CRISPR-Based Transcriptional Activation in Drosophila.

Overexpression is one of the classical approaches to study pleiotropic functions of genes of interest. To achieve overexpression, we often increase the transcription by introducing genes on exogenous vectors or by using the CRISPR/dCas9-based transcriptional activation system. To date, the most efficient CRISPR/dCas9-based transcriptional activator is the Synergistic Activation Mediator (SAM) system whereby three different transcriptional activation domains are directly fused to dCas9 and MS2 phage Coat Protein (MCP), respectively, and the system in Drosophila is named flySAM. Here we describe the effective and convenient transcriptional activation system, flySAM, starting from vector construction, microinjection, and transgenic fly selection to the phenotypic analysis.

HP1c regulates development and gut homeostasis by suppressing Notch signaling through Su(H).

Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.

SPATA4 improves aging-induced metabolic dysfunction through promotion of preadipocyte differentiation and adipose tissue expansion.

Spermatogenesis-associated protein 4 (SPATA4) is conserved across multiple species. However, the function of this gene remains largely unknown. In this study, we generated Spata4 transgenic mice to explore tissue-specific function of SPATA4. Spata4 overexpression mice displayed increased subcutaneous fat tissue compared with wild-type littermates at an old age, while this difference was not observed in younger mice. Aging-induced ectopic fat distribution, inflammation, and insulin resistance were also significantly attenuated by SPATA4. In vitro, SPATA4 promoted preadipocyte differentiation through activation of the ERK1/2 and C/EBPß pathway and increased the expression of adipokines. These data suggest SPATA4 can regulate lipid accumulation in a tissue-specific manner and improve aging-induced dysmetabolic syndromes. Clarifying the mechanism of SPATA4 functioning in lipid metabolism might provide novel therapeutic targets for disease interventions.

Katanin p60-like 1 sculpts the cytoskeleton in mechanosensory cilia.

Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.

Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila.

The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.

The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription.

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.

Zinc Finger RNA-Binding Protein Zn72D Regulates ADAR-Mediated RNA Editing in Neurons.

Adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, alters RNA sequences from those encoded by DNA. These editing events are dynamically regulated, but few trans regulators of ADARs are known in vivo. Here, we screen RNA-binding proteins for roles in editing regulation with knockdown experiments in the Drosophila brain. We identify zinc-finger protein at 72D (Zn72D) as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and fly mobility. Furthermore, Zn72D's regulatory role in RNA editing is conserved because the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary neurons. The broad and conserved regulation of ADAR editing by Zn72D in neurons sustains critically important editing events.

Premature termination codon readthrough in Drosophila varies in a developmental and tissue-specific manner.

Despite their essential function in terminating translation, readthrough of stop codons occurs more frequently than previously supposed. However, little is known about the regulation of stop codon readthrough by anatomical site and over the life cycle of animals. Here, we developed a set of reporters to measure readthrough in Drosophila melanogaster. A focused RNAi screen in whole animals identified upf1 as a mediator of readthrough, suggesting that the stop codons in the reporters were recognized as premature termination codons (PTCs). We found readthrough rates of PTCs varied significantly throughout the life cycle of flies, being highest in older adult flies. Furthermore, readthrough rates varied dramatically by tissue and, intriguingly, were highest in fly brains, specifically neurons and not glia. This was not due to differences in reporter abundance or nonsense-mediated mRNA decay (NMD) surveillance between these tissues. Readthrough rates also varied within neurons, with cholinergic neurons having highest readthrough compared with lowest readthrough rates in dopaminergic neurons. Overall, our data reveal temporal and spatial variation of PTC-mediated readthrough in animals, and suggest that readthrough may be a potential rescue mechanism for PTC-harboring transcripts when the NMD surveillance pathway is inhibited.

Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies.

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.

A high-fat diet reverses metabolic disorders and premature aging by modulating insulin and IGF1 signaling in SIRT6 knockout mice.

Mammalian sirtuin 6 (SIRT6) is involved in the regulation of many essential processes, especially metabolic homeostasis. SIRT6 knockout mice undergo premature aging and die at age ~4 weeks. Severe glycometabolic disorders have been found in SIRT6 knockout mice, and whether a dietary intervention can rescue SIRT6 knockout mice remains unknown. In our study, we found that at the same calorie intake, a high-fat diet dramatically increased the lifespan of SIRT6 knockout mice to 26 weeks (males) and 37 weeks (females), reversed multi-organ atrophy, and reduced body weight, hypoglycemia, and premature aging. Furthermore, the high-fat diet partially but significantly normalized the global gene expression profile in SIRT6 knockout mice. Regarding the mechanism, excessive glucose uptake and glycolysis induced by the SIRT6 deficiency were attenuated in skeletal muscle through inhibition of insulin and IGF1 signaling by the high-fat diet. Similarly, fatty acids but not ketone bodies inhibited glucose uptake, glycolysis, and senescence in SIRT6 knockout fibroblasts, whereas PI3K inhibition antagonized the effects of a high-fatty-acid medium in vitro. Overall, the high-fat diet dramatically reverses numerous consequences of SIRT6 deficiency through modulation of insulin and IGF1 signaling, providing a new basis for elucidation of SIRT6 and fatty-acid functions and supporting novel therapeutic approaches against metabolic disorders and aging-related diseases.

Perspectives on gene expression regulation techniques in Drosophila.

Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.

The exocyst functions in niche cells to promote germline stem cell differentiation by directly controlling EGFR membrane trafficking.

The niche controls stem cell self-renewal and differentiation in animal tissues. Although the exocyst is known to be important for protein membrane trafficking and secretion, its role in stem cells and niches has never been reported. Here, this study shows that the exocyst functions in the niche to promote germline stem cell (GSC) progeny differentiation in the Drosophila ovary by directly regulating EGFR membrane trafficking and signaling. Inactivation of exocyst components in inner germarial sheath cells, which form the differentiation niche, causes a severe GSC differentiation defect. The exocyst is required for maintaining niche cells and preventing BMP signaling in GSC progeny by promoting EGFR membrane targeting and signaling through direct association with EGFR. Finally, it is also required for EGFR membrane targeting, recycling and signaling in human cells. Therefore, this study reveals a novel function of the exocyst in niche cells to promote stem cell progeny differentiation by directly controlling EGFR membrane trafficking and signaling in vivo, and also provides important insight into how the niche controls stem cell progeny differentiation at the molecular level.

Spectraplakin Shot Maintains Perinuclear Microtubule Organization in Drosophila Polyploid Cells.

Polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis as part of their differentiation programs. Upon cell-cycle exit and differentiation, non-centrosomal sites govern microtubule distribution in most cells. Little is known on how polyploid cells, differentiated but cycling, organize their microtubules. We show that microtubules in Drosophila adipocytes and other polyploid tissues form a dense perinuclear cortex responsible for nuclear size and position. Confirming a relation between this perinuclear cortex and the polyploid endocycle, polyploidization of normally diploid cells was sufficient for cortex formation. A critical component of the perinuclear microtubule organizer (pnMTOC) is Shot, absence of which caused collapse of the perinuclear network into a condensed organizer through kinesin-dependent microtubule sliding. Furthermore, this ectopic organizer was capable of directing partial assembly of a deeply disruptive cytokinesis furrow. In all, our study revealed the importance of perinuclear microtubule organization for stability of endocycling Drosophila cells.

Defining gene networks controlling the maintenance and function of the differentiation niche by an in vivo systematic RNAi screen.

In the Drosophila ovary, escort cells (ECs) extrinsically control germline stem cell (GSC) maintenance and progeny differentiation. However, the underlying mechanisms remain poorly understood. In this study, we identified 173 EC genes for their roles in controlling GSC maintenance and progeny differentiation by using an in vivo systematic RNAi approach. Of the identified genes, 10 and 163 are required in ECs to promote GSC maintenance and progeny differentiation, respectively. The genes required for progeny differentiation fall into different functional categories, including transcription, mRNA splicing, protein degradation, signal transduction and cytoskeleton regulation. In addition, the GSC progeny differentiation defects caused by defective ECs are often associated with BMP signaling elevation, indicating that preventing BMP signaling is a general functional feature of the differentiation niche. Lastly, exon junction complex (EJC) components, which are essential for mRNA splicing, are required in ECs to promote GSC progeny differentiation by maintaining ECs and preventing BMP signaling. Therefore, this study has identified the major regulators of the differentiation niche, which provides important insights into how stem cell progeny differentiation is extrinsically controlled.

CRISPR-Cas9 Mediated Genome Editing in Drosophila.

In recent years, great progress has been made in the research of genome editing systems, one of which is the CRISPR-Cas9 system, a powerful technology that is applied to edit animal genome. Here, we describe a CRISPR-Cas9 mediated mutation protocol for efficiently and specifically editing genes in Drosophila. In this optimized system, the mutant progeny can be generated by only injecting a DNA plasmid encoding synthetic guide RNA (sgRNA) under the control of the U6b promoter into transgenic fly embryos in which Cas9 is specifically expressed in the progenitor cells, thus the gene of interest can be edited by the CRISPR in germ cells, with high rate of heritable mutations and few side effects.

flySAM Transgenic CRISPRa System Manual.

Powerful and general methods that can enhance gene expression are useful to systematically study gene function. To date, compared with the methods in generating loss-of-function mutants, methods to achieve gain-of-function are limited. The entire field in Drosophila has relied heavily on the Gal4/UAS:cDNA overexpression system developed over two decades ago. It is laborious and expensive to clone the coding DNA sequence (CDS) of a gene, especially those of large size. In addition, side effects of this method are often observed because of the ectopic expression. Also, simultaneous activation of two genes with the traditional method is often time-consuming, and few are achievable for three or more genes. In this protocol, we describe how to build an effective and convenient targeting activator system, flySAM, to activate endogenous genes in Drosophila melanogaster based on the structure-guided engineering of CRISPR-Cas9 complex.

pNP Transgenic RNAi System Manual in Drosophila.

Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority choice to study gene functions due to its simple and practical characteristics in Drosophila. We established a novel system that works well in both soma and germ cells which is efficient and specific. With this system, we can precisely and efficiently modulate highly expressed genes, and simultaneously knock down multiple genes in one step. In this study, we provide a detailed protocol of the pNP system, which replaces other transgenic systems, and expect it can provide some help to researchers who are using this system.

Erratum: Correction Notice: CRISPR-Cas9 Mediated Genome Editing in Drosophila.

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An efficient and multiple target transgenic RNAi technique with low toxicity in Drosophila.

Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, we develop a next generation transgenic RNAi system. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, we increase versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, we have generated a collection of transgenic RNAi lines for those genes that are highly homologous to human disease genes.

The Lysine Demethylase dKDM2 Is Non-essential for Viability, but Regulates Circadian Rhythms in Drosophila.

Post-translational modification of histones, such as histone methylation controlled by specific methyltransferases and demethylases, play critical roles in modulating chromatin dynamics and transcription in eukaryotes. Misregulation of histone methylation can lead to aberrant gene expression, thereby contributing to abnormal development and diseases such as cancer. As such, the mammalian lysine-specific demethylase 2 (KDM2) homologs, KDM2A and KDM2B, are either oncogenic or tumor suppressive depending on specific pathological contexts. However, the role of KDM2 proteins during development remains poorly understood. Unlike vertebrates, Drosophila has only one KDM2 homolog (dKDM2), but its functions in vivo remain elusive due to the complexities of the existing mutant alleles. To address this problem, we have generated two dKdm2 null alleles using the CRISPR/Cas9 technique. These dKdm2 homozygous mutants are fully viable and fertile, with no developmental defects observed under laboratory conditions. However, the dKdm2 null mutant adults display defects in circadian rhythms. Most of the dKdm2 mutants become arrhythmic under constant darkness, while the circadian period of the rhythmic mutant flies is approximately 1 h shorter than the control. Interestingly, lengthened circadian periods are observed when dKDM2 is overexpressed in circadian pacemaker neurons. Taken together, these results demonstrate that dKdm2 is not essential for viability; instead, dKDM2 protein plays important roles in regulating circadian rhythms in Drosophila. Further analyses of the molecular mechanisms of dKDM2 and its orthologs in vertebrates regarding the regulation of circadian rhythms will advance our understanding of the epigenetic regulations of circadian clocks.