In the past two years, genome-wide association (GWA) studies for disease-related metabolic and cardiovascular traits have successfully identified more than 70 loci containing dozens of DNA variants strongly confirmed to be associated with a trait, although a likely functional variant has been implicated for very few loci. Successful identification of these underlying variants will facilitate understanding of biological pathways and disease-related mechanisms of gene regulation. Knowledge of variants increasing susceptibility to type 2 diabetes, obesity, unhealthy cholesterol levels and related traits would substantially impact public health by providing biological and clinical data about development and treatment of cardiovascular disease and by advising specific lifestyle changes in at-risk individuals. We hypothesize that for numerous loci, functional variants are present in non-coding regulatory elements such as promoters, enhancers, repressors, and insulators. Substantial evidence indicates that many regulatory elements are located in regions of open chromatin, not bound by nucleosomes when in an active state. Successful genome-wide experimental approaches to identify open chromatin include DNase hypersensitivity (DHS), formaldehyde-assisted identification of regulatory elements (FAIRE), markers of histone modification, and chromatin immunoprecipitation (ChIP) with regulatory proteins. Importantly, these data are now available for several medically relevant cell types. In this application, we focus initially on loci for which pancreatic islets and hepatocytes are most likely to be implicated, and for these loci we propose to analyze existing resequencing data to develop comprehensive lists of potential functional variants, prioritize the variants based on sequence annotation and genome-wide maps of open chromatin from relevant tissues, and test high priority predicted regulatory variants for allele-specific effects on promoter, enhancer, repressor, or insulator activity. Allele- specific binding to transcription factors and other regulatory proteins will be validated using electrophoretic mobility shift (EMSA) and supershift assays and allele-specific ChIP. Variants exhibiting an allele-specific effect on transcription will be tested for differential representation in regions of open chromatin. Through this work we expect to identify likely functional regulatory variants associated with metabolic and cardiovascular traits and develop testable hypotheses for assessing the biological impact in future animal model and human physiology studies of metabolic and cardiovascular traits.
We propose to identify DNA variants that influence inter-individual variation in cardiovascular and metabolic traits by altering gene regulation.
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