DNA double-strand breaks (DSBs) are involved in many cellular mechanisms, including
replication, transcription, and genome rearrangements. The recent observation that hot spots of
DSBs in human chromosomes delimit DNA domains that possess coordinately expressed genes
suggests a strong relationship between the organization of transcription patterns and hot spots of
DSBs. In this study, we performed mapping of hot spots of DSBs in a human 43-kb ribosomal
DNA (rDNA) repeated unit. We observed that rDNA units corresponded to the most fragile sites
in human chromosomes and that these units possessed at least nine specific regions containing
clusters of extremely frequently occurring DSBs, which were located exclusively in non-coding
intergenic spacer (IGS) regions. The hot spots of DSBs corresponded to only a specific subset of
DNase-hypersensitive sites, and coincided with CTCF, PARP1, and HNRNPA2B1 binding sites,
and H3K4me3 marks. Our rDNA-4C data indicate that the regions of IGS containing the hot
spots of DSBs often form contacts with specific regions in different chromosomes, including the
pericentromeric regions, as well as regions that are characterized by H3K27ac and H3K4me3
marks, CTCF binding sites, ChIA-PET and RIP signals, and high levels of DSBs. The data
suggest a strong link between chromosome breakage and several different mechanisms of
epigenetic regulation of gene expression.