Functional characterization of cardiovascular disease associated cis-regulatory regions in vivo

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Cardiovascular diseases are one of the major clinical challenges of our time. Precise spatio-temporal control of gene expression is required for proper cardiac development and heart function. Enhancers are cis-regulatory elements in non-coding regions of the genome that regulate gene expression in a cell-type specific manner. Single nucleotide polymorphisms (SNP) associated with cardiac diseases including congenital heart diseases and cardiac hypertrophy are enriched in enhancers. However, the functional role of specific enhancers is still mostly unknown. The overall aim of my project will be to characterize the molecular function of cardiomyocyte enhancers in vivo. I hypothesize that disease triggering enhancers affect gene expression of essential regulators of cardiomyocyte differentiation and function. The specific aims are:1) Characterization of the dynamics of gene regulatory promoter-enhancer networks during differentiation of cardiomyocytes from human embryonic stem cells (hESC) will be identified using high throughput analyses to study the 3D genome architecture.2) To determine enhancers and cardiac disease associated SNPs crucial for gene expression in human cardiomyocytes, I will employ the CRISPR/Cas9 genome-editing technology to manipulate the hESC genome. Resulting gene expression changes in hESC-derived cardiomyocytes will be examined to reveal functionally important sequence variants.3) To validate enhancer function in vivo, conserved regions of essential cardiomyocyte enhancers will be deleted in mouse embryonic stem cells using the CRISPR/Cas9 genome editing technology. These cells will be used for tetraploid complementation assays to generate genetic modified mouse lines. Finally, the cardiac phenotype as well as gene expression changes will be investigated.My work will uncover the molecular function of non-coding genomic elements in vivo and contribute to a better understanding of cardiac development and function. These results might pave the way to new therapeutic strategies for complex cardiac diseases by inferring with gene regulation processes.