RESUMEN
Chromatin architecture facilitates accurate transcription at a number of loci, but it remains unclear how much chromatin architecture is involved in global transcriptional regulation. Previous work has shown that rapid depletion of the architectural protein CTCF in cell culture strongly alters chromatin organization but results in surprisingly limited gene expression changes. This discrepancy has also been observed when other architectural proteins are depleted, and one possible explanation is that full transcriptional changes are masked by cellular heterogeneity. We tested this idea by performing multi-omics analyses with sorted post-mitotic mouse rods, which undergo synchronized development, and identified CTCF-dependent regulation of global chromatin accessibility and gene expression. Depletion of CTCF leads to dysregulation of â¼20% of the entire transcriptome (>3,000 genes) and â¼41% of genome accessibility (>26,000 sites), and these regions are strongly enriched in euchromatin. Importantly, these changes are highly enriched for CTCF occupancy, suggesting direct CTCF binding and transcriptional regulation at these active loci. CTCF mainly promotes chromatin accessibility of these direct binding targets, and a large fraction of these sites correspond to promoters. At these sites, CTCF binding frequently promotes accessibility and inhibits expression, and motifs of transcription repressors are found to be significantly enriched. Our findings provide different and often opposite conclusions from previous studies, emphasizing the need to consider cell heterogeneity and cell type specificity when performing multi-omics analyses. We conclude that the architectural protein CTCF binds chromatin and regulates global chromatin accessibility and transcription during rod development.
RESUMEN
Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the center of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments co-orienting these separated kinetochores. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.
Asunto(s)
Bombyx , Segregación Cromosómica , Meiosis , Mariposas Nocturnas , Espermatogénesis , Animales , Segregación Cromosómica/genética , Mariposas Nocturnas/genética , Mariposas Nocturnas/fisiología , Masculino , Espermatogénesis/genética , Meiosis/genética , Bombyx/genética , Bombyx/fisiología , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Microtúbulos/genética , Emparejamiento Cromosómico/genética , Cromosomas de Insectos/genética , Hibridación Fluorescente in Situ , Metafase , Telómero/genética , Telómero/metabolismo , CinéticaRESUMEN
Chromatin insulators are DNA-protein complexes that promote specificity of enhancer-promoter interactions and maintain distinct transcriptional states through control of 3D genome organization. In this review, we highlight recent work visualizing how mammalian CCCTC-binding factor acts as a boundary to dynamic DNA loop extrusion mediated by cohesin. We also discuss new studies in both mammals and Drosophila that elucidate biological redundancy of chromatin insulator function and interplay with transcription with respect to topologically associating domain formation. Finally, we present novel concepts in spatiotemporal regulation of chromatin insulator function during differentiation and development and possible consequences of disrupted insulator activity on cellular proliferation.
Asunto(s)
Cromatina , Genoma , Elementos Aisladores , Animales , Cromatina/genética , Cromatina/metabolismo , Elementos Aisladores/genética , Genoma/genética , Humanos , Cohesinas , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas , Drosophila/genética , Drosophila/metabolismo , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genéticaRESUMEN
Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , ADN/metabolismo , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Elementos Aisladores/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismoRESUMEN
MOTIVATION: ChIP-seq detects protein-DNA interactions within chromatin, such as that of chromatin structural components and transcription machinery. ChIP-seq profiles are often noisy and variable across replicates, posing a challenge to the development of effective algorithms to accurately detect differential peaks. Methods have recently been designed for this purpose but sometimes yield conflicting results that are inconsistent with the underlying biology. Most existing algorithms perform well on limited datasets. To improve differential analysis of ChIP-seq, we present a novel Differential analysis method for ChIP-seq based on Limma (DiffChIPL). RESULTS: DiffChIPL is adaptive to asymmetrical or symmetrical data and can accurately report global differences. We used simulated and real datasets for transcription factors (TFs) and histone modification marks to validate and benchmark our algorithm. DiffChIPL shows superior performance in sensitivity and false positive rate in different simulations and control datasets. DiffChIPL also performs well on real ChIP-seq, CUT&RUN, CUT&Tag and ATAC-seq datasets. DiffChIPL is an accurate and robust method, exhibiting better performance in differential analysis for a variety of applications including TF binding, histone modifications and chromatin accessibility. AVAILABILITY AND IMPLEMENTATION: https://github.com/yancychy/DiffChIPL. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación de Cromatina/métodos , Análisis de Secuencia de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Cromatina , AlgoritmosRESUMEN
Chromatin insulators are DNA-protein complexes localized throughout the genome capable of establishing independent transcriptional domains. It was previously reported that the Drosophila su(Hw) mRNA physically associates with the gypsy chromatin insulator protein complex within the nucleus and may serve a noncoding function to affect insulator activity. However, how this mRNA is recruited to the gypsy complex is not known. Here, we utilized RNA-affinity pulldown coupled with mass spectrometry to identify a novel RNA-binding protein, Isha (CG4266), that associates with su(Hw) mRNA in vitro and in vivo. Isha harbors a conserved RNA recognition motif and RNA Polymerase II C-terminal domain-interacting domain (CID). We found that Isha physically interacts with total and elongating Polymerase II and associates with chromatin at the 5' end of genes in an RNA-dependent manner. Furthermore, ChIP-seq analysis reveals Isha overlaps particularly with the core gypsy insulator component CP190 on chromatin. Depletion of Isha reduces enhancer-blocking and barrier activities of the gypsy insulator and disrupts the nuclear localization of insulator bodies. Our results reveal a novel factor Isha that promotes gypsy insulator activity that may act as a nuclear RNA-binding protein adapter for su(Hw) noncoding mRNA.
Asunto(s)
Proteínas de Drosophila , Animales , Proteínas Portadoras/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
SignificanceGenes on sex chromosomes (i.e. human chX) are regulated differently in males and females to balance gene expression levels between sexes (XY vs. XX). This sex-specific regulation is called dosage compensation (DC). DC is achieved by altering the shape and compaction of sex chromosomes specifically in one sex. In this study, we use Oligopaints to examine DC in silkworms. This study visualizes this phenomenon in a species with ZW sex chromosomes, which evolved independently of XY. Our data support a long-standing model for how DC mechanisms evolved across species, and we show potential similarity between DC in silkworms and nematodes, suggesting that this type of DC may have emerged multiple independent times throughout evolution.
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Bombyx/genética , Cromosomas de Insectos/genética , Compensación de Dosificación (Genética) , Cromosomas Sexuales/genética , AnimalesRESUMEN
Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To address this question, we quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. We quantified TLs in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when small RNA and RNA interference (RNAi) pathways are compromised. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, we show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. Our study suggests that in addition to a possible influence by different genetic backgrounds, small RNA and RNAi mechanisms may mitigate genomic TL expansion despite the increase in TE transcripts during aging.
Asunto(s)
Elementos Transponibles de ADN , Drosophila , Envejecimiento/genética , Animales , Elementos Transponibles de ADN/genética , Drosophila/genética , Genómica/métodos , ARNRESUMEN
During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation.
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Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Plasticidad Neuronal/fisiología , Animales , Cromatina/química , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Animales Modificados Genéticamente , Línea Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Proteínas de Drosophila/genética , Técnicas de Silenciamiento del Gen , Genoma de los Insectos , Elementos Aisladores/genética , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , RNA-Seq , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
Accurate chromosome segregation during meiosis is essential for reproductive success. Yet, many fundamental aspects of meiosis remain unclear, including the mechanisms regulating homolog pairing across species. This gap is partially due to our inability to visualize individual chromosomes during meiosis. Here, we employ Oligopaint FISH to investigate homolog pairing and compaction of meiotic chromosomes and resurrect a classical model system, the silkworm Bombyx mori. Our Oligopaint design combines multiplexed barcoding with secondary oligo labeling for high flexibility and low cost. These studies illustrate that Oligopaints are highly specific in whole-mount gonads and on meiotic squashes. We show that meiotic pairing is robust in both males and females and that pairing can occur through numerous partially paired intermediate structures. We also show that pairing in male meiosis occurs asynchronously and seemingly in a transcription-biased manner. Further, we reveal that meiotic bivalent formation in B. mori males is highly similar to bivalent formation in C. elegans, with both of these pathways ultimately resulting in the pairing of chromosome ends with non-paired ends facing the spindle pole. Additionally, microtubule recruitment in both C. elegans and B. mori is likely dependent on kinetochore proteins but independent of the centromere-specifying histone CENP-A. Finally, using super-resolution microscopy in the female germline, we show that homologous chromosomes remain associated at telomere domains in the absence of chiasma and after breakdown and modification to the synaptonemal complex in pachytene. These studies reveal novel insights into mechanisms of meiotic homolog pairing both with or without recombination.
Asunto(s)
Bombyx/genética , Emparejamiento Cromosómico/genética , Telómero/genética , Animales , Proteínas de Ciclo Celular/genética , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Cromosomas/genética , ADN/genética , Femenino , Masculino , Meiosis/genética , Microtúbulos/metabolismo , Complejo Sinaptonémico/metabolismoRESUMEN
Chromatin insulators are DNA-protein complexes that play a crucial role in regulating chromatin organization. Within the past two years, a plethora of genome-wide conformation capture studies have helped reveal that insulators are necessary for proper genome-wide organization of topologically associating domains, which are formed in a manner distinct from that of compartments. These studies have also provided novel insights into the mechanics of how CTCF/cohesin-dependent loops form in mammals, strongly supporting the loop extrusion model. In combination with single-cell imaging approaches in both Drosophila and mammals, the dynamics of insulator-mediated chromatin interactions are also coming to light. Insulator-dependent structures vary across individual cells and tissues, highlighting the need to study the regulation of insulators in particular temporal and spatial contexts throughout development.
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Cromatina/química , Cromatina/genética , Elementos Aisladores , Animales , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Drosophila/genética , Regulación de la Expresión Génica , Humanos , Sistema Nervioso/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , CohesinasRESUMEN
Chromatin insulators are DNA-protein complexes that establish independent higher-order DNA domains to influence transcription. Insulators are functionally defined by two properties: they can block communication between an enhancer and a promoter, and also act as a barrier between heterochromatin and euchromatin. In Drosophila, the gypsy insulator complex contains three core components; Su(Hw), CP190 and Mod(mdg4)67.2. Here, we identify a novel role for Chromatin-linked adaptor for MSL proteins (CLAMP) in promoting gypsy chromatin insulator function. When clamp is knocked down, gypsy-dependent enhancer-blocking and barrier activities are strongly reduced. CLAMP associates physically with the core gypsy insulator complex, and ChIP-seq analysis reveals extensive overlap, particularly with promoter-bound CP190 on chromatin. Depletion of CLAMP disrupts CP190 binding at a minority of shared sites, whereas depletion of CP190 results in extensive loss of CLAMP chromatin association. Finally, reduction of CLAMP disrupts CP190 localization within the nucleus. Our results support a positive functional relationship between CLAMP and CP190 to promote gypsy chromatin insulator activity.
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Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Cromatina/genética , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Técnicas de Silenciamiento del Gen , Complejos Multiproteicos , Unión Proteica , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Dedos de Zinc/genéticaRESUMEN
Chromatin insulators are DNA-protein complexes that regulate chromatin structure and gene expression in a wide range of organisms. These complexes also harbor enhancer blocking and barrier activities. Increasing evidence suggests that RNA molecules are integral components of insulator complexes. However, how these RNA molecules are involved in insulator function remains unclear. The Drosophila RNA-binding protein Shep associates with the gypsy insulator complex and inhibits insulator activities. By mutating key residues in the RRM domains, we generated a Shep mutant protein incapable of RNA-binding, and this mutant lost the ability to inhibit barrier activity. In addition, we found that one of many wildtype Shep isoforms but not RRM mutant Shep was sufficient to repress enhancer blocking activities. Finally, wildtype Shep rescued synthetic lethality of shep, mod(mdg4) double-mutants and developmental defects of shep mutant neurons, whereas mutant Shep failed to do so. These results indicate that the RNA-binding ability of Shep is essential for its ability to antagonize insulator activities and promote neuronal maturation. Our findings suggest that regulation of insulator function by RNA-binding proteins relies on RNA-mediated interactions.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Drosophila melanogaster/genética , Femenino , ARN/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Topoisomerases solve topological problems during DNA metabolism, but whether they participate in RNA metabolism remains unclear. Top3ß represents a family of topoisomerases carrying activities for both DNA and RNA. Here we show that in Drosophila, Top3ß interacts biochemically and genetically with the RNAi-induced silencing complex (RISC) containing AGO2, p68 RNA helicase, and FMRP. Top3ß and RISC mutants are similarly defective in heterochromatin formation and transcriptional silencing by position-effect variegation assay. Moreover, both Top3ß and AGO2 mutants exhibit reduced levels of heterochromatin protein HP1 in heterochromatin. Furthermore, expression of several genes and transposable elements in heterochromatin is increased in the Top3ß mutant. Notably, Top3ß mutants defective in either RNA binding or catalytic activity are deficient in promoting HP1 recruitment and silencing of transposable elements. Our data suggest that Top3ß may act as an RNA topoisomerase in siRNA-guided heterochromatin formation and transcriptional silencing.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Drosophila melanogaster/enzimología , Heterocromatina/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Heterocromatina/genética , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño , Complejo Silenciador Inducido por ARN/genéticaRESUMEN
Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Remarkably, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (NELF) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
Asunto(s)
Proteínas Argonautas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , ARN Polimerasa II/genética , Elongación de la Transcripción Genética , Animales , Proteínas Argonautas/metabolismo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Quinasa 9 Dependiente de la Ciclina/genética , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Técnicas de Silenciamiento del Gen , Hemocitos/citología , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression.
Asunto(s)
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/fisiología , Lamina Tipo B/metabolismo , Laminas/metabolismo , Animales , Proteínas Argonautas/genética , Línea Celular , Inmunoprecipitación de Cromatina , Cromatografía de Afinidad/métodos , Proteínas de Drosophila/genética , Drosophila melanogaster , Hibridación Fluorescente in Situ , Lamina Tipo B/genética , Laminas/genética , Espectrometría de MasasRESUMEN
Neuronal remodeling is crucial for formation of the mature nervous system and disruption of this process can lead to neuropsychiatric diseases. Global gene expression changes in neurons during remodeling as well as the factors that regulate these changes remain poorly defined. To elucidate this process, we performed RNA-seq on isolated Drosophila larval and pupal neurons and found upregulated synaptic signaling and downregulated gene expression regulators as a result of normal neuronal metamorphosis. We further tested the role of alan shepard (shep), which encodes an evolutionarily conserved RNA-binding protein required for proper neuronal remodeling. Depletion of shep in neurons prevents the execution of metamorphic gene expression patterns, and shep-regulated genes correspond to Shep chromatin and/or RNA-binding targets. Reduced expression of a Shep-inhibited target gene that we identified, brat, is sufficient to rescue neuronal remodeling defects of shep knockdown flies. Our results reveal direct regulation of transcriptional programs by Shep to regulate neuronal remodeling during metamorphosis.