Increased DNA Methyltransferase 3b (Dnmt3b)-Mediated CpG Island Methylation Stimulated by Oxidative Stress Inhibits Expression of a Gene Required for Neural Tube and Neural Crest Development in Diabetic Pregnancy

Previous studies have shown that diabetic embryopathy results from impaired expression of genes that are required for formation of embryonic structures. We have focused on Pax3, a gene that is expressed in embryonic neuroepithelium and is required for neural tube closure. Pax3 expression is inhibited in embryos of diabetic mice due to hyperglycemia-induced oxidative stress. DNA methylation silences developmentally expressed genes before differentiation. We hypothesized that hypomethylation of Pax3 upon neuroepithelial differentiation may be inhibited by hyperglycemia-induced oxidative stress. We tested this using embryos of pregnant hyperglycemic mice and mouse embryonic stem cells (ESC). Methylation of a Pax3 CpG island decreased upon neurulation of embryos and formation of neuronal precursors from ESC. In ESC, this was inhibited by oxidative stress. Use of short hairpin RNA in ESC demonstrated that DNA methyltransferase 3b (Dnmt3b) was responsible for methylation and silencing of Pax3 before differentiation and by oxidative stress. Although expression of Dnmt3b was not affected by oxidative stress, DNA methyltransferase activity was increased. These results indicate that hyperglycemia-induced oxidative stress stimulates Dnmt3b activity, thereby inhibiting chromatin modifications necessary for induction of Pax3 expression during neurulation and thus providing a molecular mechanism for defects caused by Pax3 insufficiency in diabetic pregnancy.