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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
We generated a library of ~1,000stocks in which we inserted a construct in the intron of genes allowing expression ofunder 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 singlecDNA construct. Third, loss-of-function phenotypes associated with manyinsertions can be reverted by excision with. Fourth,drivenreports 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 withor any DNA. These stocks comprise a powerful resource for assessing gene function.
In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in bothandApplication of a JH mimic on ex vivo cultured PGs fromandlarvae inducesexpression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown ofpromotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication inPG cells and that the reduction of PG size mediated byoverexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone.
-methyladenosine (mA), the most abundant chemical modification in eukaryotic mRNA, has been implicated insex determination by modifying() pre-mRNA and facilitating its alternative splicing. Here, we identify a sex determination gene,, and rename itaccording to its loss-of-function female-to-male transformation phenotype.encodes a conserved ubiquitous nuclear protein of unknown function. We show that Xio colocalizes and interacts with all previously known mA writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss ofis associated with phenotypes that resemble other mA factors, such as sexual transformations,splicing defect, held-out wings, flightless flies, and reduction of mA levels. Thus, Xio encodes a member of the mA methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several mA writer factors, the function of Xio in the mA pathway is likely evolutionarily conserved.
Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Calevels, and increases in cytosolic Caresemble the Piezo overexpression phenotype, suggesting that Piezo functions through Casignalling. Further studies suggest that Casignalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Cain response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.
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 '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 'omics datasets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.
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 '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.
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.
The mechanistic target of rapamycin complex 1 (mTORC1) supports proliferation through parallel induction of key anabolic processes, including protein, lipid, and nucleotide synthesis. We hypothesized that these processes are coupled to maintain anabolic balance in cells with mTORC1 activation, a common event in human cancers. Loss of the tuberous sclerosis complex (TSC) tumor suppressors results in activation of mTORC1 and development of the tumor syndrome TSC. We find that pharmacological inhibitors of guanylate nucleotide synthesis have selective deleterious effects on TSC-deficient cells, including in mouse tumor models. This effect stems from replication stress and DNA damage caused by mTORC1-driven rRNA synthesis, which renders nucleotide pools limiting. These findings reveal a metabolic vulnerability downstream of mTORC1 triggered by anabolic imbalance.
Adipocytes sense systemic nutrient status and systemically communicate this information by releasing adipokines. The mechanisms that couple nutritional state to adipokine release are unknown. Here, we investigated how Unpaired 2 (Upd2), a structural and functional ortholog of the primary human adipokine leptin, is released from Drosophila fat cells. We find that Golgi reassembly stacking protein (GRASP), an unconventional secretion pathway component, is required for Upd2 secretion. In nutrient-rich fat cells, GRASP clusters in close proximity to the apical side of lipid droplets (LDs). During nutrient deprivation, glucagon-mediated increase in calcium (Ca(2+)) levels, via calmodulin kinase II (CaMKII) phosphorylation, inhibits proximal GRASP localization to LDs. Using a heterologous cell system, we show that human leptin secretion is also regulated by Ca(2+) and CaMKII. In summary, we describe a mechanism by which increased cytosolic Ca(2+) negatively regulates adipokine secretion and have uncovered an evolutionarily conserved molecular link between intracellular Ca(2+) levels and energy homeostasis.