Research Project
10245030
PROJECT SUMMARY/ABSTRACT PROJECT 2 Congenital heart defects (CHDs) are among the most common and devastating birth defects in humans, occurring in about 1% of live births and resulting in significant mortality and morbidity. We have very little understanding of the processes that, when gone awry, cause CHDs. Human genetics have uncovered mutations in transcription factors (TFs) and chromatin remodeling factors as the underlying etiology of several CHDs. Thus, understanding the function of these critical regulators of cell identity and function is crucial. The function of TFs is intimately related to and regulated by the status of the chromatin at their target binding sites. Chromatin structure affects the accessibility and activity of TFs. Therefore, the regulation of chromatin structure is critical for controlling transcription. Chromatin remodeling occurs largely via ATP-dependent chromatin remodeling complexes, which alter the structure of histone-associated DNA to allow access to the transcriptional machinery. By immunoprecipitation of complexes followed by mass spectrometry (IP-MS) we identified the stoichiometrically regulated components of dynamic BAF complexes during in vitro cardiac differentiation. BAF60c and BAF170 are preferably included in cardiac BAF complexes, as are newly identified subunits such as WDR5, which is mutated in human CHD. Genetic ablation of BRG1, BAF60c, or BAF170 revealed essential roles in activating and maintaining the cardiac program. We further examined BAF60c- and BAF170-containing complexes, and identified an additional subset of complexes that include the alternate ATPase, BRM. We find that BRM is strikingly essential for activation of the cardiogenic program: In the absence of BRM, CP transcriptional regulators fail to be activated, leading to catastrophic failure of cardiogenesis, with cells instead adopting alternate fates. We hypothesize that specific BAF complexes form dynamically to coordinate regulation of distinct aspects of cardiac morphogenesis and lineage decisions. We will test this hypothesis in three Specific Aims: 1. To uncover the molecular mechanisms by which dynamic BAF complex subunits regulate specific gene expression programs. 2. To determine the functional importance of the BAF complex subunit switch in cardiomyocyte differentiation. 3. To decipher the redundant and specific roles played by the interchangeable BAF complex subunits. The knowledge generated from this study, integrating the data generated in the other PPG projects, will be crucial to our understanding of the transcriptional regulation of cardiac development, and mechanisms underlying CHDs. Importantly, we will gain significant insight into the basis of the integration of cardiac TFs with chromatin remodeling complexes, and the broad set of essential interactions that lead to finely tuned regulation of cardiac gene expression.
