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Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

ABSTRACT: CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, though the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we have shown that the DEAD box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but it does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF, by binding to both, and is required for proper insulator function.

ORGANISM(S): Homo sapiens

PROVIDER: GSE24126 | GEO | 2010/11/16

SECONDARY ACCESSION(S): PRJNA130117

REPOSITORIES: GEO

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Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA

Project description:CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, though the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we have shown that the DEAD box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but it does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF, by binding to both, and is required for proper insulator function. Identification of p68-binding sites in Hela cells using ChIP-Seq.

2010-11-16 | E-GEOD-24126 | biostudies-arrayexpress

Cohesin mediates transcriptional insulation by CCCTC-binding factor

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Transcription Across the H19/Igf2 Imprinting Control Region Initiates Paternally Methylated Imprinting in Mouse Prospermatogonia (ChIP-Seq)

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Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for proper CTCF function

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Genomic context sensitizes regulatory elements to genetic disruption [DNAse-seq]

Project description:Enhancer function is frequently investigated piecemeal using truncated reporter assays or single deletion analysis, thus it remains unclear to what extent their function is influenced by surrounding genomic context. Using our Big-IN technology for targeted integration of large DNAs, we analyzed the regulatory architecture of the Igf2/H19 locus, a paradigmatic model of enhancer selectivity. We assembled payloads containing a 157-kb functional Igf2/H19 locus and engineered mutations to genetically direct CTCF occupancy at the imprinting control region (ICR) that switches the target gene of the H19 enhancer cluster. Contrasting the activity of payloads delivered to the endogenous locus or to a safe harbor locus (Hprt) revealed that the functional elements comprising the Igf2/H19 locus are highly sensitive to their native context. Exchanging components of the Igf2/H19 locus with the well-studied Sox2 locus showed that the H19 enhancer cluster in particular functioned poorly out of context, and required its native surroundings to activate Sox2 expression. Conversely, the Sox2 locus control region (LCR) could activate both Igf2 and H19 outside its native context, but its activity was only partially modulated by CTCF occupancy at the ICR. Analysis of regulatory DNA actuation across different cell types revealed that, while the H19 enhancers are tightly coordinated within their native locus, the Sox2 LCR acts more independently. We show that these enhancer clusters typify broader classes of loci genome-wide. Our results show that unexpected dependencies may influence even the most studied functional elements, and our synthetic regulatory genomics approach permits large-scale manipulation of complete loci to investigate how locus architecture relates to function.

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Genomic context sensitizes regulatory elements to genetic disruption [DNA]

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Genomic context sensitizes regulatory elements to genetic disruption [CUT&RUN]

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Transcription Across the H19/Igf2 Imprinting Control Region Initiates Paternally Methylated Imprinting in Mouse Prospermatogonia (RNA-Seq)

Project description:Silver-Russell syndrome(SRS, OMIM 180860) is an imprinting disordermanifesting in fetal and postnatal growth retardation. Loss of methylation (LOM) is detected in the imprinting control region 1 (ICR1) that controls the H19/IGF2 domain, in about 65% of SRS cases. The cause of this epigenetic deficiency is unknown. It may affect the process of imprint establishment --de novo methylation of ICR1 in the male germ line. In the paralogous mouse locus this process involves DNA methyltransferase DNMT3A and H3K36me2 histone methyltransferase NSD1, but the mechanism that targets these enzymes to this ICR is unknown. Based on our genome-wide deep-sequencing data of transcription and DNA methylation, we hypothesized that low-level transcription is required in prospermatogonia for targeting H3K36me2 and de novo methylation to ICR. We truncated this transcript at the entry point to ICR by gene-targeting. In response, the establishment of DNA methylation imprint was incomplete. The sporadic LOM persisted from the male germ line into the paternal allele of the soma, resulting in reduced Igf2, and increased, biallelic H19 RNA in the fetal organs, consistent with the insulator model. Fetus weight was also variably reduced upon paternal transmission of this mutation, consistent with the role of IGF2 as a fetal growth factor. We identify low-level transcription-through as the mechanism that initiates paternal imprint establishment at the H19/Igf2 ICR in the male germ line. The findings predict that RNA-dependent imprint establishment may be affected in human fetuses resulting in sporadic hypomethylation of the ICR1 in SRS.

2023-08-02 | GSE202875 | GEO

Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF (ChIP-Seq)

Project description:Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and transcription factor binding, including the insulator protein CTCF. To define which subcomplex mediate these diverse functions we phenotyped a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of either CERF, RSF1, ACF, WICH or NoRC subcomplexes cause only moderate effects on the chromatin landscape, while removal of the NURF specific subunit BPTF leads to drastic reduction in chromatin accessibility and Snf2h ATPase localization around CTCF sites. While this reduces distances to the adjacent nucleosomes it only modestly impacts CTCF binding itself. In absence of accessibility bound CTCF is nevertheless impaired in function resulting in lower occupancy of cohesin and cohesin-loading factors, and reduced insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function. Our comprehensive analysis reveals a specific role for NURF in mediating Snf2h localization and chromatin opening at bound CTCF sites showing that local accessibility is critical for cohesin binding and insulator function.

2024-03-11 | GSE250226 | GEO

Systematic assessment of ISWI subunits reveals that NURF creates local accessibility for CTCF (RNA-Seq)

Project description:Catalytic activity of the ISWI family of remodelers is critical for nucleosomal organization and transcription factor binding, including the insulator protein CTCF. To define which subcomplex mediate these diverse functions we phenotyped a panel of isogenic mouse stem cell lines each lacking one of six ISWI accessory subunits. Individual deletions of either CERF, RSF1, ACF, WICH or NoRC subcomplexes cause only moderate effects on the chromatin landscape, while removal of the NURF specific subunit BPTF leads to drastic reduction in chromatin accessibility and Snf2h ATPase localization around CTCF sites. While this reduces distances to the adjacent nucleosomes it only modestly impacts CTCF binding itself. In absence of accessibility bound CTCF is nevertheless impaired in function resulting in lower occupancy of cohesin and cohesin-loading factors, and reduced insulation at these sites, highlighting the need of NURF-mediated remodeling for open chromatin and proper CTCF function. Our comprehensive analysis reveals a specific role for NURF in mediating Snf2h localization and chromatin opening at bound CTCF sites showing that local accessibility is critical for cohesin binding and insulator function.

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