ABSTRACT Congenital heart defects (CHDs) are seen in ~70% of individuals with 1p36 deletion syndrome and are a major cause of morbidity and mortality. RERE and SPEN are nuclear receptor coregulator encoding genes located in CHD critical regions on chromosome 1p36. We have shown that their haploinsufficiencies contribute to the development of 1p36-related CHD and individually cause syndromic forms of CHD with ventricular septal defects (VSDs) being particularly common. In this application, we will elucidate the morphogenetic and molecular mechanisms by which RERE and SPEN function during cardiac development. Using our unique mouse resources, we have shown that systemic or endocardial-specific RERE deficiency causes endocardial cushion hypoplasia and VSDs by decreasing levels of endothelial-to-mesenchymal transition (EndMT) and mesenchymal cell proliferation in the atrioventricular (AV) canal. Our published and preliminary data suggest that RERE functions in the AV canal by positively regulating the expression of Spen, Gata4, Bmp6, and Bmp7, all of which have been implicated in the development of VSDs. Similarly, we have shown that systemic or endocardial-specific SPEN deficiency causes endocardial cushion hypoplasia and VSDs, and that Rere and Spen interact genetically in VSD formation. These results lead us to hypothesize that SPEN deficiency triggers morphogenetic changes in the developing AV canal that are similar to those caused by RERE deficiency. Since SPEN and RERE encode nuclear receptor coregulators and interact with similar proteins including RARα and HDAC1, we also hypothesize that they function within the same pathway to regulate Gata4, Bmp6, and Bmp7 transcription in the developing AV canal. To test these hypotheses, we propose two Specific Aims. In Specific Aim #1, we will determine the morphogenic mechanisms by which SPEN deficiency causes VSDs using AV canal explant and immunohistochemical studies. Specifically, we will determine if SPEN deficiency leads to decreased levels of EndMT and mesenchymal cell proliferation, and if it also triggers increased levels of mesenchymal cell death not seen in RERE deficient embryos. In Specific Aim #2, we will determine the molecular mechanisms by which SPEN deficiency causes VSDs and how RERE and SPEN interact using a variety of molecular techniques. Specifically, we will determine if SPEN functions similarly to RERE in regulating the expression of Gata4, Bmp6, and Bmp7, if RERE can activate the SPEN promoter, and if RERE and SPEN interact in a complex to drive the transcription of their target genes. The knowledge gained from these experiments will serve as a foundation from which therapeutic and preventative interventions can be developed, not only for individuals with 1p36 deletions or pathogenic variants in RERE and SPEN, but also for a much larger group of individuals at risk for VSDs. The innovative mouse models used in this proposal will provide a platform on which these intervention...