landry SD, et al. iProteinDB: An Integrative Database of Post-translational Modifications. G3 (Bethesda). 2018;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
2018_G3_Hu.pdf Supplement.zip
Okamoto N, Viswanatha R, Bittar R, Li Z, Haga-Yamanaka S, Perrimon N, et al. A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila. Dev Cell. 2018;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.
2018_Dev Cell_Okamoto.pdf 2018_Dev Cell_Okamoto_Supplement.pdf Table S1.xlsx
Branon TC, Bosch JA, Sanchez AD, Udeshi ND, Svinkina T, Carr SA, et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol. 2018;Abstract
Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 h of labeling time or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-min PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.
2018_Nat Biotech_Branon.pdf Supplemental Info.pdf Supp. Tables.zip
Viswanatha R, Li Z, Hu Y, Perrimon N. Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in cells. Elife. 2018;7. 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.
2018_eLife_Viswanatha.pdf
Ewen-Campen B, Perrimon N. ovoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila. G3 (Bethesda). 2018;Abstract
Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, In this system (" co-selection"), the only functional germ cells in injected females are those that have been edited at the locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that co-selection reduces the amount of screening necessary to isolate desired CRISPR events in
2018_G3_Ewen-Campen.pdf Supplemental Files.zip
Rodriguez-Muela N, Parkhitko A, Grass T, Gibbs RM, Norabuena EM, Perrimon N, et al. Blocking p62/SQSTM1-dependent SMN degradation ameliorates Spinal Muscular Atrophy disease phenotypes. J Clin Invest. 2018;Abstract
Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease caused by loss of functional SMN protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for SMA patients. The ubiquitin/proteasome system is known to regulate SMN protein levels; however whether autophagy controls SMN levels remains poorly explored. Here we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increase SMN levels, while induction of autophagy decreases SMN. SMN degradation occurs via its interaction with the autophagy adapter p62/SQSTM1. We also show that SMA neurons display reduced autophagosome clearance, increased p62/ubiquitinated protein levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival and extends lifespan in fly and mouse SMA models revealing p62 as a new potential therapeutic target to treat SMA.
2018_JCI_Rodriguez.pdf Supplement.pdf
Jia Y, Xu R-G, Ren X, Ewen-Campen B, Rajakumar R, Zirin J, et al. Next-generation CRISPR/Cas9 transcriptional activation in using flySAM. Proc Natl Acad Sci U S A. 2018;Abstract
CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.
2018_PNAS_Jia.pdf Supplemental Figures.pdf Supplemental Tables.zip
Hong S, Song W, Zushin P-JH, Liu B, Jedrychowski MP, Mina AI, et al. Phosphorylation of Beta-3 adrenergic receptor at serine 247 by ERK MAP kinase drives lipolysis in obese adipocytes. Mol Metab. 2018;Abstract
OBJECTIVE: The inappropriate release of free fatty acids from obese adipose tissue stores has detrimental effects on metabolism, but key molecular mechanisms controlling FFA release from adipocytes remain undefined. Although obesity promotes systemic inflammation, we find activation of the inflammation-associated Mitogen Activated Protein kinase ERK occurs specifically in adipose tissues of obese mice, and provide evidence that adipocyte ERK activation may explain exaggerated adipose tissue lipolysis observed in obesity. METHODS AND RESULTS: We provide genetic and pharmacological evidence that inhibition of the MEK/ERK pathway in human adipose tissue, mice, and flies all effectively limit adipocyte lipolysis. In complementary findings, we show that genetic and obesity-mediated activation of ERK enhances lipolysis, whereas adipose tissue specific knock-out of ERK2, the exclusive ERK1/2 protein in adipocytes, dramatically impairs lipolysis in explanted mouse adipose tissue. In addition, acute inhibition of MEK/ERK signaling also decreases lipolysis in adipose tissue and improves insulin sensitivity in obese mice. Mice with decreased rates of adipose tissue lipolysis in vivo caused by either MEK or ATGL pharmacological inhibition were unable to liberate sufficient White Adipose Tissue (WAT) energy stores to fuel thermogenesis from brown fat during a cold temperature challenge. To identify a molecular mechanism controlling these actions, we performed unbiased phosphoproteomic analysis of obese adipose tissue at different time points following acute pharmacological MEK/ERK inhibition. MEK/ERK inhibition decreased levels of adrenergic signaling and caused de-phosphorylation of the β3-adrenergic receptor (β3AR) on serine 247. To define the functional implications of this phosphorylation, we showed that CRISPR/Cas9 engineered cells expressing wild type β3AR exhibited β3AR phosphorylation by ERK2 and enhanced lipolysis, but this was not seen when serine 247 of β3AR was mutated to alanine. CONCLUSION: Taken together, these data suggest that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity.
2018_Mol Metab_Hong.pdf Supplemental Methods.pdf Supplemental Figures.pdf Supplemental Data.xlsx
Tang H-W, Hu Y, Chen C-L, Xia B, Zirin J, Yuan M, et al. The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming. Cell Metab. 2018;Abstract
Nutrient 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.
2018_Cell Metab_Tang.pdf Supplemental Figures.pdf Supplemental Tables.zip

Pages