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CTCF is required to form regulatory and physical boundaries at limited sites in the Drosophila nervous system [ChIP-seq]

ABSTRACT: CCCTC-binding factor (CTCF) is a conserved protein able to block communication between regulatory elements and gene promoters in flies and mammals. Here, we studied CTCF function in the Drosophila central nervous system (CNS) in which we find CTCF plays a vital role. Chromatin immunoprecipitation (ChIP)-sequencing of CTCF and its partner protein Centrosomal protein 190 kDa (Cp190) in CNSs of wildtype (WT), CTCF[0] mutants completely lacking CTCF and Cp190[KO] mutants with a CRISPR/Cas9-mediated deletion of the Cp190 open reading frame identified CTCF and Cp190 peaks defined as enriched in WT relative to each respective mutant control. These experiments also revealed that CTCF recruits Cp190 to CTCF's binding sites, but Cp190 is dispensable for CTCF binding to most of CTCF's sites.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE146749 | GEO | 2021/01/18

REPOSITORIES: GEO

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CTCF is required to form regulatory and physical boundaries at limited sites in the Drosophila nervous system [RNA-seq]

Project description:CCCTC-binding factor (CTCF) is a conserved protein able to block communication between regulatory elements and gene promoters in flies and mammals. Here, we studied CTCF function in the Drosophila central nervous system (CNS) in which we find CTCF plays a vital role. RNA-sequencing of CNSs of CTCF[0] mutants that completely lack CTCF identified putative targets that are expressed in altered patterns. We find that CTCF recruits Centrosomal protein 190 kDa (Cp190) to CTCF's binding sites to co-regulate a substantial fraction of common genes that we find are also altered in CNSs of Cp190[KO] mutants carrying a CRISPR/Cas9-mediated deletion of the Cp190 open reading frame.

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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)

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CTCF is required to form regulatory and physical boundaries at limited sites in the Drosophila nervous system [Hi-C]

2021-01-18 | GSE146750 | GEO

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

2011-08-15 | GSE22623 | GEO

Expression analyses in Drosophila ovaries

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Expression analyses in Drosophila ovaries

Project description:Suppressor of Hairy-wing [Su(Hw)] is a multi-zinc finger DNA binding factor required for gypsy insulator function and female germline development in Drosophila. The enhancer-blocking and barrier functions of the gypsy retrotransposon involve Su(Hw) binding to twelve clustered Su(Hw) binding sites (SBSs) and recruitment of the Centrosomal Protein of 190 kD (CP190) and Modifier of mdg4 67.2 kD isoform (Mod67.2) insulator proteins. In contrast, the Su(Hw) germline function involves binding to non-clustered genomic SBSs and does not require CP190 or Mod67.2. Here, we use genome-wide expression analyses in the ovary to identify the first Su(Hw) regulated target genes. Ovaries for RNA isolation were dissected from 4-6 hour old virgin females of wild type (Canton S and BL15598) and su(Hw) null sterile mutants (su(Hw)A2663/v and su(Hw)Pb/2) Drosophila melanogaster. At this stage of development, ovaries only contain egg chamber stages 1-8. Loss of Su(Hw) causes apoptosis at stage 9. Thus, the experimental design compares transcriptional changes in the ovary prior to induction nof apoptosis in su(Hw) mutants.

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Analysis of genome-wide binding of the insulator factors CTCF and CP190

Project description:We identified about 3000 CTCF sites in the genome. In addition to binding to CTCF sites we found CP190 to bind to transcriptional start sites of actively transcribed genes where it inversely correlates with nucleosome occupancy. Keywords: ChIP-chip CTCF and CP190 ChIP. 2 biological replicates per experiment. dye-swaps as indicated in sample description.

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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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DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries

Project description:The interplay of DNA (de)methylation and the architectural chromatin protein CTCF is an important regulator of cellular differentiation, but molecular mechanisms determining differential CTCF binding are not well understood. Mouse embryonic stem cells (ESCs) contain up to 10% of CpG residues in 5-hydroxymethylated form (5hmC) and smaller amounts in 5-formylated or 5-carboxylated forms (5fC, 5caC). Here, we studied double knock out ESCs (DKO) deficient for TET1 and TET2 enzymes which are responsible for the conversion of 5mC to 5hmC, 5fC and 5caC. For both wild type (WT) and DKO cells, we measured changes in nucleosome positions, CTCF binding, DNA methylation and gene expression. Our integrative data analysis combined with biophysical modelling suggests that both differential (de)methylation and CTCF binding are to a large degree predictable from the DNA sequence. Common CTCF sites that remained bound in DKO cells were significantly enriched at unmethylated CpG islands, while CTCF loss was associated with differential DNA methylation. 5hmCs and 5fCs had the opposite effects in terms of CTCF priming: CTCF was preferentially lost from sites that were marked in WT cells by 5hmCs but not 5fCs. Interestingly, the losses of CTCF at TAD boundaries lead to aberrant spreading of DNA methylation, as well as downregulation of neighbouring genes. Overall, our study clarifies the fundamental mechanism of differential CTCF binding and provides evidence of DNA sequence-encoded temporal chromatin changes during cell differentiation.

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