Abstract 614: from Genotype to Phenotype: Molecular and Functional Characterization of the CVD-associated SNP Rs11574 in ID3

Atherosclerosis is the primary pathological process of CVD and is strongly controlled by heritable factors; however, the mechanisms and pathways through which many of these genetic components regulate plaque development are poorly understood. Recently, a SNP in the coding region of the gene inhibitor of differentiation 3 ( ID3 ) at rs11574 was identified in multiple studies to be associated with CVD. Mutation of rs11574 from the major allele (G) to the minor allele (A) changes the 105 th amino acid of ID3 from an alanine to a threonine and is associated with an attenuated ability of ID3 to antagonize bHLH transcription factors and to prevent them from binding to DNA and activating transcription. The current study utilized co-immunoprecipitation to demonstrate that the minor allele of rs11574 specifically impairs the ability of ID3 to bind to and sequester E12 and no other member of the bHLH family. ChIP studies further demonstrated that the impaired regulatory ability of the ID3 minor allele variant increases E12 occupancy at promoter regions and enhances transcription of E12 target genes including smooth muscle alpha actin ( Acta2 ) and p21 in murine vascular smooth muscle cells (VSMCs). To study the role of this SNP in human cells in intact chromatin under endogenous regulation, we genome edited human 293T cells using CRISPR/Cas9 to produce the allelic variants of rs11574 in the ID3 gene (GG, AG, and AA). Edited cells showed significant changes in cellular proliferation, gene expression, and promoter occupancy depending upon genotype at rs11574. Cells containing the minor allele of rs11574 displayed significantly increased p21 expression and proliferated more slowly than cells containing the major allele of rs11574; whereas, cells heterozygote at rs11574 displayed an intermediate phenotype. These data implicate rs11574 in the regulation of cellular proliferation and mature VSMC marker expression, key phenotypes regulated in the processes of lesion development and formation.