School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences , Beijing 100049, China.
State Key Laboratory of Environmental Chemistry and Ecotoxicoogy, Research Center for Eco-Environmental Sciences , Beijing 100085, China.
Anal Chem. 2017 Jun 6;89(11):5702-5706. doi: 10.1021/acs.analchem.7b01428. Epub 2017 May 22.
DNA 5-hydroxymethylcytosine (5hmC) is an important epigenetic modification found in various mammalian cells. Immunofluorescence imaging analysis essentially provides visual pictures for the abundance and distribution of DNA 5hmC in single cells. However, nuclear DNA is usually wrapped around nucleosomes, packaged into chromatins, and further bound with many functional proteins. These physiologically relevant events would generate barriers to the anti-5hmC antibody to selectively recognize 5hmC in DNA. By taking advantage of these naturally generated barriers, here, we present a strategy to evaluate the accessibility of DNA 5hmC in chromatins in situ. We demonstrate that a few of the 5hmC sites in DNA are exposed or accessible to anti-5hmC antibody under nondenaturing conditions, suggesting that these 5hmC sites are not covered by functional DNA-binding proteins in mouse embryonic stem cells. Consistently, these 5hmC foci were distributed in open euchromatin regions as revealed by the 4',6-diamidino-2-phenylindole (DAPI) staining. By overexpressing TET1 catalytic domain (responsible for oxidation 5mC to produce 5hmC) in human MCF-7 cells, we observed a significant increase in accessible 5hmC along with an increase in total 5hmC sites. Collectively, by the use of the nondenaturing immunofluorescence imaging approach, we could obtain a visual landscape on the accessibility of DNA 5hmC in chromatins.
DNA 5-羟甲基胞嘧啶（5hmC）是各种哺乳动物细胞中一种重要的表观遗传修饰。免疫荧光成像分析基本上为单个细胞中 DNA 5hmC 的丰度和分布提供了直观的图片。然而，核 DNA 通常围绕核小体缠绕，包装成染色质，并进一步与许多功能蛋白结合。这些与生理相关的事件会为抗 5hmC 抗体对 DNA 中 5hmC 的选择性识别制造障碍。利用这些天然产生的障碍，我们提出了一种策略来评估染色质中 DNA 5hmC 的可及性。我们证明，在非变性条件下，DNA 中的几个 5hmC 位点暴露或可被抗 5hmC 抗体识别，这表明这些 5hmC 位点在小鼠胚胎干细胞中没有被功能 DNA 结合蛋白覆盖。一致地，这些 5hmC 焦点分布在开放的常染色质区域，如 4'，6-二脒基-2-苯基吲哚（DAPI）染色所揭示的。通过在人 MCF-7 细胞中转染 TET1 催化结构域（负责将 5mC 氧化生成 5hmC），我们观察到可及性 5hmC 随着总 5hmC 位点的增加而显著增加。总之，通过使用非变性免疫荧光成像方法，我们可以获得染色质中 DNA 5hmC 可及性的直观图谱。
