Identification of the elementary structural units of the DNA damage response

• Published in: Nature communications Vol. 8; no. 1; p. 15760
• Main Authors: Natale, Francesco, Rapp, Alexander, Yu, Wei, Maiser, Andreas, Harz, Hartmann, Scholl, Annina, Grulich, Stephan, Anton, Tobias, Hörl, David, Chen, Wei, Durante, Marco, Taucher-Scholz, Gisela, Leonhardt, Heinrich, Cardoso, M Cristina
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 12.06.2017; Nature Portfolio
• Subjects: Cell cycle; Chromatin - chemistry; Chromatin - metabolism; Chromosomes; CRISPR; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Epigenesis, Genetic; Genomes; Histones - metabolism; Kinases; Microscopy; Models, Genetic; Phosphorylation; Germany
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms15760

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.