Nitric oxide inhibits ten-eleven translocation DNA demethylase activity to regulate 5mC and 5hmC across the genome [RNA-seq]

DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and Human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom in place of O2 to form a dinitrosyliron complex (DNIC). In cancer cells treated with exogeneous NO, or cells endogenously synthesizing NO, there was a global increase in DNA 5mC, a substrate for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing mouse xenograft and patient derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC at gene-regulatory loci which correlated to the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO-signaling and represents a novel epigenetic regulatory mechanism of NO.