Transcriptional regulation of chamber selective gene expression

Project Summary How the same genome is parsed by the transcriptional machinery to yield hundreds of distinct cell types remains one of biology’s enduring mysteries. Within the heart, atrial and ventricular cardiomyocytes (aCM and vCMs) are examples of distinct but related cell types with unique expression profiles. These cell-type-specific expression profiles are essential to normal heart function, as their perturbation leads to arrhythmias and contractile dysfunction. Our studies in the prior funding period identified three transcriptional regulators, TBX5, CHD4, and ESRRA/G that are essential to maintain aCM or vCM identity and whose inactivation causes atrial fibrillation and cardiomyopathy. Here we continue the analysis of chamber-selective gene expression by investigating two key questions: (1) what are the rules that govern enhancer and promoter compatibility and how do these rules contribute to tissue-specific expression, specifically to aCM and vCM identity? (2) what are the trans-activing factors responsible for chamber-selective expression? We propose the following Specific Aims: (1) To evaluate the compatibility between tissue-selective enhancers and promoters. Using massively parallel reporter assays performed within intact animals to assess combinatorial interaction of hundreds of thousands of tissue-selective enhancers and promoters, and to dissect the sequences that encode enhancer- promoter compatibility. Based on these data, we will generate novel chamber-selective regulatory elements for research and clinical gene therapy applications. (2) To identify trans-acting factors responsible for chamber- selective expression. We will use computational prediction of aCM and vCM identity regulators and a novel in vivo forward genetic screen to identify candidate transcriptional regulators that govern chamber-selective gene expression. We will characterize the functional role of selected novel regulators on heart morphology, gene expression, and chromatin landscape. Together, these studies will yield fundamental insights into cardiomyocyte gene regulatory mechanisms that will inform our understanding of atrial fibrillation, cardiomyopathy, and other cardiac pathologies.