Background CGGBP1 is a repeat-binding protein with diverse functions in the regulation of gene expression, cytosine methylation, repeat silencing and genomic integrity. CGGBP1 has also been identified as a cooperator of histone-modifying enzymes and as a component of CTCF-containing complexes that regulate the enhancer–promoter looping. CGGBP1–CTCF cross talk in chromatin regulation has been hitherto unknown. Results Here, we report that the occupancy of CTCF at repeats depends on CGGBP1. Using ChIP-sequencing for CTCF, we describe its occupancy at repetitive DNA. Our results show that endogenous level of CGGBP1 ensures CTCF occupancy preferentially on repeats over canonical CTCF motifs. By combining CTCF ChIP-sequencing results with ChIP sequencing for three different kinds of histone modifications (H3K4me3, H3K9me3 and H3K27me3), we show that the CGGBP1-dependent repeat-rich CTCF-binding sites regulate histone marks in flanking regions. Conclusion CGGBP1 affects the pattern of CTCF occupancy. Our results posit CGGBP1 as a regulator of CTCF and its binding sites in interspersed repeats.

Background The human CGGBP1 binds to GC-rich regions and interspersed repeats, maintains homeostasis of stochastic cytosine methylation and determines DNA-binding of CTCF. Interdependence between regulation of cytosine methylation and CTCF occupancy by CGGBP1 remains unknown. Results By analyzing methylated DNA-sequencing data obtained from CGGBP1-depleted cells, we report that some transcription factor-binding sites, including CTCF, resist stochastic changes in cytosine methylation. By analysing CTCF-binding sites we show that cytosine methylation changes at CTCF motifs caused by CGGBP1 depletion resist stochastic changes. These CTCF-binding sites are positioned at locations where the spread of cytosine methylation in cis depends on the levels of CGGBP1. Conclusion Our findings suggest that CTCF occupancy and functions are determined by CGGBP1-regulated cytosine methylation patterns.

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones which show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription termination sequence required for the upstream transcription seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

Binding sites of the chromatin regulator protein CTCF function as important landmarks in the human genome. The recently characterized CTCF-binding sites at LINE-1 repeats depend on another repeat-regulatory protein CGGBP1. These CGGBP1-dependent CTCF-binding sites serve as potential barrier elements for epigenetic marks such as H3K9me3. Such CTCF-binding sites are associated with asymmetric H3K9me3 levels as well as RNA levels in their flanks. The functions of these CGGBP1-dependent CTCF-binding sites remain unknown. By performing targeted studies on candidate CGGBP1-dependent CTCF-binding sites cloned in an SV40 promoter-enhancer episomal system we show that these regions act as inhibitors of ectopic transcription from the SV40 promoter. CGGBP1-dependent CTCF-binding sites that recapitulate their genomic function of loss of CTCF binding upon CGGBP1 depletion and H3K9me3 asymmetry in immediate flanks are also the ones that show the strongest inhibition of ectopic transcription. By performing a series of strand-specific reverse transcription PCRs we demonstrate that this ectopic transcription results in the synthesis of RNA from the SV40 promoter in a direction opposite to the downstream reporter gene in a strand-specific manner. The unleashing of the bidirectionality of the SV40 promoter activity and a breach of the transcription barrier seems to depend on depletion of CGGBP1 and loss of CTCF binding proximal to the SV40 promoter. RNA-sequencing reveals that CGGBP1regulated CTCF-binding sites act as barriers to transcription at multiple locations genome-wide. These findings suggest a role of CGGBP1-dependent binding sites in restricting ectopic transcription.

Genome-wide occupancy of several histone modifications in various cell types has been studied using chromatin immunoprecipitation (ChIP) sequencing. Histone occupancy depends on DNA sequence features like inter-strand symmetry of base composition and periodic occurrence of TT/AT. However, whether DNA sequence motifs act as an additional effector of histone occupancy is not known. We have analyzed the presence of DNA sequence motifs in publicly available ChIP-sequence datasets for different histone modifications. Our results show that DNA sequence motifs are associated with histone occupancy, some of which are different between primary and transformed cells. The motifs for primary and transformed cells showed different levels of GC-richness and proximity to transcription start sites (TSSs). The TSSs associated with transformed or primary cell-specific motifs showed different levels of TSS flank transcription in primary and transformed cells. Interestingly, TSSs with a motif-linked occupancy of H2AFZ, a component of positioned nucleosomes, showed a distinct pattern of RNA Polymerase II (POLR2A) occupancy and TSS flank transcription in primary and transformed cells. These results indicate that DNA sequence features dictate differential histone occupancy in primary and transformed cells, and the DNA sequence motifs affect transcription through regulation of histone occupancy.