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Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila

ABSTRACT: Chromatin insulators are functionally conserved DNA-protein complexes that are situated throughout the genome and organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the mechanisms by which RNA affects insulator activity are not yet understood. Here we identify the exosome, the highly conserved major cellular 3’ to 5’ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. High resolution genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32, and CTCF insulator proteins. Colocalization occurs mainly at promoters but also well-characterized boundary elements, such as scs, scs’, Mcp, and Fab-8. Surprisingly, exosome associates primarily with promoters but not gene bodies, arguing against simple cotranscriptional recruitment to RNA substrates. We find that exosome is recruited to chromatin in a transcription dependent manner, preferentially to highly transcribed genes. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is specifically required for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel relationship between exosome and chromatin insulators throughout the genome.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE41950 | GEO | 2013/01/29

SECONDARY ACCESSION(S): PRJNA178575

REPOSITORIES: GEO

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Genome-wide localization of exosome components to active promoters and chromatin insulators in Drosophila

Project description:Chromatin insulators are functionally conserved DNA-protein complexes that are situated throughout the genome and organize independent transcriptional domains. Previous work implicated RNA as an important cofactor in chromatin insulator activity, although the mechanisms by which RNA affects insulator activity are not yet understood. Here we identify the exosome, the highly conserved major cellular 3’ to 5’ RNA degradation machinery, as a physical interactor of CP190-dependent chromatin insulator complexes in Drosophila. High resolution genome-wide profiling of exosome by ChIP-seq in two different embryonic cell lines reveals extensive and specific overlap with the CP190, BEAF-32, and CTCF insulator proteins. Colocalization occurs mainly at promoters but also well-characterized boundary elements, such as scs, scs’, Mcp, and Fab-8. Surprisingly, exosome associates primarily with promoters but not gene bodies, arguing against simple cotranscriptional recruitment to RNA substrates. We find that exosome is recruited to chromatin in a transcription dependent manner, preferentially to highly transcribed genes. Similar to insulator proteins, exosome is also significantly enriched at divergently transcribed promoters. Directed ChIP of exosome in cell lines depleted of insulator proteins shows that CTCF is specifically required for exosome association at Mcp and Fab-8 but not other sites, suggesting that alternate mechanisms must also contribute to exosome chromatin recruitment. Taken together, our results reveal a novel relationship between exosome and chromatin insulators throughout the genome. ChIP-seq of exosome components. RNA-seq after control and exosome subunit knockdown in Drosophila cell lines.

2013-01-29 | E-GEOD-41950 | biostudies-arrayexpress

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

Project description:Chromatin insulators and Polycomb group (PcG) complexes control nuclear organization to effect changes in gene expression. In Drosophila, RNA silencing pathways influence long range interactions mediated by PcG proteins and nuclear localization of the gypsy insulator; however, the underlying mechanisms are unknown. Here, we identify a singular requirement for Argonaute2 (AGO2) for the activity of the CCCTC-binding factor (CTCF)/Centrosomal protein 190 (CP190) dependent Fab-8 insulator. AGO2 and CP190 interact physically, and genome wide localization of AGO2 by chromatin immunoprecipitation and sequencing (ChIP-seq) reveals extensive colocalization of AGO2 with insulators and Polycomb Response Elements (PREs) but minimal overlap with regions of endogenous small interfering RNA (endo-siRNA) production. Finally, depletion of either CTCF or CP190 results in loss of AGO2 association with insulators, PREs, and other cis-regulatory regions. Our findings suggest that Dicer-independent recruitment of AGO2 to chromatin by insulator proteins promotes the definition of transcriptional domains throughout the genome. ChIP-seq of AGO2 in two Drosophila cell types (S2 and S3)

2011-08-15 | E-GEOD-22623 | biostudies-arrayexpress

RNAi-independent role for Argonaute2 in CTCF/CP190 chromatin insulator function

2011-08-15 | GSE22623 | GEO

NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization

Project description: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 their normal localization is correlated with proper 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) are also required for recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy binding site DNA, 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.

2022-10-14 | PXD032165 | Pride

Genomewide analysis of Insulator protein binding sites in Drosophila embryos at E0-12

Project description:Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins – dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF – to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all previously known insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw) respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters, divide differentially expressed, alternative, and divergent promoters, act as chromatin boundaries, are associated with chromosomal breakpoints among species, and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units, and a framework for understanding insulator function during the development and evolution of Drosophila. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

2009-05-29 | GSE16245 | GEO

CP190 and CTCF mediated enhancer blocking is augmented by SUMOylation

Project description:Chromatin insulators shield promoters and chromatin domains from neighbouring enhancers or chromatin regions with opposing activities. Insulator binding proteins and their cofactors mediate the boundary function. In general, covalent modification of proteins by the Small Ubiqutin-like Modifier (SUMO) is an important mechanism to control the interaction of proteins within complexes. Here we addressed the impact of SUMO in respect to insulator function, chromatin binding of insulator factors and formation of insulator speckles. SUMOylation augments the enhancer blocking function of four different insulator sequences and increases the genome-wide binding of the insulator cofactor CP190. A model is discussed with enhanced chromatin binding of SUMOylated CP190 causing fusion of insulator speckles, which may allow for more efficient insulation.

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M1BP cooperates with CP190 to activate transcription at TAD borders and promote chromatin insulator activity

Project description: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.

2021-06-07 | PXD026497 | Pride

The transcription factor M1BP targets CP190 to chromatin to regulate transcription and chromatin insulator activity (RNA-Seq)

Project description:Chromatin insulators are DNA-protein complexes that establish distinct higher order transcriptional domains. In Drosophila, CP190 is the only common factor for all the DNA-binding insulator protein complex. Insulators harbour 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. In our studies, using mass-spec analysis and of immunopurified complexes from Drosophila embryonic nuclear extracts, we identified the transcription factor Motif 1 Binding Protein (M1BP) associated with CP190 and verified this interaction by coimmunoprecipitation. Furthermore, depletion of M1BP results in loss of both enhancer-blocking and barrier activities, also disrupts the nuclear localization of CP190-marked insulator bodies within the developing fly. ChIP-seq analysis of both factors in Kc167 cultured hemocytes revealed extensive overlap of the two factors, particularly at Motif 1 containing promoters. Depletion of M1BP results in extensive loss of CP190 chromatin association, and depletion of CP190 also greatly affects M1BP chromatin association. Moreover, EU-seq analysis after depletion of either CP190 or M1BP suggests they regulate gene expression in similar fashion. Further analysis using reporter assays verifies that CP190-dependent gene expression changes are dependent on the presence of Motif 1. Our results suggest a novel mechanistic relationship between CP190 and M1BP function with respect to transcriptional regulation and higher order chromatin organization.

2020-10-27 | GSE142532 | GEO

The transcription factor M1BP targets CP190 to chromatin to regulate transcription and chromatin insulator activity (ChIP-Seq)

2020-10-27 | GSE142531 | GEO

Genome-wide binding of two new insulator-like proteins Pita and ZIPIC

Project description:Insulators are multiprotein–DNA complexes that regulate nuclear architecture. The Drosophila CP190 protein is a common cofactor of two main insulator complexes formed by the DNA-binding proteins Su(Hw) and dCTCF. Here, we have identified two new DNA-binding proteins, Pita and ZIPIC, that interact with CP190. In vitro, CP190 interacts with Pita through the BTB domain and with ZIPIC through the centrosomal targeting domain. The artificial binding sites for Pita or ZIPIC partially block enhancers and protect gene expression from PRE-mediated silencing in transgenic lines. Pita binds adjacent to dCTCF in the well-studied Mcp insulator and is required for its activity, which indicates that two insulator proteins can cooperate in organizing a functional insulator. Pita and ZIPIC preferentially bind to the promoter regions and extensively colocalize with dCTCF and BEAF. These results suggest that insulator proteins can fulfill multiple functions in chromosome architecture and gene expression.

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