ABSTRACT Deciphering the molecular circuitries that guide cardiac progenitor cell (CPC) differentiation will contribute to understand the causes of congenital heart disease (CHD). Vitamin A/Retinoic Acid (RA) signaling is a master regulator of cell specification in the heart. RA induces posterior specification of the cardiac progenitor cells (CPCs) of the Second Heart Field (pSHF), the pool that populates the atria and sinus venosus structures. Consequently, VitaminA/RA deficiency and excess in this developmental window lead to structural abnormalities in the heart. Thus, understanding the mechanisms that regulate RA signaling homeostasis is a major goal in the heart field. Here, we adopted a functional genomic approach at single-cell resolution to investigate the Retinoic Acid- regulatory network in human embryonic stem cell-derived CPCs (hESC-CPCs). Our preliminary data suggest that the Hippo effectors YAP1 and TEAD4 are non-canonical effectors of RA signaling critical for atrial cardiomyocyte differentiation. Our studies show that YAP deletion affects the expression of RA-target genes and phenotypic properties of atrial cardiomyocytes (CMs). Furthermore, our single-cell ATAC-seq analysis in WT versus YAP KO CPCs suggest that YAP facilitates accessibility of key atrial transcription factors (TFs) to the chromatin, including MEIS2, HOXA1 and GATA6, supporting a pivotal role of YAP in the TF composition of atrial enhancers (Aim1). Interestingly, our ongoing studies show that RA signaling induces genome-wide de-novo recruitment of YAP:TEAD4 to atrial enhancers in CPCs, independently of the activity of the Hippo-kinases. Our findings suggest that RA recruits YAP to the chromatin through the RA-activated steroid nuclear receptor NR2F2. NR2F2 is a key RA-effector, that regulates the acquisition of atrial lineages in hESC-CPCs and pSHF progenitors. Moreover human NR2F2 mutations lead to CHD. Therefore, we will investigate a novel non-canonical function of YAP and TEAD4 as NR2F2 cofactors in regulating atrial specification, highlighting the potential of YAP/TEAD as possible CHD modifiers (Aim2). Accordingly, our ongoing studies in mouse models and iPSCs revealed that the 22q11.2 genetic deletion or DiGeorge Syndrome (DGS) lead to the inactivation of YAP signaling. DGS is one of the main causes of human CHD, which is largely attributed to the haploinsufficiency in TBX1 in concert with downstream pathways, such as Vitamin A/RA. It has been shown that TBX1 loss in the SHF lead to unbalanced Vitamin A signaling, which evokes in erratic specification of these cells and CHD. Therefore, YAP inactivation in DGS may contribute to abnormal RA signaling in SHF cells and yet, CHD. Thus, we will examine whether restoring the activity of YAP in a mouse model of DGS improves CHD phenotypes. Finally, in collaboration with the UCLA CHD Biocore, we will validate the impact of the Retinoic Acid and YAP pathways in the phenotypes of biopsies of infants with CHD and DGS (A...