5-羟甲基胞嘧啶
达皮
化学
染色质
DNA
常染色质
免疫荧光
细胞生物学
组蛋白
核小体
5-甲基胞嘧啶
分子生物学
表观遗传学
DNA甲基化
生物
异染色质
基因表达
抗体
生物化学
基因
遗传学
细胞凋亡
作者
Shangwei Zhong,Zhe Li,Ting Jiang,Xiangjun Li,Hailin Wang
标识
DOI:10.1021/acs.analchem.7b01428
摘要
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.
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