Disruptions in cardiac development cause congenital heart disease, the most prevalent and deadly congenital malformation. Genetic and environmental factors are thought to contribute to these defects, however molecular mechanisms remain largely undefined. Recent work highlighted potential roles of chromatin- modifying enzymes in congenital heart disease pathogenesis. Histone deacetylases, a class of chromatin-modifying enzymes, have developmental importance and recognized roles in the mature heart. This thesis aimed to characterize functions of Hdac3 in cardiac development. We found loss of Hdac3 in the primary heart field causes precocious progenitor cell differentiation, resulting in hypoplastic ventricular walls, ventricular septal defect, and mid- gestational lethality. In primary heart field progenitors, Hdac3 interacts with, deacetylates, and functionally suppresses transcription factor Tbx5. Furthermore, a disease-associated Tbx5 mutation disrupts this interaction, rendering Tbx5 hyperacetylated and hyperactive. By contrast, deletion of Hdac3 in second heart field progenitors bypasses these defects, instead causing malformations in the outflow tract and semilunar valves, with lethality prior to birth. Affected semilunar valves and outflow tract vessels exhibit extracellular matrix and EndMT defects and activation of the Tgfβ1 signaling pathway. In normal second heart field development, Hdac3 represses Tgfβ1 transcription, independent of its deacetylase activity, by recruiting the PRC2 methyltransferase complex to methylate the Tgfβ1 promoter. Importantly, knockouts of Hdac3 in differentiated cardiac cells do not fully recapitulate the progenitor-specific knockout phenotypes. These results illustrate spatiotemporal roles of Hdac3, both deacetylase-dependent and deacetylase-independent, in cardiac development, suggesting that dysregulation of Hdac3 in cardiac progenitor cells could be a contributing factor in congenital heart disease pathogenesis.

About two-thirds of human congenital heart disease (CHD) involves second heart field (SHF) derived structures. Histone-modifying enzymes, histone deacetylases (HDACs), regulate the epigenome; however, their functions within the second heart field remain elusive. Here we demonstrate that histone deacetylase 3 (Hdac3) orchestrates epigenetic silencing of Tgfβ1, a causative factor in CHD pathogenesis, in a deacetylase-independent manner to regulate development of SHF-derived structures. In murine embryos lacking Hdac3 in the SHF, increased Tgfβ1 bioavailability is associated with ascending aortic dilatation, outflow tract malrotation, overriding aorta, double outlet right ventricle, aberrant semilunar valve development, bicuspid aortic valve, ventricular septal defects, and embryonic lethality. Activation of Tgfβ signaling causes aberrant endothelial-to-mesenchymal transition (EndMT) and altered extracellular matrix homeostasis in Hdac3-null outflow tracts and semilunar valves and pharmacological inhibition of Tgfβ rescues these defects. Hdac3 recruits components of PRC2 complex, methyltransferase Ezh2, Eed, and Suz12 to the Ncor complex to enrich trimethylation of lys27 on histone H3 at the Tgfβ1 regulatory region and thereby maintains epigenetic silencing of Tgfβ1 specifically within the SHF-derived mesenchyme. Wild-type Hdac3 or catalytically-inactive Hdac3 expression rescue aberrant EndMT and epigenetic silencing of Tgfβ1 in Hdac3-null outflow tracts and semilunar valves. These findings reveal that epigenetic dysregulation within the SHF is a predisposing factor for CHD.

Congenital heart defects often result from improper differentiation of cardiac progenitor cells. Although transcription factors involved in cardiac progenitor cell differentiation have been described, the associated chromatin modifiers in this process remain largely unknown. Here we show that mouse embryos lacking the chromatin-modifying enzyme histone deacetylase 3 (Hdac3) in cardiac progenitor cells exhibit precocious cardiomyocyte differentiation, severe cardiac developmental defects, upregulation of Tbx5 target genes and embryonic lethality. Hdac3 physically interacts with Tbx5 and modulates its acetylation to repress Tbx5-dependent activation of cardiomyocyte lineage-specific genes. These findings reveal that Hdac3 plays a critical role in cardiac progenitor cells to regulate early cardiogenesis.