Molecular Regulation of Low-Density Lipoprotein Receptor Expression

Atherosclerotic cardiovascular disease (ASCVD) refers to a collection of conditions including myocardial infarction and ischemic stroke that are driven by the formation of cholesterol-rich plaques in arterial walls. Cholesterol circulates in the bloodstream in the form of lipoproteins, which are classified according to their density and biochemical composition. An elevated level of circulating low-density lipoproteins (LDL) is a key risk factor for atherosclerosis. The major regulator of circulating LDL is the hepatic LDL receptor (LDLR), which removes LDL from the bloodstream through receptor-mediated endocytosis. Our lab previously performed a series of functional genomic CRISPR screens to identify genetic modulators of LDL uptake and LDLR abundance in hepatocytes. These studies identified around 100 genes with potential roles in hepatic LDL uptake. The work reported here characterizes how two of these genes, CHD7 and PROX1, promote LDL uptake through distinct mechanisms. My findings suggest that PROX1 acts as a direct hepatic LDLR transcriptional regulator whereas CHD7 promotes LDL clearance in an LDLR-independent manner. I further investigated the molecular basis of LDLR transcriptional regulation by extending our CRISPR screening approach to the noncoding genome. A new CRISPR library containing 12,372 unique gRNAs was designed and synthesize targeting candidate cis-regulatory elements (CREs) near the LDLR locus. These target sites were nominated based on several potential predictors of CRE activity from existing public datasets - open chromatin regions, histone modifications associated with enhancer activity, proximity to common genetic variants associated with LDL cholesterol levels by human genome-wide association studies, and evolutionary conservation. Analysis of gRNA enrichment revealed a clear identification of internal control sequences as well as several noncoding regions that were not previously recognized to regulate LDLR expression. This latter group included a region in the proximal first intron of the LDLR gene that was also associated with biochemical hallmarks of enhancer activity and is highly conserved across vertebrates. Taken together, the findings reported here provide novel insights into the molecular basis of hepatic LDLR gene expression, LDL metabolism, and ASCVD risk.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/175599/1/cserran_1.pd