Abstract Body: Introduction: Genome-wide association studies (GWAS) have identified thousands of non-coding variants associated with coronary artery disease (CAD), yet the mechanisms linking these loci to pathogenesis remain undefined. Here, we aim to characterize the functional role of a high-priority CAD risk SNP rs4266144. While TIPARP is a known responder to xenobiotic stress via the Aryl Hydrocarbon Receptor, its regulation by the Hippo/YAP/TEAD pathway in vascular smooth muscle cells (SMCs) has not been explored. We hypothesized that the CAD-risk SNP rs4266144 disrupts a critical TEAD binding motif within a distal TIPARP enhancer, reducing TEAD-induced TIPARP expression and destabilizing protective vascular SMC networks.

Methods: We utilized epigenomic (ATAC-seq, H3K27ac/H3K4me1 ChIP-seq) data from human coronary artery SMCs (HCASMC) for functional annotation. For validation, we performed dual-luciferase assays and CRISPR interference (CRISPRi) targeting the identified enhancer (R1) in HCASMCs. Downstream effects of TIPARP loss were assessed via siRNA knockdown and RNA-sequencing followed by DESeq2 and GSEA analysis.

Results: The risk allele of lead SNP (G allele; rs4266144) in the 3q25.31 region significantly correlates with CAD (p<10−11) and reduced TIPARP expression in vascular tissue (p<3.4×10−6). The SNP is located within an active enhancer region marked by ATAC-seq and H3K27ac/H3K4me1 peaks and is physically linked to the TIPARP promoter via Hi-C chromatin looping. ChromBPNet analysis predicted the lead SNP significantly impairs TEAD binding in SMCs, and single-cell RNA-seq confirmed robust TIPARP expression within SMCs of human and murine atherosclerotic lesions. Luciferase assays revealed a robust response to TEAD+YAP activation with the reference allele, which was muted by the risk allele (p<0.0001), and CRISPRi silencing of the enhancer in HCASMCs reduced TIPARP mRNA by 20% relative to non-targeting controls (p<0.05), both independent of xenobiotic exposures. RNA-sequencing following siTIPARP knockdown in HCASMCs revealed significant enrichment of TNFalpha signaling via NFkB and Interferon Gamma Response, concomitant with the suppression of structural integrity networks including Apical Junctions.

Conclusions: The rs4266144 risk allele acts as a molecular switch that disrupts novel TEAD-mediated TIPARP regulation in SMCs, driving a loss of structural integrity and heightened inflammatory signaling independent of traditional xenobiotic response.