Study of epigenetic regulation of the embryonic gene, PAX3, by diabetic pregnancy-induced oxidative stress

Abstract

Maternal diabetes effects on congenital malformations are due to hyperglycemia-induced oxidative stress, and that oxidative stress inhibits expression of genes that are essential for early organogenesis in the embryo. In particular, research team from Joslin Diabetes Center_Harvard Medical School has shown that oxidative stress inhibits Pax3 expression, which encodes a protein that is required for closure of the neural tube and septation of cardiac outflow tracts. We hypothesize that during normal embryogenesis, increasing oxidative metabolism is coupled to Pax3 induction, in part due to chromatin modifications (histone methylation and acetylation, and DNA methylation) that permit Pax3 transcription, and that excessive free radical production due to hyperglycemia prevents the normal chromatin modifications during differentiation. Thus, this project intends to study chromatin modifications in embryos before and after Pax3 induction (E3.5 and E8.5), and in E8.5 embryos during diabetic pregnancy or transient induction of oxidative stress, and in embryonic stem cells (ESC). This period will study due to that ESC may grow as undifferentiated cells (similar to the inner cell mass of E3.5 embryos) or as neuronal precursors (similar to E8.5 embryos). We will test this hypothesis using embryos and embryonic stem cells (ESC) from laboratory animals as a cell culture model of differentiating neuroepithelium. If the hypothesis is correct, we expect to see epigenetic modifications associated with gene silencing in blastocyst and undifferentiated ESC, and epigenetic modifications associated with gene expression in E8.5 embryos and differentiating ESC; furthermore, we expect that oxidative stress will inhibit differentiation-induced epigenetic modifications and that antioxidants will prevent the oxidative stress effects. After the first experiments, all steps of the methodology will be detailed by the student in scientific report. Furthermore, the fellow will be able to perform these techniques and to explain to other undergraduate and graduate in Brazil. (AU)