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Genomes of somatic cells in culture are prone to spontaneous mutations due to errors in replication and DNA repair. Some of these errors, such as chromosomal fusions, are not rectifiable and subject to selection or elimination in growing cultures. Somatic cell cultures are thus expected to generate background levels of potentially stable chromosomal chimeras. A description of the landscape of such spontaneously generated chromosomal chimeras in cultured cells will help us understand the factors affecting somatic mosaicism. Here we show that short homology-associated non-homologous chromosomal chimeras occur in normal human fibroblasts and HEK293T cells at genomic repeats. The occurrence of chromosomal chimeras is enhanced by heat stress and depletion of a repeat regulatory protein CGGBP1. We also present evidence of homologous chromosomal chimeras between allelic copies in repeat-rich DNA obtained by methylcytosine immunoprecipitation. The formation of homologous chromosomal chimeras at Alu and L1 repeats increases upon depletion of CGGBP1. Our data are derived from de novo sequencing from three different cell lines under different experimental conditions and our chromosomal chimera detection pipeline is applicable to long read as well as short read sequencing platforms. These findings present significant information about the generation, sensitivity and regulation of somatic mosaicism in human cell cultures.

Background Inhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins. Methods We have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen. Results Our primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding. Conclusions The assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

Objectives Although CpG methylation is well studied, mechanisms of non-CpG methylation in mammals remains elusive. Studying proteins with non-CpG cytosine methylation-sensitive DNA-binding, such as human CGGBP1, can unveil cytosine methylation regulatory mechanisms. Here we have resequenced a published genome-wide bisulfite sequencing library and analyzed it at base level resolution. CpG, CHG and CHH (where H is any nucleotide other than G) methylation states in non-targeting or CGGBP1-targeting shmiR lentivirus-transduced cells have been analyzed to identify how CGGBP1 regulates CpG and non-CpG methylation. Results We report that CGGBP1 acts as a dynamic bimodal balancer of methylation. Both gain and loss of methylation observed upon CGGBP1 depletion were spatially overlapping at annotated functional regions and not identifiable with any sequence motifs but clearly associated with GC-skew. CGGBP1 depletion caused clustered methylation changes in cis, upstream of R-loop forming promoters. This was complemented by clustered occurrences of methylation changes in proximity of transcription start sites of known cytosine methylation regulatory genes, altered expression of which can regulate cytosine methylation in trans. Despite low coverage, our data provide reliable estimates of the spectrum of methylation changes regulated by CGGBP1 in all cytosine contexts genome-wide through a combination of cis and trans-acting mechanisms. Electronic supplementary material The online version of this article (10.1186/s13104-018-3516-1) contains supplementary material, which is available to authorized users.

DNA cytosine methylation occurs in all three sequence contexts: CpG and CHG (symmetrically on the two strands) and CHH (asymmetrically on one strand). While mechanisms of CpG methylation regulation are well studied; the non-CpG methylation is beginning to be understood. Cytosine methylation patterns genome-wide are required for gene expression regulation, allele-specific functions of genomic loci, genomic integrity and silencing of repetitive elements. Studying proteins that exhibit cytosine methylation methylation-sensitive DNA-binding can unveil novel cytosine methylation regulatory mechanisms and their consequences. One such recently reported cytosine methylation regulator protein is human CGGBP1 that has CpG and non-CpG cytosines both in its binding sites. CGGBP1 depletion affects CpG methylation at repetitive elements. Normal human foreskin fibroblasts 1064Sk cells were stably transduced with lentivirally expressed CGGBP1 shmiR and control shmiR. Genomic DNA was isolated followed by bisulfite treatment and subjected to deep sequencing (Illumina). Cytosine methylation state in presence and absence of CGGBP1 was ascertained by Bismark. Through genome-wide bisulfite sequencing, we describe the effects of CGGBP1 loss-of-function on CpG, CHG and CHH methylation at a base level resolution. We observed a dynamic bimodal balancing of methylation upon CGGBP1 depletion; as it causes both gain and loss of methylation, with spatial overlap at annotated functional regions and not identifiable with any sequence motifs. However, we observed a clear association of methylation changes with GC skew genome-wide. CGGBP1 depletion causes clustered methylation changes in cis, upstream of GC skew promoters complemented by clustered occurrences of methylation changes in proximity of transcription start sites of known cytosine methylation regulatory genes, altered expression of which can regulate cytosine methylation in trans. CGGBP1 maintains a balance between pro- and anti-cytosine methylation mechanisms independent of the nucleotide sequences, but acts at the higher level of DNA structure, nucleotide composition bias and secondary structure formation ability of the strands. Our findings collectively convey that CGGBP1 is a regulator of cytosine methylation in all sequence contexts, including CHG and CHH, genome-wide through a combination of cis and trans-acting mechanisms.

Introduction: Histone modification, a covalent post-translational modification of histone proteins, influences gene expression by altering chromatin structure giving rise to key characteristics to various cell types. While interaction of histone proteins with DNA is not known to be dependent on DNA sequence features, histone modifications are highly regulated in certain genomic regions. This specificity is required in normal cells to prevent genome instability, chromosome segregation defects and to maintain cellular homeostasis and is altered in cancer. Identifying [1] if specific sequence features are associated with certain histone modifications and [2] if these associations are consistently disrupted in transformed cells, will establish links between the genotype and epigenotype giving us valuable predictability about histone code. In this study, we have analyzed ChIP-seq data of different histone modifications in human across primary and immortalized cell lines from ENCODE database. Experiments and key results findings: We have identified unique as well as common genomic regions that carry histone marks commonly in primary and immortal cell lines. Additionally, motif finding analysis indeed shows certain modifications are associated with crucial genes required for cell survival and function; some uniquely in transformed cell lines. The regions with consistently different histone marks are currently being studied to check whether they are associated with certain cytosine methylation profile too. Our results suggest a genotypic predisposition for epigenotype.

Genome organisation and gene expression is regulated by specific DNA sequences that include “insulator elements”. Insulator proteins, such as CTCF bind to insulator elements to block spreading of silent chromatin in-cis or inhibit interactions between transcriptional enhancers and promoters. By binding to insulators in a methylation-sensittive manner, CTCF establishes and maintains contrasting transcription patterns on either side of the insulator elements [1]. Though details of CTCF-insulator activities have been worked out, mechanisms of regulation of insulator activity by other proteins is unknown. CTCF-binding insulators are retrotransposon-derived, the same elements to which CGGBP1 binds making CGGBP1 a candidate insulator regulator factor [2]. Objective is to explore role of CGGBP1-CTCF dynamics in regulation of insulator activity. 1064Sk skin fibroblasts were grown in presence or absence of CGGBP1 in growth stimulated or starved condition. ChIP-seq was performed to identify CGGBP1-binding DNA sequence motifs [3]. We have observed a strong overlap between binding sites of CTCF and CGGBP1 [4, 5]. CGGBP1 and CTCF seem to share the retrotransposons-derived M1 and M2 motifs. Unlike in quiescent cells, growth factor-stimulation increased CGGBP1 binding to CTCF-CGGBP1 binding sites with decreased CTCF insulator activity. The distance between CGGBP1 M1 and M2 motifs was longer in quiescent cells as compared to growth stimulated cells. Our results suggest that CGGBP1 negatively regulates CTCF insulator activity in normal cells in a growth signal-dependent manner.