Poster #50 - Pooja Sonawane

Characterizing Regulatory Regions of Key Cardiac Transcription Factors Using Lentiviral Based Massively Parallel Reporter Assays

Pooja Sonawane1,2, Justin Cotney2,3 Affiliations 1 University of Connecticut Health Center, Farmington, CT 2 Department of Surgery, Children's Hospital of Philadelphia, Philadelphia, PA 3 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

Congenital Heart Defects (CHDs) are the most prevalent form of structural birth defects, affecting approximately 1% of live births worldwide. While genetic factors are strongly implicated in CHD etiology, underlying causes remain largely unknown, with 60% of cases lacking a defined explanation. One prevailing hypothesis is that noncoding changes in gene regulatory sequences contribute to these undiagnosed cases. Whole-genome sequencing of CHD trios suggests that de novo noncoding variants may be enriched in cardiac regulatory regions. However, among the limited number of regulatory sequences functionally interrogated to date, target genes and extent of enhancer activity remain unclear. To address this, a comprehensive understanding of noncoding regulatory landscape during heart development is essential. Our previous work profiled gene expression and chromatin states from 4 to 8 weeks post-conception, identifying over 150,000 putative enhancers near genes active during cardiac organogenesis. However, the functional potential of these enhancers has not yet been systematically evaluated. To meet the scale of this challenge, we utilize lentiviral-based massively parallel reporter assays (LentiMPRA), which enable high-throughput functional testing of candidate regulatory elements in a chromatin-integrated context and can be tested in diverse cell types. We have assayed 1200 candidate cis-regulatory elements (CREs) within a one megabase window around key cardiac transcription factors including TBX5, TBX20, NKX2-5, NKX2-6, HAND1, HAND2, ERBB4, ZEB1, GATA4 and GATA6. These were tested in embryonic stem cell-derived cardiomyocytes (ESC-CMs), revealing that most cardiac enhancers exhibited higher activity in ESC-CMs compared to undifferentiated ESCs, whereas enhancers near pluripotency genes such as POU5F1 and SOX2 showed higher activity in ESCs. Of the total enhancers tested, about 45% of the CREs were active in ESC-CMs. Together, these studies demonstrate that LentiMPRA in ESC-derived cardiomyocytes provides a scalable framework to functionally evaluate human cardiac enhancers and their variants, advancing our understanding of the regulatory basis of CHDs.