| DNA methylation is carried out by DNA methyltransferases. It is involved in gene expression, parental imprinting, X chromosome inactivation, and protection from retroviruses and transposons. While DNA methylation is essential for mammalian embryonic development and differentiation, alterations in genomic methylation patterns are involved in cancer initiation and progression. Hypermethylation of gene promoter CpG islands results in transcriptional silencing and loss of gene expression of tumor suppressor genes. In this dissertation, we use multiple techniques to examine the direct modification of DNA methyltransferase 1 (DNMT1) by nitric oxide. Using a novel HPLC/MS/MS technique we identify multiple cysteines, which are susceptible to S-nitrosylation including cysteine 41, cysteine 62, and cysteine 1001. Endogenous S-nitrosylation is detected in numerous cancer cell lines. Inhibition of nitric oxide synthases, which decrease nitric oxide levels, reduces global DNA methylation levels, decreases methylation of CpG-rich gene regulatory regions and relieves transcriptional silencing of certain tumor suppressor genes. Treatment of exogenous nitric oxide with nitric oxide synthase inhibitors rescues methylation levels. Incubation of purified recombinant DNMT1 with nitric oxide increases enzyme activity, whereas incubation of mutant DNMT1s---DNMT1-C41S, DNMT1-C62S, and DNMT1-C1001S---with nitric oxide does not modulate methyltransferase activity, which suggests that nitric oxide may regulate DNMT1 through direct and indirect methods. We hope these findings elucidate the mechanisms by which DNA methyltransferase modifications may lead to aberrant regulation of enzyme activity. |
