Bleomycin-induced γH2AX foci map preferentially to replicating domains in CHO9 interphase nuclei.

Exposure to DNA damaging agents triggers phosphorylation of histone variant H2AX (generating γH2AX) in large chromatin regions flanking DNA lesions, allowing their immunodetection as nuclear foci. Even though a predominance of γH2AX foci in euchromatin has been postulated, foci positioning when DNA insult occurs in replicating eu- or heterochromatin regions has not been extensively explored. Labeling of interphase nuclei with 5-ethynyl-2'-deoxyuridine (EdU) pulses has revealed that DNA replication is temporarily and spatially regulated: euchromatin replicates in early S (ES) and heterochromatin along mid and late S (MS/LS) phases. In order to map DNA damage with respect to replicating domains, the distribution of γH2AX foci induced by the radiomimetic agent bleomycin was analyzed in CHO9 interphase nuclei by delineating euchromatic (H3K4me3+) and replicating (EdU+) regions. Quantification of overlapping pixels and 3D inter-object overlap in binary masks revealed colocalization between γH2AX foci and EdU +  domains both in ES and MS/LS nuclei, indicating that primary damage distribution is modulated by DNA synthesis. Further, we verified that EdU incorporation by itself did not influence BLEO-induced γH2AX nuclear patterns. Our results also revealed a repeated localization of γH2AX foci in replicating/nonreplicating interfaces which could reflect short-range chromatin migration following DNA insult.