Causal variant association mechanisms in TCF21 binding coronary disease loci

Stanford University, Stanford CA

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Abstract

Building on the extensive genome wide association studies (GWAS) of coronary artery disease (CAD), and single cell characterization of atherosclerosis, we have shown that the smooth muscle cell (SMC) lineage harbors much of the risk for vascular disease. Further, these data indicate that SMC can assume two disease- related transition states linked to disease risk. We identified TCF21 as a protective CAD GWAS gene and showed that it regulates a disease-related transition of medial SMC to a fibroblast-like phenotype, producing cells we term “fibromyocytes” (FMC). Further, we and others have found that SMC can also transition to a second phenotype, characterized by expression of genes known for their role in endochondral bone formation and intimal vascular calcification. Mouse genetic models have indicated that this chondrogenic process, which gives rise to cells we term “chondromyocytes” (CMC), is promoted by CAD GWAS causal factors Pdgfd and Twist1, and actively inhibited by protective TCF21 target CAD GWAS signaling molecules ZEB2 and SMAD3. Further, recent transcriptomic and epigenomic (multi-ome) single cell studies indicate that FMC and CMC are the end products of independent trajectories that SMC can differentially traverse in the disease setting. We thus hypothesize that CAD protective GWAS genes TCF21 and ZEB2 promote disease protective FMC and inhibit disease promoting CMC trajectories through regulation of critical enhancers and their regulatory transcription factors (TFs) that underlie the fundamental mechanisms of CAD genetic risk in the SMC lineage. Algorithms that link disease variation and disease transcriptomic data at the whole genome level show that disease genetic risk resides in the cell state changes from SMC to FMC and SMC to CMC. We now propose to investigate the enhancers, related TFs and their target genes (regulons) that mediate these phenotype transitions, through the following Aims. In Aim 1, we will perform time-course studies with mouse disease models targeted for disease inhibiting CAD associated genes Tcf21 and Zeb2 to map the cis- and trans-acting factors and disease-related regulons that determine the disease trajectories and directionality of disease risk. Findings from these studies will be mapped onto multi-omic single cell assay data of human coronary lesions to establish that orthologous enhancers and molecular SMC mechanisms mediate phenotype transitions in human disease. In Aim 2, we will address the hypothesis that over-expression of Tcf21 in medial SMC will promote increased transition to the FMC phenotype, as indicated by epigenetic and gene regulatory network modules, as well as increased fibrous cap formation and additional risk protective features. In Aim 3, CRISPRi PerturbSeq experiments in human coronary artery smooth muscle cells will validate the results from previous Aims, and further characterize downstream enhancers, related TFs, and regulatory networks. The proposed studies will identify cellular and molecular mechanisms that mediate SMC transitions to FMC and CMC, and the relationship of these transitions to vascular calcification and disease risk.