PROJECT ABSTRACT Members of SET and MYND domain-containing protein (SMYDs) family are unique lysine methyltransferases that play important roles during embryogenesis via the regulation of histone H3 lysine residues that impart both transcriptional activation and repression functions. SMYD1 is known for its function in regulating cardiomyocyte differentiation and maturation. Our previous study on the role of SMYD4 in Zebrafish suggesting that smyd4 is critical to early embryonic development and cardiogenesis. Our recent transcriptomic analysis of smyd4-ablated zebrafish blastula and early gastrula embryos has revealed significant changes in gene regulatory networks involved in the specification of mesendoderm and the differentiation of cardiac progenitor cells (CPCs). These data suggest that smyd4 may be involved in cardiogenic-lineage specification and differentiation prior to overt heart formation. Importantly, we demonstrate that de novo SMYD4 genetic variants are associated with congenital heart defects (CHDs). To establish the mechanistic basis for the genetic association of SMYD4 to CHDs, we have analyzed the temporal expression pattern of SMYD4 within human embryonic stem cells (hESCs) and their induced differentiation into cardiomyocytes. We find that SMYD4 is expressed throughout the differentiation process from hESCs to cardiomyocytes. Genetic ablation of SMYD4 in hESCs (hESC-SMYD4KO) resulted in dramatically compromised mesodermal specification and CPC differentiation, thus phenocopying the early developmental and cardiac defects observed in smyd4 mutant zebrafish. Additional transcriptomic analysis further supports that induced cardiomyogenic differentiation of hESCs-SMYD4KO is severely altered, where hESCs-SMYD4KO adopt a more neuroectodermal lineage fate. Quantitative mass spectrometry analysis reveals that BACH1, a critical transcriptional regulator of stem cell pluripotency and lineage specification, as a strong SMYD4 binding partner. Based on these preliminary data, we hypothesize that SMYD4 functions as a critical epigenetic regulator for mesendoderm specification and cardiac development via its interaction with BACH1. In this project, we propose 3 specific aims to define the biological and molecular function of SMYD4 in mesendoderm specification and cardiac development. Aim 1 tests the hypothesis that SMYD4 is critical to mesoderm specification and cardiogenic differentiation by performing genetic rescue experiment and a series of genome-wide integrated analyses, including the single-cell RNA-seq and DNA occupancy analyses, to define the potentially altered histone epigenetic landscape during cardiomyocyte differentiation in mutant hESCs. Aim 2 tests the hypothesis that BACH1-SMYD4 complexes are critical to the epigenetic regulation of mesoderm specification and cardiogenic differentiation by determining the protein-protein interaction domains and BACH1- SMYD4 specific targeted genes using CUT&RUN analysis. Aim 3 tests the hypot...