RESUMEN
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.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas Cromosómicas no Histona/genética , Drosophila/genética , Proteínas de Drosophila/genética , Heterocromatina , Homeostasis , Humanos , Receptores Notch/genéticaRESUMEN
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.
Asunto(s)
Ciclina C/genética , Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Transcripción/metabolismo , Animales , Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Drosophila , Regulación del Desarrollo de la Expresión Génica , Haploinsuficiencia , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster 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.
Asunto(s)
Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Proteínas de Drosophila , Regulación de la Expresión Génica/genética , Factores de Transcripción , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma , Genotipo , Larva , ARN/genética , ARN/metabolismoRESUMEN
Drosophila ovary is recognized as one of the best model systems to study stem cell biology in vivo. We had previously identified an autonomous role of the histone H1 in germline stem cell (GSC) maintenance. Here, we found that histone H1 depletion in escort cells (ECs) resulted in an increase of spectrosome-containing cells (SCCs), an ovary tumor-like phenotype. Further analysis showed that the Dpp pathway is excessively activated in these SCC cells, while the expression of bam is attenuated. In the H1-depleted ECs, both transposon activity and DNA damage had increased dramatically, followed by EC apoptosis, which is consistent with the role of H1 in other somatic cells. Surprisingly, H1-depleted ECs acquired cap cell characteristics including dpp expression, and the resulting abnormal Dpp level inhibits SCC further differentiation. Most interestingly, double knockdown of H1 and dpp in ECs can reduce the number of SCCs to the normal level, indicating that the additional Dpp secreted by ECs contributes to the germline tumor. Taken together, our findings indicate that histone H1 is an important epigenetic factor in controlling EC characteristics and a key suppressor of germline tumor.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Células Germinativas/patología , Histonas/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Animales , Apoptosis , Recuento de Células , Daño del ADN , Elementos Transponibles de ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Modelos Biológicos , Fenotipo , Transducción de Señal , Transcripción Genética , Regulación hacia ArribaRESUMEN
microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.
Asunto(s)
Drosophila melanogaster/inmunología , Ecdisona/inmunología , Inmunidad Innata/inmunología , MicroARNs/inmunología , Transducción de Señal , Animales , Northern Blotting , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/inmunología , Modelos Animales de Enfermedad , Proteínas de Drosophila/inmunología , Técnicas de Inactivación de Genes , Inmunoprecipitación , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunología , Factores de Transcripción/inmunología , Proteínas Supresoras de Tumor/inmunologíaRESUMEN
The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.
Asunto(s)
Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animales , Animales Modificados Genéticamente , Ciclina C/genética , Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisteroides/biosíntesis , Femenino , Privación de Alimentos , Regulación de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutación , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Dynamic regulation of chromatin structure is required to modulate the transcription of genes in eukaryotes. However, the factors that contribute to the plasticity of heterochromatin structure are elusive. Here, we report that cyclin-dependent kinase 12 (CDK12), a transcription elongation-associated RNA polymerase II (RNAPII) kinase, antagonizes heterochromatin enrichment in Drosophila chromosomes. Notably, loss of CDK12 induces the ectopic accumulation of heterochromatin protein 1 (HP1) on euchromatic arms, with a prominent enrichment on the X chromosome. Furthermore, ChIP and sequencing analysis reveals that the heterochromatin enrichment on the X chromosome mainly occurs within long genes involved in neuronal functions. Consequently, heterochromatin enrichment reduces the transcription of neuronal genes in the adult brain and results in a defect in Drosophila courtship learning. Taken together, these results define a previously unidentified role of CDK12 in controlling the epigenetic transition between euchromatin and heterochromatin and suggest a chromatin regulatory mechanism in neuronal behaviors.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Drosophila/genética , Epigénesis Genética/fisiología , Heterocromatina/fisiología , Aprendizaje/fisiología , Animales , Secuencia de Bases , Western Blotting , Ensamble y Desensamble de Cromatina/genética , Inmunoprecipitación de Cromatina , Drosophila/fisiología , Heterocromatina/genética , Inmunoprecipitación , Datos de Secuencia Molecular , Octoxinol , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/anatomía & histología , Glándulas Salivales/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.
Asunto(s)
Sistemas CRISPR-Cas/genética , Drosophila melanogaster/genética , Ingeniería Genética/métodos , Genómica/métodos , Células Germinativas , Animales , Animales Modificados Genéticamente , Bases de Datos Genéticas , Proteínas de Drosophila/genética , Mutagénesis/genética , Regiones Promotoras Genéticas/genética , Proteínas de Unión al ARN/genéticaRESUMEN
microRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.
Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , MicroARNs/genética , MicroARNs/metabolismo , Animales , Bases de Datos Genéticas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Perfilación de la Expresión Génica , Genoma , Masculino , Modelos Biológicos , Fenotipo , Receptores Notch/metabolismo , Transducción de Señal , Transgenes , Alas de Animales/fisiologíaRESUMEN
Core histone modifications play an important role in chromatin remodeling and transcriptional regulation. Histone acetylation is one of the best-studied gene modifications and has been shown to be involved in numerous important biological processes. Herein, we demonstrated that the depletion of histone deacetylase 3 (Hdac3) in Drosophila melanogaster resulted in a reduction in body size. Further genetic studies showed that Hdac3 counteracted the organ overgrowth induced by overexpression of insulin receptor (InR), phosphoinositide 3-kinase (PI3K) or S6 kinase (S6K), and the growth regulation by Hdac3 was mediated through the deacetylation of histone H4 at lysine 16 (H4K16). Consistently, the alterations of H4K16 acetylation (H4K16ac) induced by the overexpression or depletion of males-absent-on-the-first (MOF), a histone acetyltransferase that specifically targets H4K16, resulted in changes in body size. Furthermore, we found that H4K16ac was modulated by PI3K signaling cascades. The activation of the PI3K pathway caused a reduction in H4K16ac, whereas the inactivation of the PI3K pathway resulted in an increase in H4K16ac. The increase in H4K16ac by the depletion of Hdac3 counteracted the PI3K-induced tissue overgrowth and PI3K-mediated alterations in the transcription profile. Overall, our studies indicated that Hdac3 served as an important regulator of the PI3K pathway and revealed a novel link between histone acetylation and growth control.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Especificidad de Órganos , Fosfatidilinositol 3-Quinasas/metabolismo , Acetilación , Animales , Tamaño Corporal , Tamaño de la Célula , Proteínas de Drosophila/deficiencia , Drosophila melanogaster/citología , Drosophila melanogaster/ultraestructura , Femenino , Histona Acetiltransferasas/metabolismo , Histona Desacetilasas/deficiencia , Insulina/metabolismo , Masculino , Proteínas Nucleares/metabolismo , Receptor de Insulina/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Drosophila melanogaster/genética , Genoma de los Insectos , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Técnicas Genéticas , Vectores Genéticos , MicroARNs/genética , Oogénesis/genética , ARN Interferente Pequeño/genéticaRESUMEN
Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1) is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, we showed that conditional depletion of HP1 in transgenic flies resulted in preferential lethality in male flies. Cytological analysis of mitotic chromosomes showed that HP1 depletion caused sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, were preferentially increased in males as well. Expression analysis showed that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes showed greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Cromatina/química , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Genes de Insecto , Genes Letales , Humanos , Masculino , Mitosis/genética , Datos de Secuencia Molecular , Factores Sexuales , Telómero/metabolismo , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Drosophila , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Drosophila/genética , Drosophila/metabolismo , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Marcación de Gen/métodos , Vectores Genéticos/genética , Interferencia de ARN , AnimalesRESUMEN
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.
Asunto(s)
Codón de Terminación , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Biosíntesis de Proteínas , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero/citología , Femenino , Perfilación de la Expresión Génica , Masculino , Especificidad de ÓrganosRESUMEN
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.
Asunto(s)
Adenosina Desaminasa/genética , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Neuronas/fisiología , Edición de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Adenosina Desaminasa/metabolismo , Animales , Animales Modificados Genéticamente , Encéfalo/citología , Encéfalo/metabolismo , Encéfalo/fisiología , Proteínas Portadoras/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
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.
Asunto(s)
Drosophila melanogaster , Drosophila , Animales , Composición de Base , Sistemas CRISPR-Cas , Drosophila/genética , Drosophila melanogaster/genética , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/genética , Sitio de Iniciación de la TranscripciónRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Katanina/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Poliploidía , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citocinesis , Proteínas de Drosophila/genética , Femenino , Katanina/genética , Masculino , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Huso AcromáticoRESUMEN
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.