Congenital malformations are significantly increased in the offspring of women with pregestational diabetes. Oxidative stress, resulting from excessive glucose metabolism by the embryo, early during embryonic development, is responsible for these malformations. We have shown that oxidative stress inhibits expression of genes in the embryo that control essential developmental processes. In particular, expression of Pax3, which is required for neural tube and neural crest development, is significantly reduced in embryos of diabetic mice, and antioxidants block the inhibition of Pax3 as well as the associated neural tube and neural crest defects. While several of the biochemical processes leading to oxidative stress in embryos of diabetic mothers have been determined, it is not known how oxidative stress disrupts embryo gene expression. The central hypothesis in this proposal is that epigenetic regulation of Pax3 that is associated with increasing aerobic metabolism during early embryonic development is abrogated by oxidative stress resulting from maternal hyperglycemia. This hypothesis is based upon the recognition that fuel metabolism by the early embryo is highly glycolytic and becomes more oxidative as cells differentiate, and that genes that are expressed in the early embryo are dependent upon chromatin modifications. There are three Specific Aims: (1) Test whether oxidative stress blocks epigenetic modifications of the Pax3 transcription regulatory element that are necessary for differentiation-induced gene expression. (2) Test whether mediators of chromatin modifications, which are responsive to changes in oxidation or cellular redox status, change with differentiation, whether they are inhibited by oxidative stress, and whether they regulate Pax3 expression. (3) Test whether AMP-activated protein kinase (AMPK), which is activated in embryos and embryonic stem cells (ESC) in response to hypoxia and oxidative stress, mediates the effects of oxidative stress to block differentiation- induced chromatin modifications. Our approach is to study histone methylation and acetylation and DNA methylation in ESC, and Pax3 expression in mouse embryos and in ESC. ESC will be induced to differentiate into neuronal precursors, and oxidative stress will be induced in ESC or embryos, and the effects of stimulating or inhibiting pathways that potentially modify chromatin in response to oxidative stress will be examined. The proposed study is significant because it will determine the mechanisms by which abnormal fuel metabolism caused by maternal diabetes disrupts the activation of embryonic gene expression, thereby leading to malformations. This knowledge may lead to new strategies to normalize embryo gene expression during diabetic pregnancy. This proposal is innovative because the concept that early embryonic gene expression is developmentally regulated by signals generated by fuel metabolism that alter chromatin structure has not previously been investigated.
The risk for congenital malformation is significantly increased when a mother has diabetes (either type 1 or type 2). In this proposal, we will test the hypothesis that free radicals, produced by excess glucose metabolism, interfere with modifications of DNA and of DNA-binding proteins, called histones, which are necessary for proper activation of embryo gene expression. The results obtained from these experiments may lead to the development of new treatments to prevent congenital malformations during diabetic pregnancy.
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