CGGBP1 regulates CTCF occupancy at repeats

(2019) 12:57 Patel et al. Epigenetics & Chromatin https://doi.org/10.1186/s13072-019-0305-6 Epigenetics & Chromatin Open Access RESEARCH CGGBP1 regulates CTCF occupancy at repeats Divyesh Patel†, Manthan Patel†, Subhamoy Datta and Umashankar Singh* Abstract 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 histonemodifying 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. Introduction Human CGGBP1 is a ubiquitously expressed protein with important functions in stress response, cell growth, proliferation and mitigation of endogenous DNA damage [1–5]. The CGGBP1 gene is conserved only in the amniotes with more than 98% similarity across the homeotherms [1]. Yet, the involvement of CGGBP1 in widely conserved cellular processes, such as cell cycle, maintenance of genomic integrity and cytosine methylation regulation, suggests that CGGBP1 fine-tunes these processes in homeothermic organisms to meet the challenges of their terrestrial habitats. CGGBP1 has no known paralogs in the human genome and is widely expressed in human tissues [6]. RNAi against CGGBP1 causes G1/S or G2/M arrest [3] and heat shock response-like gene expression changes with variable effects in different cell lines [3, 7]. CGGBP1 acts as a cis-regulator of transcription for tRNA genes, Alu elements [4], FMR1, CDKN1A, HSF1 [8, 9] and cytosine methylation-regulatory genes *Correspondence: † Divyesh Patel and Manthan Patel contributed equally to this work. HoMeCell Lab, Biological Engineering, Indian Institute of Technology Gandhinagar, Gandhinagar, Gujarat 382355, India including DNMT1 [9]. However, none of these functions explain the widespread effect that CGGBP1 depletion has on the global transcriptome. In cultured normal human fibroblasts, CGGBP1 depletion results in gene expression shutdown in a manner that resembles the effects of serum starvation [4]. The mechanisms through which CGGBP1 regulates the genome and the transcriptome remain enigmatic. Recent reports have shown that CGGBP1 regulates cytosine methylation genome-wide with the maximum methylation-regulatory effects at Alu and LINE elements in the CpG context [9]. The highly prevalent CHH cytosines, however, show a CGGBP1-dependent methylation pattern at GC-skew regions, insulators and enhancers [10]. Interestingly, CGGBP1 depletion causes an increase in CHH methylation at insulators (characterized as the CTCF-binding sites) and a decrease at enhancers. These findings suggest a cross talk between CGGBP1 and CTCF. There are additional evidences to support the possibility that CGGBP1 regulates the activities of insulators and enhancers. Most prominently, a targeted identification of proteins which structure the enhancer–promoter loops identified CGGBP1 as a partner of CTCF and YY1 © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Patel et al. Epigenetics & Chromatin (2019) 12:57 [11]. Despite its ubiquitous expression, CGGBP1 has not been studied further by Weintraub and co-workers because, unlike YY1, CGGBP1 has not been identified as a hit in a screen for essential genes [12]. Canonically, CTCF binds with high affinity to well-defined sequence motifs [13–15]. However, it has been reported that CTCF is also associated with additional genomic elements that do not contain these motifs. Interspersed repeats SVA and Alu-SINEs [16] as well as microsatellite repeats serve as binding sites for CTCF [17–19] and CGGBP1. Even CTCF-binding sites that contain the CTCF-binding motifs, and are not repeats per se, have evolved from Alu-SINEs and related repetitive elements [20]. Additionally, CGGBP1 and CTCF both exhibit cytosine methylation-sensitive DNA binding at GC-rich sequences [21]. Further indications of cross talk between CTCF and CGGBP1 are derived from findings that both of these proteins interact with a crucial tumor suppressor factor NPM1 [22, 23]. CGGBP1 is a proven regulator of rRNA genes that contain CGG triplet repeats and localize to the nucleoli [8]. NPM1–CTCF interactions determine the organization of chromatin in the nucleolus. CTCF and NPM1 establish transcriptionally silent chromatin domains in the nucleolar periphery [23]. NPM1–CTCF interaction is required for insulator activity at many sites in the genome. Notably, NPM1 also complexes with CGGBP1 [22]. CGGBP1 drives rRNA synthesis upon growth factor stimulation by silencing Alu repeats [4]. Both CGGBP1 and CTCF are reported to have nuclear expression in interphase cells and midbody expression in mitotic cells [3, 24]. Collectively, these facts further strengthen the possibility of a functional cross talk between CGGBP1 and epigenomic regulator factors such as CTCF. In this study, we have investigated this functional cross talk between CTCF and CGGBP1. We show that a fraction of endogenously expressed CGGBP1 and CTCF interact with each other. A systematic analysis of published ChIP-seq data for CGGBP1 and CTCF ChIP-seq data (ENCODE) show that the binding sites for the two proteins are in close proximity. Our co-immunoprecipitation (co-IP) assays lend support to these findings. Subsequently, by studying CTCF occupancy genome-wide through ChIP-seq under conditions of normal CGGBP1 expression, CGGBP1 knockdown and overexpression, we show that CTCF binds to both repeats and canonical CTCF motifs. Our analysis reveals that CGGBP1 levels determine the CTCF-binding preference between repeats and canonical CTCF motifs. By combining CTCF ChIPseq with histone modification ChI (...truncated)