Booth Michael J, Balasubramanian Shankar
Department of Chemistry, University of Oxford, Oxford, UK.
Department of Chemistry, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2021;2272:3-12. doi: 10.1007/978-1-0716-1294-1_1.
The generation of tools to study mammalian epigenetics is vital to understanding normal biological function and to identify how it is dysregulated in disease. The well-studied epigenetic DNA modification 5-methylcytosine can be enzymatically oxidized to 5-formylcytosine (5fC) in vivo. 5fC has been demonstrated to be an intermediate in demethylation, but recent evidence suggests that 5fC may have an epigenetic function of its own. We have developed reduced bisulfite sequencing (redBS-seq), which can quantitatively locate 5fC bases at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), 5fC is converted to uracil, as happens to unmodified cytosine (C), and thus cannot be discriminated from C. However, in redBS-seq, a specific reduction of 5fC to 5-hydroxymethylcytosine (5hmC) stops this conversion, allowing its discrimination from C. 5fC levels are inferred by comparison of a redBS-Seq run with a BS-seq run.
开发用于研究哺乳动物表观遗传学的工具对于理解正常生物学功能以及确定其在疾病中如何失调至关重要。经过充分研究的表观遗传DNA修饰5-甲基胞嘧啶在体内可被酶氧化为5-甲酰基胞嘧啶（5fC）。5fC已被证明是去甲基化过程中的一个中间体，但最近的证据表明5fC可能具有其自身的表观遗传功能。我们开发了亚硫酸氢盐还原测序法（redBS-seq），它可以在基因组DNA中以单碱基分辨率定量定位5fC碱基。在亚硫酸氢盐测序法（BS-seq）中，5fC会像未修饰的胞嘧啶（C）一样被转化为尿嘧啶，因此无法与C区分开来。然而，在redBS-seq中，5fC特异性还原为5-羟甲基胞嘧啶（5hmC）会阻止这种转化，从而使其能够与C区分开来。通过将redBS-Seq运行结果与BS-seq运行结果进行比较来推断5fC水平。
