Bleomycin-induced γH2AX foci map preferentially to replicating domains in CHO9 interphase nuclei
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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.
KeywordsDNA damage γH2AX foci DNA replication EdU eu-/heterochromatin H3K4me3
Histone H2AX phosphorylated on serine 139
Chinese hamster ovary cells
Euchromatic damage distribution index
Full width at half maximum
Mid and late S
Point spread function
Replication-related damage distribution index
Regions of increased gene expression
Region of interest
Replication slow zones
TNF-related apoptosis-inducing ligand
We are indebted to Thomas Cremer, Marion Cremer and Areli Cárdenas for helpful suggestions on the manuscript as well as to Katrin Pfleghaar and Yolanda Markaki for technical advice. The work was financed by the Alexander von Humboldt Foundation (AvH) and partly by the Programa de Desarrollo en Ciencias Básicas (PEDECIBA, Uruguay) together with the Agencia Nacional de Investigación e Innovación (ANII, Uruguay). PL is a former Fellow of the AvH Förderung Program at the Ludwig-Maximilians-Universität Biozentrum (Munich). Research in SCIAN-Lab is funded by BNI ICM P09-015-F (SH, MC, JJ), FONDECYT 1120579 (SH, MC, JJ), US-LACRN (MC, JJ), FONDEF D07I1019 (SH, JJ), FONDECYT 3140447 (MC), the U. Chile “U-Redes Project: BioMed-HPC” (SH), a CONICYT doctoral fellowship (JJ), and AI•BI (www.aibi.cl).
Conflict of interest
Pablo Liddle, Laura Lafon-Hughes, María Vittoria Di Tomaso, Ana Laura Reyes-Ábalos, Jorge Jara, Mauricio Cerda, Steffen Härtel and Gustavo Folle declare that they have no conflict of interest.
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