Çelik Selcen, Li Yan, O'Neill Chris
Human Reproduction Unit, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney 2065, Australia.
Human Reproduction Unit, Kolling Institute for Medical Research, Sydney Medical School, University of Sydney, Sydney 2065, Australia.
Exp Cell Res. 2015 Nov 15;339(1):20-34. doi: 10.1016/j.yexcr.2015.08.017. Epub 2015 Sep 26.
The methylation of cytosine at CpG dinucleotides (5 meC) is an important epigenetic mechanism that governs genome stability and gene expression. Important ontological and pathological transitions are associated with marked global changes in detectable levels of methylation. We have previously found two pools of immune-detectable 5 meC exist within cells, a pool that can be detected after acid treatment of fixed cells to denature chromatin and another large but variable pool that requires a further tryptic digestion step for complete epitope retrieval. The trypsin-sensitive pool has been shown to be largely associated with the heterochromatic fraction (by a heterochromatin marker, HP1-β) of the genome, and the size of this pool varies with the growth disposition of cells. Since DNA damage imposes large changes on chromatin structure the present study analyzed how such changes influences the faithful immunological detection of 5 meC within mouse embryonic fibroblasts. DNA damage was induced by either UV-irradiation or doxorubicin treatment, each of which resulted in increased levels of immune-detectable 5 meC at 24-48 h after treatment. There was a marked trypsin-sensitive pool of 5 meC in these cells which was significantly increased after DNA damage. The increased levels of 5 meC staining predominantly co-located with heterochromatic foci within nuclei, as assessed by HP1-β staining. The relative amount of masked 5 meC after DNA damage was positively associated with increased levels of HP1-β. The methyl binding protein, MBD1, was a less reliable measure of changes in 5 meC, with a significant fraction of 5 meC not being marked by MBD1. The cyto-epigenetic approaches used here reveal dynamism in the levels and localization of immune-detectable 5 meC within the nuclei of fibroblasts in response to DNA damage.
CpG二核苷酸处胞嘧啶的甲基化（5-甲基胞嘧啶）是一种重要的表观遗传机制，它控制着基因组稳定性和基因表达。重要的本体和病理转变与可检测的甲基化水平的显著全局变化相关。我们之前发现细胞内存在两群可通过免疫检测到的5-甲基胞嘧啶，一群是在对固定细胞进行酸处理使染色质变性后可检测到的，另一大群但数量可变的群体则需要进一步的胰蛋白酶消化步骤以完全检索表位。已证明对胰蛋白酶敏感的群体在很大程度上与基因组的异染色质部分（通过异染色质标记物HP1-β）相关，并且该群体的大小随细胞的生长状态而变化。由于DNA损伤会对染色质结构造成巨大变化，本研究分析了此类变化如何影响小鼠胚胎成纤维细胞内5-甲基胞嘧啶的可靠免疫检测。通过紫外线照射或阿霉素处理诱导DNA损伤，每种处理在处理后24至48小时均导致可通过免疫检测到的5-甲基胞嘧啶水平升高。这些细胞中存在明显的对胰蛋白酶敏感的5-甲基胞嘧啶群体，DNA损伤后其显著增加。通过HP1-β染色评估，5-甲基胞嘧啶染色水平的增加主要与细胞核内的异染色质灶共定位。DNA损伤后被掩盖的5-甲基胞嘧啶的相对量与HP1-β水平的增加呈正相关。甲基结合蛋白MBD1对5-甲基胞嘧啶变化的测量不太可靠，很大一部分5-甲基胞嘧啶未被MBD1标记。此处使用的细胞表观遗传学方法揭示了成纤维细胞核内可通过免疫检测到的5-甲基胞嘧啶水平和定位在响应DNA损伤时的动态变化。
