Endogenous hot spots of DNA double-strand breaks (DSBs) are tightly linked with transcription patterns and cancer genomics1,2. There are nine hot spots of DSBs located in human rDNA units3–6. Here we describe that the profiles of these hot spots coincide with the profiles of γ-H2AX or H2AX, strongly suggesting a high level of in vivo breakage inside rDNA genes. The data were confirmed by microscopic observation of the largest γ-H2AX foci inside nucleoli in interphase chromosomes. In metaphase chromosomes, we observed that only some portion of rDNA clusters possess γ-H2AX foci and that all γ-H2AX foci co-localize with UBF-1 binding sites, which strongly suggests that only active rDNA units possess the hot spots of DSBs. Both γ-H2AX and UBF-1 are epigenetically inherited and thus indicate the rDNA units that were active in the previous cell cycle. These results have implications for diverse fields, including epigenetics and cancer genomics.

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).

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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.