Congenital Heart Defect-Associated Enhancers Shape Human Cardiomyocyte Lineage Commitment

Advancements in whole genome sequencing have identified thousands of disease-associated variants which land within enhancer boundaries. As enhancers play critical roles in orchestrating gene networks throughout development, variants which disrupt enhancer function have been shown to contribute to developmental defects. However studying enhancer variants within a developmental context has been limited by a few key challenges. First, thousands of enhancer variants have been identified which could be causal for disease. Thus, a high-throughput approach is necessary to feasibly interrogate these elements. Second, enhancers function in a cell-type specific and spatiotemporal manner to regulate target gene expression. Perturbation of these enhancers thus requires an in vitro model that can phenocopy the lineage and context in which they are active. Addressing these points, I first identify 25 putative cardiac enhancers harboring variants identified in patients with congenital heart defects (CHD). Using a CRISPRi repression system, I perturb these putative enhancers in human embryonic stem cells (hESC) followed by differentiation towards cardiomyocytes (CM). This allows for the study of enhancer activity throughout the specification of the vital muscle cells of the cardiac system. I then perform single-cell RNA sequencing to identify diverse CM cell populations and assess the impact of enhancer perturbations on lineage specification. My analysis revealed 16 enhancers of known cardiac genes which, when perturbed, result in deficient CM differentiation. Genetic knockouts of two enhancers near TBX5 phenocopied the single-cell data and revealed enrichment of early CM populations resulting from depletion of later stages. My thesis provides a framework for single-cell enhancer screens within a developmental context and provides support for the biological relevance of the approach. I expect that the throughput of this methodology and the ease at which it can be adapted towards diverse developmental systems will provide an invaluable tool for future studies.