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CTCF Loss Potentiates P53 Mediated Gene Transcription in Breast Tissue

ABSTRACT: A single copy loss of CTCF is found in about 50% of breast cancer patients. Based on clinical TCGA data we hypothesized that the loss of CTCF may potentiate TP53 target gene expression in patients. Using MCF10A cells as a model, we deleted a single copy of CTCF using CRISPR/Cas9. We found, using qPCR and RNA-seq, that cells carrying low CTCF displayed an enhanced TP53 response after exposure to chemotherapeutics. Using ATAC-seq, we aimed to explore whether the elevated induction of TP53 target gene transcription was associated with changes in open or closed chromatin structure. Specifically, we were interested in comparing chromatin accessibility at TP53 target genes within MCF10A CTCF +/- cells compared to control cells following the induction of DNA damage (6uM cisplatin for 8h). We discovered that accessibility of the transcription start site is associated with heightened gene expression in CTCF+/- compared to the control. Interestingly, for a subset of TP53 response genes, there is increased accessibility on both transcriptional start sites and termination sites following the induction of DNA damage. The importance of chromatin accessibility at these two regions is still under investigation. Additionally, accessible gene regions at both sites also appear to have greater enrichment within TADS in the CTCF+/- cell compared to the control. We propose that the increased accessibility of TP53 target genes following damage represents a mechanism enhancing the efficacy of the TP53-regulated DNA damage response.

ORGANISM(S): Homo sapiens

PROVIDER: GSE217698 | GEO | 2023/11/10

REPOSITORIES: GEO

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Project description:Chromatin-organizing factors, like CTCF and cohesins, have been implicated in the control of complex viral regulatory programs. We investigated the role of CTCF and cohesin in the control of the latent to lytic switch for Kaposi's Sarcoma-Associated Herpesvirus (KSHV). We found that cohesin subunits, but not CTCF, were required for the repression of KSHV immediate early gene transcription. Depletion of cohesin subunits Rad21, SMC1, or SMC3 resulted in lytic cycle gene transcription and viral DNA replication. In contrast, depletion of CTCF failed to induce lytic transcription or DNA replication. ChiP-Seq analysis revealed that cohesins and CTCF bound to several sites within the immediate early control regions for ORF50 and more distal 5' sites that also regulate the divergently transcribed ORF45-46-47 gene cluster. Rad21 depletion led to a robust increase in ORF45 and ORF47 transcripts, with similar kinetics to that observed with chemical induction by sodium butyrate. During latency, the chromatin between the ORF45 and ORF50 transcription start sites was enriched in histone H3K4me3 with elevated H3K9ac at the ORF45 promoter and elevated H3K27me3 at the ORF50 promoter. A paused form of RNA pol II was loosely associated with the ORF45 promoter region during latency, but was converted to an active elongating form upon reactivation induced by Rad21 depletion. Butyrate-induced transcription of ORF45 and ORF47 was resistant to cyclohexamide, suggesting that these genes have immediate early features similar to ORF50. Butyrate-treatment caused the rapid dissociation of cohesins and loss of CTCF binding at the immediate early gene locus, suggesting that cohesins may be a direct target of butyrate-mediated lytic induction. Our findings implicate cohesins as a major repressor of KSHV lytic gene activation, and function coordinately with CTCF to regulate the switch between latent and lytic gene activity. Study of chromatin-organizing factors, like CTCF and cohesins.

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CTCF Loss Potentiates P53 Mediated Gene Transcription in Breast Tissue

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