New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA.
J Biol Chem. 2011 Jul 15;286(28):24685-93. doi: 10.1074/jbc.M110.217083. Epub 2011 May 24.
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.
脊椎动物基因组中的胞嘧啶残基被酶修饰为 5-甲基胞嘧啶，它在发育和疾病进展过程中参与基因转录抑制。5-甲基胞嘧啶可以被 TET 家族的甲基胞嘧啶双加氧酶进一步酶修饰为 5-羟甲基胞嘧啶。5-甲基胞嘧啶和 5-羟甲基胞嘧啶的分析很复杂，因为即使通过亚硫酸氢盐转化进行补充，这些修饰也无法通过传统的测序方法区分。在这里，我们展示了一种简单的酶促方法，该方法涉及在小鼠和人类基因组中的各种 CCGG 基因座中克隆、鉴定和定量 5-羟甲基胞嘧啶。5-羟甲基胞嘧啶在人类和小鼠大脑和心脏基因组 DNA 的几个区域中普遍存在。培养的细胞系 NIH3T3 和 HeLa 在检测到的基因座处均显示出非常低或无法检测到的 5-羟甲基胞嘧啶。有趣的是，在分化为胚状体的过程中，小鼠胚胎干细胞 DNA 中的 5-羟甲基胞嘧啶水平先增加然后缓慢减少，而 5-甲基胞嘧啶水平随时间逐渐增加。最后，使用定量 PCR 方法，我们确定了人类组织 DNA 样本中 VANGL1 和 EGFR 基因体甲基化的一部分确实是羟甲基化。
