Fukuzawa Seketsu, Takahashi Saori, Tachibana Kazuo, Tajima Shoji, Suetake Isao
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; CREST, Japan Agency for Medical Research and Development (AMED), 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Bioorg Med Chem. 2016 Sep 15;24(18):4254-4262. doi: 10.1016/j.bmc.2016.07.016. Epub 2016 Jul 11.
Oxidation of 5-methylcytosine (5mC) is catalyzed by ten-eleven translocation (TET) enzymes to produce 5-hydroxymethylcytosine (5hmC) and following oxidative products. The oxidized nucleotides were shown to be the intermediates for DNA demethylation, as the nucleotides are removed by base excision repair system initiated by thymine DNA glycosylase. A simple and accurate method to determine initial oxidation product 5hmC at single base resolution in genomic DNA is necessary to understand demethylation mechanism. Recently, we have developed a new catalytic oxidation reaction using micelle-incarcerated oxidants to oxidize 5hmC to form 5-formylcytosine (5fC), and subsequent bisulfite sequencing can determine the positions of 5hmC in DNA. In the present study, we described the optimization of the catalytic oxidative bisulfite sequencing (coBS-seq), and its application to the analysis of 5hmC in genomic DNA at single base resolution in a quantitative manner. As the oxidation step showed quite low damage on genomic DNA, the method allows us to down scale the sample to be analyzed.
5-甲基胞嘧啶（5mC）的氧化由10-11易位（TET）酶催化，生成5-羟甲基胞嘧啶（5hmC）及后续氧化产物。氧化后的核苷酸被证明是DNA去甲基化的中间体，因为这些核苷酸会被胸腺嘧啶DNA糖基化酶启动的碱基切除修复系统去除。为了理解去甲基化机制，需要一种简单准确的方法来在单碱基分辨率下测定基因组DNA中的初始氧化产物5hmC。最近，我们开发了一种新的催化氧化反应，使用胶束包裹的氧化剂将5hmC氧化形成5-甲酰基胞嘧啶（5fC），随后的亚硫酸氢盐测序可以确定DNA中5hmC的位置。在本研究中，我们描述了催化氧化亚硫酸氢盐测序（coBS-seq）的优化，及其在单碱基分辨率下以定量方式分析基因组DNA中5hmC的应用。由于氧化步骤对基因组DNA的损伤相当低，该方法使我们能够缩小待分析样本的规模。
