Liddle Pablo, Lafon-Hughes Laura, Di Tomaso María Vittoria, Reyes-Ábalos Ana Laura, Jara Jorge, Cerda Mauricio, Härtel Steffen, Folle Gustavo A
Departamento de Genética, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay,
Chromosome Res. 2014 Dec;22(4):463-81. doi: 10.1007/s10577-014-9433-9. Epub 2014 Jul 18.
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.
暴露于DNA损伤剂会触发DNA损伤侧翼大片染色质区域中组蛋白变体H2AX的磷酸化（产生γH2AX），从而使其能够作为核灶进行免疫检测。尽管有人推测常染色质中γH2AX灶占优势，但当DNA损伤发生在复制的常染色质或异染色质区域时，灶的定位尚未得到广泛研究。用5-乙炔基-2'-脱氧尿苷（EdU）脉冲标记间期核已表明DNA复制是暂时和空间调控的：常染色质在S期早期（ES）复制，异染色质在S期中期和后期（MS/LS）复制。为了绘制相对于复制结构域的DNA损伤图谱，通过描绘常染色质（H3K4me3+）和复制（EdU+）区域，分析了放射模拟剂博来霉素诱导的γH2AX灶在CHO9间期核中的分布。对二元掩码中重叠像素和三维物体间重叠的定量分析揭示了γH2AX灶与ES和MS/LS核中EdU+结构域的共定位，表明原发性损伤分布受DNA合成调节。此外，我们证实EdU自身掺入并不影响博来霉素诱导的γH2AX核模式。我们的结果还揭示了γH2AX灶在复制/非复制界面的重复定位，这可能反映了DNA损伤后短程染色质迁移。
