Role of Polycomb group proteins in the DNA damage response – a reassessment. Homo sapiens

We have investigated whether components of Polycomb repressive complex 1 (PRC1) are recruited to double-strand breaks (DSBs) generated by inducible expression of the AsiSI restriction enzyme in normal human fibroblasts. 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 DNA repair 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. Overall design: Single chromatin preparations from treated and untreated cells (+/- OHT), immunoprecipitated with two antibodies (pDNA-PKcs and MEL18) were sequenced as technical replicates, along with the corresponding input DNAs

本研究以正常人类成纤维细胞为研究对象，探究多梳阻遏复合体1（Polycomb repressive complex 1, PRC1）的组分是否会被招募至由限制性内切酶AsiSI诱导表达所产生的双链断裂（double-strand breaks, DSBs）位点。本研究采用染色质免疫共沉淀联合高通量测序（chromatin immunoprecipitation and deep sequencing, ChIP-seq）技术——该技术可在全基因组普遍存在的位点检测PRC1蛋白——未检测到PRC1组分被招募至AsiSI诱导产生的DSBs位点的相关证据。与之相反，DNA依赖蛋白激酶催化亚基（DNA-PKcs）的S2056磷酸化形式以及其他DNA修复蛋白，可在部分AsiSI切割位点被检测到，这些位点主要位于转录活跃基因的5'端。本研究数据对"多梳家族蛋白招募可介导双链断裂修复与转录抑制之间的关联"这一假说提出了质疑。
实验设计概述：分别从经处理与未处理的细胞（±OHT）中制备单份染色质样品，使用两种抗体（pDNA-PKcs与MEL18）进行免疫沉淀，将免疫沉淀产物与对应的输入DNA一同作为技术重复进行测序。