美国加利福尼亚大学的研究人员近日绘制出第一张人胚胎干细胞中5-羟甲基胞嘧啶（5hmC）的基因组分布图，发现5hmC集中在增强子以及基因本身，表明了5hmC在基因调控中的潜在作用。该研究结果发表在6月20日的《Genome Biology》上。
近两年，研究人员发现5hmC在某些细胞类型中大量存在，包括胚胎干细胞。同时，越来越多的证据表明，将5-甲基胞嘧啶（5mC）转化成5hmC的TET蛋白扮演着重要的生物角色。为了进一步了解5hmC的功能，研究人员着手分析它在基因组中的分布。
在这项研究中，研究人员使用了hmeDIP-seq技术，即先开展羟甲基DNA免疫沉淀，之后在Illumina Genome Analyzer平台上进行大规模并行测序。在这之前，人们富集5hmC主要是通过化学反应添加标签，再亲和纯化。而现在，5hmC特异抗体的上市，使得5hmC的直接免疫沉淀成为可能。他们使用的是Active Motif和Diagenode这两家公司的抗体。
通过数据分析，研究人员发现5hmC主要集中在增强子以及基因本身，表明了5hmC在基因调控中的潜在作用。与它在增强子中的定位一致，5hmC还大量集中在与增强子相关的组蛋白修饰位点，如H3K4me1和H3K27ac。同时，5hmC还集中在其他的蛋白-DNA相互作用位点，如OCT4和NANOG结合位点。
此结果与之前报道的小鼠胚胎干细胞中5hmC的基因组分布相似。据5月份的《Nature》杂志报道，波士顿儿童医院的研究人员发现，5hmC片段主要集中在外显子和转录起始位点附近，尤其是H3K27me3和H3K4me3位点。
同时，研究人员还发现5hmC区域富含GC，但GC含量有差异。在5’端，G多过C，而3’端刚好相反，C多过G。这种现象在与基因和增强子重叠的5hmC区域以及未与上述元件重叠的区域都观察到，表明这种序列组成是所有5hmC区域的共同特征。
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5-hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells
Abstract
Background
5-Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types, including embryonic stem cells. There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.
Results
Here, we have generated a genome-wide map of 5hmC in human embryonic stem cells by hmeDIP-seq, in which hydroxymethyl-DNA immunoprecipitation is followed by massively parallel sequencing. We found that 5hmC is enriched in enhancers as well as in gene bodies, suggesting a potential role for 5hmC in gene regulation. Consistent with localization of 5hmC at enhancers, 5hmC was significantly enriched in histone modifications associated with enhancers, such as H3K4me1 and H3K27ac. 5hmC was also enriched in other protein-DNA interaction sites, such as OCT4 and NANOG binding sites. Furthermore, we found that 5hmC regions tend to have an excess of G over C on one strand of DNA.
Conclusions
Our findings suggest that 5hmC may be targeted to certain genomic regions based both on gene expression and sequence composition.
