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The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

ABSTRACT: The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

PROVIDER: PRJNA1123167 | ENA |

REPOSITORIES: ENA

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The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression (HTS)

Project description:Truncated forms of CGGBP1 with (N-term) or without (C-term) the DNA-binding domain (DBD) have been used to to assay global cytosine methylation. HEK293T cells with endogenous CGGBP1 knocked down were used to over-express truncated forms of CGGBP1 followed by MeDIP-Seq analysis. Genome-wide analyses of cytosine methylation and binding of CGGBP1 DBD show that CGGBP1 restricts cytosine methylation in a manner that depends on its DBD and its DNA-binding. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss. A superimposition of our results and evolution of CGGBP1 suggests that mitigation of cytosine methylation is majorly achieved by its N-terminal DBD. Our results position CGGBP1 DNA-binding as a major evolutionarily acquired mechanism through which it keeps cytosine methylation under check and regulates TFBS retention.

2024-11-17 | GSE269791 | GEO

The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression

Project description:Truncated forms of CGGBP1 with (N-term) or without (C-term) the DNA-binding domain (DBD) have been used to to assay global gene expression. HEK293T cells with endogenous CGGBP1 knocked down were used to over-express truncated forms of CGGBP1 followed by RNA extraction and one-coloured global gene expression analysis. The data suggests that while the C-term of CGGBP1 is the major repressor of transcription, just the N-term containing the DBD fails to achieve so. Proximal promoters of CGGBP1-repressed genes, although significantly GC-poor, contain GC-rich transcription factor binding motifs and exhibit base compositions indicative of low C-T transition rates due to targeted prevention of cytosine methylation. Our findings suggest that CGGBP1 protects transcription factor binding sites (TFBS) from cytosine methylation-associated loss and thereby regulates gene expression. By analysing orthologous promoter sequences, we show that protection from cytosine methylation is a function of CGGBP1 progressively acquired during vertebrate evolution.

2024-11-17 | GSE269654 | GEO

CGGBP1-regulated cytosine methylation at CTCF-binding motifs resists stochasticity

Project description:To study if regulation of cytosine methylation and CTCF occupancy are interdependent and governed by the levels of CGGBP1, we specifically pulled-down methylated cytosines and found that some transcription factor binding sites including that of CTCF held out against the cytosine methylation changes. The cytosine methylation at CTCF-binding repeat-free motifs show a non-stochastic depdendence on CGGBP1 and occur at sites that mark cytosine methylation transition boundaries. We also show that allelic imbalance dictates stochastic methylation changes due to CGGBP1 depletion.

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The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression (HTS)

Project description:The amniote-conserved DNA-binding domain of CGGBP1 restricts cytosine methylation of transcription factor binding sites in proximal promoters to regulate gene expression (HTS)

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CGGBP1 inhibits cytosine methylation at repetitive DNA sequences

Project description:By depleting CGGBP1 in normal human fibroblasts and by performing genome-wide sequencing (with and without bisulfite conversion) we show that upon CGGBP1 depletion cytosine methylation increases significantly at repeat regions. Using Pacbio sequencing of Alu and LINE-1 repeats amplified genome-wide from bisulfite converted DNA, we further establish the cytosine methylation-inhibitory functions of CGGBP1.

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Project description:CGGBP1-regulated cytosine methylation at CTCF-binding motifs resists stochasticity

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CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

Project description:CGGBP1-dependent CTCF-binding sites identified in Patel et. al. 2019 [PMID 31547883] serve as barrier elements consistent with asymmetrical levels of H3K9me3 in the flanks and asymmetrical RNA levels at these sites in the genome. In this study we have characterised the function of such CGGBP1-depenedent CTCF binding sites which are usually repeat rich in nature. By cloning one such CTCF-binding site in an episomal system, we have studied the barrier activity of this CGGBP1-dependent CTCF-binding sites in the prsesnce and absence of CGGBP1 in HEK293T cells through various molecular assays and RNA-sequencing. Our results show that these sites act as barrier for the ectopic transcription as the depletion of CGGBP1 lead to starnd-specific bidirectional transcription as a result of loss of barrier activity concomitant with the loss of CTCF-binding. Further, analysing the RNA-seq revealed that weakly transcribed sites are flanked by the CTCF-binding at transcription start and end sites in the presence of CGGBP1. However, CGGBP1 depletion leads to loss of barrier activity maintained by CGGBP1 with dispersed CTCF binding and a loss of transcription restriction. Such CGGBP1-dependent CTCF-binding sites prevents ectopic transcription.

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Dynamic bimodal changes in CpG and non-CpG methylation genome-wide upon CGGBP1 loss-of-function

Project description:By depleting CGGBP1 in normal human fibroblasts 1064Sk using lentiviral shmiR (non-targeting or targeting CGGBP1) and by performing genome-wide sequencing (on bisulfite converted genomic DNA from both samples) we show that both gain and loss of cytosine methylation occurs upon CGGBP1 depletion. This gain and loss of methylation is clustered, spatially overlapping at major functional genomic regions and transcends all three cytosine sequence contexts. We demonstrate that CGGBP1 regulates cytosine methylation genome-wide through cis and trans-acting mechanisms. Library preparation for sequencing has been described in PMID 25981527.

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CGGBP1 regulates chromatin barrier activity and CTCF occupancy at repeats

Project description:To study the role of CGGBP1 in regulation of CTCF occupancy, we performed CTCF ChIP-seq in HEK293T cells with three different levels of CGGBP1: (i) endogenous levels of CGGBP1 (ii) CGGBP1 knockdown, and (iii) CGGBP1 overexpression. Here, we show that CTCF binds to repeats as well as canonical CTCF-motifs and CGGBP1 determines the CTCF occupancy preference for repeats over canonical CTCF-motif. By combining CTCF ChIP-seq with histone modifications ChIP-seq (for H3K4me3, H3K9me3 and H3K27me3) under conditions of normal or CGGBP1 knockdown, we demonstrate that CTCF binding sites regulated by CGGBP1 correspond to chromatin barrier elements with profound effects on H3K9me3 distribution. In conclusion, these finding shows that CGGBP1 is a regulator of CTCF occupancy and serve as a regulator of barrier functions of CTCF-binding sites.

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CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

Project description:CGGBP1-dependent CTCF-binding sites restrict ectopic transcription

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