Department of Chemistry, Institute for Biophysical Dynamics, The University of Chicago, IL 60637, USA.
Cell. 2012 Jun 8;149(6):1368-80. doi: 10.1016/j.cell.2012.04.027. Epub 2012 May 17.
The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.
5-羟甲基胞嘧啶（5hmC）的研究受到缺乏在全基因组范围内以单碱基分辨率对其进行作图方法的阻碍。基于亲和纯化的方法不能精确地定位 5hmC，也不能准确地确定每个修饰位点的相对丰度。我们在此提出了一种全基因组方法，Tet 辅助亚硫酸氢盐测序（TAB-Seq），当与传统的亚硫酸氢盐测序结合使用时，可用于以碱基分辨率作图 5hmC，并定量测定 5hmC 和 5mC 的相对丰度。该方法在胚胎干细胞中的应用不仅证实了 5hmC 在哺乳动物基因组中的广泛分布，而且还揭示了 5hmC 位点的序列偏倚和链不对称性。我们观察到在转录因子结合位点附近但不在其附近有高水平的 5hmC 和相反的低水平的 5mC。此外，5hmC 的相对丰度在不同的功能序列元件之间差异显著，表明 5hmC 沉积和维持的机制不同。
