From Genotype to Phenotype: Molecular and Functional Characterization of the Disease-associated ID3 SNP rs11574

Henderson, Christopher, Biochemistry and Molecular Genetics - School of Medicine, University of Virginia
McNamara, Coleen, Internal Medicine, University of Virginia
Owens, Gary, MD-MPHY Mole Phys & Biophysics, University of Virginia

The inhibitor of differentiation 3 (ID3) gene functions as a dominant negative regulator of a broadly expressed subgroup of basic-helix-loop-helix (bHLH) transcription factors known as E-proteins. ID3 regulates gene expression by binding to these E-proteins and preventing the formation of transcriptionally-active promoter complexes. Recently, a single nucleotide polymorphism (SNP) associated with cardiovascular disease was found in the coding region of ID3 at rs11574. Mutation of rs11574 from the major allele (C) to the minor allele (T) changes the 105th amino acid of ID3 from an alanine to a threonine. Preliminary evidence indicates that vascular smooth muscle cells (VSMCs) are a promising tissue type in which this SNP might mediate its effects. To better understand the biology of VSMCs and how ID3 and its disease-associated variant might perturb VSMC phenotypes, we begin with an overview of VSMC biology in the context of atherosclerosis. We then transition to a direct study of the ID3 gene, and demonstrate that this amino acid substitution attenuates the ability of ID3 to antagonize E12 and no other member of the E-protein family. We further show that the uniquely-reduced affinity of this ID3 variant for E12 results in increased E12-mediated promoter binding and activation of target genes. We provide the first molecular evidence identifying the critical amino acid residues in E12 which are responsible for the reduced binding affinity between the minor allele variant of ID3 and E12. Additionally, we explore the functional consequences of this dysregulated ID3:E12 transcriptional axis and show, in a genetically-engineered human cell line, that rs11574 alters basal cellular proliferation. We identify genes and pathways regulated by E12 in a VSMC model system and demonstrate that this SNP is associated with a differential proliferative response to treatment with the ID3-inducing mitogen TGF-β1 in a cohort of primary human aortic VSMCs. Finally, we describe a novel murine model system allowing for the permanent lineage tracing and deletion of Id3 specifically in VSMCs and demonstrate the utility of a novel mass cytometry by time of flight (CyTOF) panel for use in quantifying changes in aortic cell state and composition. Together, these data explain how the minor allele variant of rs11574 alters ID3:E12 binding dynamics and implicate this SNP in transcriptional dysregulation affecting myriad cellular phenotypes and pathways associated with both normal development and disease with a particular focus on the implications of this SNP for vascular smooth muscle cell biology and cardiovascular disease.

PHD (Doctor of Philosophy)
Genetics, ID3, Cardiovascular Disease, SNP, Vascular Smooth Muscle Cells, VSMC
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