Chandler Hollie, Patel Harshil, Palermo Richard, Brookes Sharon, Matthews Nik, Peters Gordon
Molecular Oncology Laboratory, Cancer Research UK London Research Institute, London, United Kingdom.
Bioinformatics and Biostatistics Service, Cancer Research UK London Research Institute, London, United Kingdom.
PLoS One. 2014 Jul 24;9(7):e102968. doi: 10.1371/journal.pone.0102968. eCollection 2014.
A growing body of evidence suggests that Polycomb group (PcG) proteins, key regulators of lineage specific gene expression, also participate in the repair of DNA double-strand breaks (DSBs) but evidence for direct recruitment of PcG proteins at specific breaks remains limited. Here we explore the association of Polycomb repressive complex 1 (PRC1) components with DSBs generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. Based on immunofluorescent staining, the co-localization of PRC1 proteins with components of the DNA damage response (DDR) in these primary cells is unconvincing. Moreover, using chromatin immunoprecipitation and deep sequencing (ChIP-seq), which detects PRC1 proteins at common sites throughout the genome, we did not find evidence for recruitment of PRC1 components to AsiSI-induced DSBs. In contrast, the S2056 phosphorylated form of DNA-PKcs and other DDR proteins were detected at a subset of AsiSI sites that are predominantly at the 5' ends of transcriptionally active genes. Our data question the idea that PcG protein recruitment provides a link between DSB repairs and transcriptional repression.
越来越多的证据表明，多梳蛋白家族（PcG）蛋白作为谱系特异性基因表达的关键调节因子，也参与DNA双链断裂（DSB）的修复，但PcG蛋白在特定断裂处直接募集的证据仍然有限。在这里，我们探讨了多梳抑制复合体1（PRC1）组分与正常人成纤维细胞中通过诱导表达AsiSI限制酶产生的DSB之间的关联。基于免疫荧光染色，在这些原代细胞中PRC1蛋白与DNA损伤反应（DDR）组分的共定位并不令人信服。此外，使用染色质免疫沉淀和深度测序（ChIP-seq）来检测全基因组常见位点的PRC1蛋白，我们没有发现PRC1组分募集到AsiSI诱导的DSB的证据。相反，在主要位于转录活性基因5'端的一部分AsiSI位点检测到了DNA-PKcs的S2056磷酸化形式和其他DDR蛋白。我们的数据对PcG蛋白募集在DSB修复和转录抑制之间建立联系这一观点提出了质疑。
