Tissue-specific distribution and dynamic changes of 5-hydroxymethylcytosine in mammalian genomes.
Tissue-specific distribution and dynamic changes of 5-hydroxymethylcytosine in mammalian genomes.
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DOI：
10.1074/jbc.m110.217083
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发表时间：
2011-07-15
期刊：
影响因子：
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通讯作者：
Pradhan S
中科院分区：
文献类型：
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作者：
Kinney SM;Chin HG;Vaisvila R;Bitinaite J;Zheng Y;Estève PO;Feng S;Stroud H;Jacobsen SE;Pradhan S
脊椎动物基因组中的胞嘧啶残基被酶促修饰为5-甲基胞嘧啶，其在发育和疾病进展期间参与基因的转录抑制。5-甲基胞嘧啶可以通过甲基胞嘧啶双加氧酶的泰特家族进一步酶促修饰为5-羟甲基胞嘧啶。5-甲基胞嘧啶和5-羟甲基胞嘧啶的分析是混淆的，因为这些修饰通过传统的测序方法是不可区分的，即使当通过亚硫酸氢盐转化补充时。在这里，我们展示了一个简单的酶的方法，涉及克隆，鉴定和定量的5-羟甲基胞嘧啶在各种CCGG基因座内的小鼠和人类基因组。5-羟甲基胞嘧啶普遍存在于人类和小鼠脑和心脏基因组DNA的几个区域。培养的细胞系NIH 3 T3和HeLa都显示出非常低或检测不到的5-羟甲基胞嘧啶的量在检查的位点。有趣的是，小鼠胚胎干细胞DNA中的5-羟甲基胞嘧啶水平在分化为胚状体后首先增加，然后缓慢下降，而5-甲基胞嘧啶水平随着时间的推移逐渐增加。最后，使用定量PCR方法，我们确定了人类组织DNA样本中VANGL 1和EGFR基因体甲基化的一部分确实是羟甲基化。
Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.