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Enhancers drive tissue specific expression, and cardiovascular (CV) enhancers are poorly conserved between humans and other species. Therefore, it is important to define enhancer function in defined human CV lineages. The hypothesis of our Main Project is that enhancers operate dynamically in a temporal and/or lineage specific manner during cardiomyocyte (CM) specification, and that mutation of enhancers driving expression of CHD relevant genes will disrupt CM lineage development. Aims are: 1) To investigate the transcriptional profile and genome-wide epigenetic landscape of human ventricular and atrial CM lineages during development; 2) To investigate enhancer-promoter interactions that regulate ventricular and atrial gene programs; 3) To examine effects of enhancer mutation on expression of cognate CHD relevant transcription factors and CM lineage development in vitro and in vivo. The Main Project will provide a CV epigenomic regulatory framework for interrogation of PDVs in noncoding regions, as proposed in the Collaborative Project. The hypothesis of our Collaborative Project is that many genetically unexplained CHDs will be due to PDVs which are located in non-coding sequences and perturb target gene expression by altering functions of cis-regulatory elements, thus leading to an overall disruption of pathways critical for normal cardiac lineage development. Aims are: 1) To identify a comprehensive set of candidate CHD non-coding PDVs through systematic analysis of Pediatric Cardiac Genetics Center (PCGC) CHD Whole Genomic Sequencing studies with Cardiovascular Development Center (CvDC) lineage specific human epigenomic datasets; 2) To investigate whether candidate CHD non-coding variants perturb enhancer activity using multiple highthroughput in vitro assays; 3) To investigate the functional significance of high confidence regulatory CHD variants in vitro and in vivo. All aims will utilize hESC models of CV lineage differentiation, and Aim 3 of each project will additionally utilize mouse models. We will establish pipelines of genomewide and highthroughput functional assays, many available through a UCSD Genomics Core. Results will be compared to patient data to investigate congruence of biochemical and morphological/physiological phenotypes. These studies represent collaboration between multiple PIs of complementary expertise, including members of the CvDC and the PCGC (see attached letters), representing an ideal training environment for junior scientists in collaborativ science. 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