Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Oslo University Hospital, Rikshospitalet, Norway.
Nucleic Acids Res. 2011 Apr;39(8):e55. doi: 10.1093/nar/gkr051. Epub 2011 Feb 7.
Recently, 5-hydroxymethylcytosine (5hmC) was identified in mammalian genomic DNA. The biological role of this modification remains unclear; however, identifying the genomic location of this modified base will assist in elucidating its function. We describe a method for the rapid and inexpensive identification of genomic regions containing 5hmC. This method involves the selective glucosylation of 5hmC residues by the β-glucosyltransferase from T4 bacteriophage creating β-glucosyl-5-hydroxymethylcytosine (β-glu-5hmC). The β-glu-5hmC modification provides a target that can be efficiently and selectively pulled down by J-binding protein 1 coupled to magnetic beads. DNA that is precipitated is suitable for analysis by quantitative PCR, microarray or sequencing. Furthermore, we demonstrate that the J-binding protein 1 pull down assay identifies 5hmC at the promoters of developmentally regulated genes in human embryonic stem cells. The method described here will allow for a greater understanding of the temporal and spatial effects that 5hmC may have on epigenetic regulation at the single gene level.
最近，5-羟甲基胞嘧啶（5hmC）在哺乳动物基因组 DNA 中被鉴定出来。这种修饰的生物学作用尚不清楚；然而，确定该修饰碱基的基因组位置将有助于阐明其功能。我们描述了一种快速且廉价的鉴定含有 5hmC 的基因组区域的方法。该方法涉及 T4 噬菌体的β-葡萄糖基转移酶对 5hmC 残基的选择性葡糖基化，产生β-葡萄糖基-5-羟甲基胞嘧啶（β-glu-5hmC）。β-glu-5hmC 修饰提供了一个靶标，可以通过与磁珠偶联的 J 结合蛋白 1 有效地选择性下拉。沉淀的 DNA 适合进行定量 PCR、微阵列或测序分析。此外，我们证明 J 结合蛋白 1 下拉测定法可以鉴定人胚胎干细胞中发育调节基因启动子上的 5hmC。这里描述的方法将有助于更好地理解 5hmC 在单个基因水平上对表观遗传调控的时间和空间影响。
