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.