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Nat Commun. 2017 Jun 12;8:15760. doi: 10.1038/ncomms15760.

Identification of the elementary structural units of the DNA damage response.

Author information

1
Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
2
Department of Biology II, Center for Integrated Protein Science Munich (CIPSM), LMU Munich, 82152 Planegg-Martinsried, Germany.
3
Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany.
4
Department of Biophysics, GSI Helmholtzzentrum für Schwerionenforschung, 64291 Darmstadt, Germany.

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.

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