Len Pennacchio - US grants

? DESCRIPTION (provided by applicant): The HBCLL center, in the CvDC, collaborates with the PCGC to form the twin pillars of the B2B Program. The B2B seeks to define the complex molecular and cell interactions that are required for normal heart development, to define genetic perturbations that cause congenital heart disease (CHD), and to elucidate the mechanisms by which these CHD mutations disrupt heart development. We will address the following unanswered questions: What are the DNA elements with which these global transcriptional regulators interact? Might mutations in these DNA elements also cause CHD? What are the genes with altered expression and how does disruption of physiologic temporal-spatial expression result in CHD? The answers to these questions will significantly expand our understanding of cardiac development as well as fundamental processes involved in cell lineage commitment, differentiation and ultimately organogenesis. Our overarching goal is to establish a blueprint of the transcriptional regulatory elements that direct cardiogenesis, and to use this information to identify the rare variants that cause CHD and to elucidate how these disrupt heart development. The HBCLL will use next generation sequencing technologies to map both chromatin structure and gene expression during cardiac development. The new maps will consist of spatiotemporal and cell type specific assessment of transcript abundance (Aim 3) and structure (Aim 1,2). We also propose a series of collaborative experiments, with the PCGC and other CvDC centers, to distinguish pathogenic human sequence variants from benign variants. We suggest that each group in CvDC and PCGC centers focus on variants that are appropriate to their expertise. As CHD-associated variants will likely impair multiple distinct developmental pathways this `bundling' of variants will facilitate analyses. For example, one group will assess the pathogenicity of approximately 150 recently discovered variants that are in transcriptional regulatory and chromatin modifying genes. Similarly, another set of variants may be involved in altering RNA splicing. Another set of variants will likely fall in non-coding regulatory regions an land on our regulatory element map; these could be characterized using cell or mouse-based models (Aim 3). Characterization of these human variants in human iPS cells and animal models will provide novel insights into developmental networks and regulatory blueprints and their perturbations in CHD. In summary we propose experiments directed towards understanding cardiovascular development and its perturbation in CHD. We have 4 specific aims: 1) Define the RNA transcriptome in human normal and CHD tissues; 2) Complete the murine cardiac transcriptome map and assess mechanisms of cardiac chamber formation by single cell analyses; 3) Annotate regulatory elements and their interacting transcriptional regulatory proteins in human and mouse genomes and 4) Perform functional analyses of human genetic variants and critical transcriptional regulatory nodes (Collaborative Project).