Abstract
Histone H2AX phosphorylation is an early signalling event triggered by DNA double-strand breaks (DSBs). To elucidate the elementary units of phospho-H2AX-labelled chromatin, we integrate super-resolution microscopy of phospho-H2AX during DNA repair in human cells with genome-wide sequencing analyses. Here we identify phospho-H2AX chromatin domains in the nanometre range with median length of ∼75 kb. Correlation analysis with over 60 genomic features shows a time-dependent euchromatin-to-heterochromatin repair trend. After X-ray or CRISPR-Cas9-mediated DSBs, phospho-H2AX-labelled heterochromatin exhibits DNA decondensation while retaining heterochromatic histone marks, indicating that chromatin structural and molecular determinants are uncoupled during repair. The phospho-H2AX nano-domains arrange into higher-order clustered structures of discontinuously phosphorylated chromatin, flanked by CTCF. CTCF knockdown impairs spreading of the phosphorylation throughout the 3D-looped nano-domains. Co-staining of phospho-H2AX with phospho-Ku70 and TUNEL reveals that clusters rather than nano-foci represent single DSBs. Hence, each chromatin loop is a nano-focus, whose clusters correspond to previously known phospho-H2AX foci.