CBP and RAD21 Proteins Bind at the Termini of Forum Domains in Human Chromosomes.

Forum domains are 50-100-kb stretches of DNA delimited by the hotspots of double-strand breaks (DSBs). These domains possess coordinately expressed genes. However, molecular mechanisms of such regulation are not clear. It is assumed that the proteins specifically binding at the termini of domains can be involved in coordinated regulation of expression. In this study, we used the results of precise mapping of hotspots of DSBs and ChIP-Seq data for ten nuclear proteins in HEK293T cell line for a search of proteins specifically binding at forum-domain termini. We detected that two proteins, CBP and RAD24, which are known to be involved in epigenetic regulation of gene expression and formation of 3D chromosomal structures, bind at the termini. We assume that these proteins may be involved in coordinated expression of genes in forum domains.

Interchromosomal Contacts of rDNA Clusters in Three Human Cell Lines Are Associated with Silencing of Genes Controlling Morphogenesis.

To study the rDNA contacts with genes in three human cell lines of different origin, we used 4C approach. Our data indicate that the same set of about five hundred genes frequently shape contacts with rDNA clusters in HEK293T, K652, and hESM01 cells. Gene ontology search suggests that the genes are involved in development and morphogenesis. Approximately one hundred of these genes are highly associated with silencing by H3K27me3 mark in different normal cells, including bronchial epithelial cells, keratinocytes, myoblasts, monocytes, endothelial cells, kidney epithelial cells, and some others. We conclude that the concerted silencing of specific group of rDNA-contacting genes controlling development occurs during differentiation. We assume that the phase separation mechanisms may be involved in the rDNA-mediated silencing of a set of genes via the contacts with inactive rDNA clusters.

Interchromosomal Contacts of rDNA Clusters with DUX Genes in Human Chromosome 4 Are Very Sensitive to Heat Shock Treatment.

In order to study the effects of heat shock treatment on the distribution of rDNA contacts at the region possessing DUX genes inside chromosome 4 we used 4C approach. Our data indicate that the treatment removes the frequent rDNA contacts in this region. The recent data on involvement of superenhancers that are decorated by broad H3K27ac marks in the phase separation mechanisms and the previous data demonstrating that these broad marks are the favorite sites of rDNA contacts taken together with our data on sensitivity of the contacts to the heat shock treatment suggest that the phase separation mechanisms are involved in the reversible rDNA-mediated regulation of gene expression via the contacts.

Mapping of genomic double-strand breaks by ligation of biotinylated oligonucleotides to forum domains: Analysis of the data obtained for human rDNA units.

DNA double-strand breaks (DSBs) are associated with different physiological and pathological processes in different organisms. To understand the role of DSBs in multiple cellular mechanisms, a robust method for genome-wide mapping of chromosomal breaks at one-nucleotide resolution is required. Many years ago, we detected large DNA fragments migrating from DNA-agarose plugs in pulsed-field gels, which we named 'forum domains' [1,2]. Recently, we developed a method for genome-wide mapping of DSBs that produces these 50-150 kb DNA domains using microarrays or 454 sequencing (Tchurikov et al., 2011; 2013). Now we have used Illumina sequencing to map DSBs in repetitive rDNA units in human HEK293T cells. Here we describe in detail the experimental design and bioinformatics analysis of the data deposited in the Gene Expression Omnibus with accession number GSE49302 and associated with the study published in the Journal of Molecular Cell Biology (Tchurikov et al., 2014).

Genome-wide mapping of hot spots of DNA double-strand breaks in human cells as a tool for epigenetic studies and cancer genomics.

Hot spots of DNA double-strand breaks (DSBs) are associated with coordinated expression of genes in chromosomal domains (Tchurikov et al., 2011 [1]; 2013). These 50-150-kb DNA domains (denoted "forum domains") can be visualized by separation of undigested chromosomal DNA in pulsed-field agarose gels (Tchurikov et al., 1988; 1992) and used for genome-wide mapping of the DSBs that produce them. Recently, we described nine hot spots of DSBs in human rDNA genes and observed that, in rDNA units, the hot spots coincide with CTCF binding sites and H3K4me3 marks (Tchurikov et al., 2014), suggesting a role for DSBs in active transcription. Here we have used Illumina sequencing to map DSBs in chromosomes of human HEK293T cells, and describe in detail the experimental design and bioinformatics analysis of the data deposited in the Gene Expression Omnibus with accession number GSE53811 and associated with the study published in DNA Research (Kravatsky et al., 2015). Our data indicate that H3K4me3 marks often coincide with hot spots of DSBs in HEK293T cells and that the mapping of these hot spots is important for cancer genomic studies.

Coexistence of different base periodicities in prokaryotic genomes as related to DNA curvature, supercoiling, and transcription.

We analyzed the periodic patterns in E. coli promoters and compared the distributions of the corresponding patterns in promoters and in the complete genome to elucidate their function. Except the three-base periodicity, coincident with that in the coding regions and growing stronger in the region downstream from the transcriptions start (TS), all other salient periodicities are peaked upstream of TS. We found that helical periodicities with the lengths about B-helix pitch ~10.2-10.5 bp and A-helix pitch ~10.8-11.1 bp coexist in the genomic sequences. We mapped the distributions of stretches with A-, B-, and Z-like DNA periodicities onto E. coli genome. All three periodicities tend to concentrate within non-coding regions when their intensity becomes stronger and prevail in the promoter sequences. The comparison with available experimental data indicates that promoters with the most pronounced periodicities may be related to the supercoiling-sensitive genes.