Project Summary/ Abstract The development of heart is a complicated developmental process that requires precise and coordinated spatial and temporal regulation in gene transcription. Epigenetic factors, such as the Ten-eleven Translocation (TET) family of dioxygenase that catalyzes 5-methyocytosine (5mC) oxidation, play indispensable roles in orchestrating the cardiac transcription network during normal heart development. Tet depletion has been shown by others and us to block the differentiation of embryonic stem cells toward cardiac progenitor ex vivo. The team has further established that cardiac specific Tet deletion could result in noncompaction cardiomyopathy (NCC) due to dysregulation of chromatin accessibility and impaired long-range enhancer-promoter looping that disrupts the transcription of genes important for cardiac development. More recently, the team further discovered that Tet deficient embryonic heart tissues exhibited abnormal expression of genes that are important for lipid metabolism, which might also count for developmental defects observed in cardiac specific Tet-deficient mouse models. Despite the knowledges obtained from these studies, two outstanding questions remain unresolved: (i) how Tet proteins acquire their specificity to precisely regulate lineage-specific enhancers; and (ii) whether and how prenatal maternal metabolic stressors influence TET-related epigenetic pathways and impact cardiac development in the progeny. In this research program, the PI will use both unique genetically modified mouse models and human embryonic stem cells differentiated toward the cardiac lineage to address these questions. In project 1, the team will investigate the pathogenic mechanisms regarding how newly discovered TET2 mutations in patients with congenital heart diseases (CHD) impair heart development and cardiac lineage specification. In project 2, the team will unravel molecular mechanisms undergirding TET-mediated enhancer recognition specificity during heart development. In project 3, the team will illuminate how prenatal maternal high fat diet as a stressor effects Tet mediated epigenetic regulation to perturb cardiac development. From a translational perspective, we will test the use of vitamin C to boost Tet activity and reduce the risk of developmental defects related to maternal high fat-diet exposure. The proposed fundamental studies are necessary to promote healthy cardiac development and illuminate previously underappreciated mechanisms underpinning CHDs and cardiovascular diseases. This proposal will also lend insight into how external metabolites influence epigenetic landscapes during heart development and how metabolic factors can be harnessed to rectify epigenetic abnormalities to restore normal cardiac development.