The multiscale distribution of radiation-induced DNA damage and its impact on local genome structure

The three-dimensional genome structure is critical for the regulation of gene expression and repair of DNA damage. While previous work has characterized genome-wide sites of DNA damage caused by etoposide or nucleases, the distribution of double-strand breaks (DSBs) caused by external radiation and how these interact with the 3D genome organization is less well understood. Here, we measure the genomic landscape of radiation-induced DNA damage using END-seq in fibroblasts and lymphoblasts after exposure to 5 Gy X-rays. We identify frequently broken regions and investigate the 3D genome properties around these breaks with Hi-C data. We observe that the distribution of robust breaks correlates with transcriptional and chromatin features of the genome. Transcriptionally active and decondensed regions, such as chromosome 19, the A compartment, and topologically associating domain (TAD) boundaries, show pronounced break probability. We also find evidence of DSB-induced loop formation in the vicinity of frequent radiation-induced breaks. Our data reveal that pre-existing 3D genome architecture influences the distribution of radiation-induced DSBs and that these breaks reshape local chromatin landscapes, advancing our understanding of genome instability. BJ-5ta adhesion cells were cultured and passaged at a density of 80%. GM12878 suspension cells were cultured and passaged at around 500,000 cells per 1 mL of medium. For irradiation experiments, BJ-5ta cells were grown to confluency, and GM12878 cells were grown to a density of 1  106 cells per 1 mL medium prior to 5 Gy X-ray exposure. END-seq experiments were performed in BJ-5ta, GM12878 on control and X-ray treated conditions. U2OS-ER-AsiSI and BJ-5ta-ER-AsiSI cells were cultured with puromycin selection. For DNA damage induction, both cells were seeded without puromycin treatment and treated with 300 nM 4-hydroxytamoxifen (4OHT) for 4 h and 6 h, respectively. END-seq experiments were performed in U2OS-ER-AsiSI and BJ-5ta-ER-AsiSI cells on untreated and treated 4OHT conditions.