Changes in DNA methylation by exposure to nitric oxide

The enzyme DNA methyltransferase (DNMT) catalyzes the methylation of the 5-carbon (C5) position of cytosine in mammals. DNA Methylation regulates gene transcription and serves a variety of physiological and pathophysiological roles. However, the chemical mechanisms regulating DNMT enzyme activity remained largely unknown. In this study, we investigated if nitrosative stress affects the DNA methylation patterns in cells. Initially, HeLa cells were treated with an NO donor for 1-3 h or transduced with NOS2 for 48h. Genomic DNA was isolated from HeLa cell and then fragmented. Targeted bisulfite sequencing libraries were prepared, followed by 101 bp paired-end sequencing on a NextSeq550 system. Ratio of the number of sequenced methylated cytosine reads to the total number of reads for each locus was evaluated.DNA甲基转移酶（DNA methyltransferase，DNMT）可催化哺乳动物胞嘧啶5位碳（C5）的甲基化修饰。DNA甲基化能够调控基因转录，并参与多种生理及病理生理过程。然而，调控DNMT酶活性的化学机制迄今仍未完全阐明。本研究旨在探究亚硝基化应激是否会影响细胞内的DNA甲基化模式。首先，我们将HeLa细胞经一氧化氮供体（NO donor）处理1~3小时，或用NOS2（nitric oxide synthase 2）转导48小时。随后从HeLa细胞中提取基因组DNA并进行片段化处理。构建靶向亚硫酸氢盐测序文库，随后在NextSeq550测序系统上开展101 bp双端测序。对每个基因组位点的甲基化胞嘧啶测序读段数与总读段数的比值进行了评估分析。