Xiangmeng Shawn Cai

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes - those with strong distal enhancers - are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

BACKGROUND: Coronary artery disease (CAD) is a complex condition and remains the leading cause of mortality worldwide. Genome-wide association studies have identified a CAD risk locus on chromosome 10q23 that is independent of traditional risk factors, providing an opportunity to uncover novel molecular mechanisms contributing to CAD pathogenesis. METHODS: Improved fine-mapping approaches were used to prioritize noncoding variants at the 10q23 locus within the intronic region of TSPAN14 (tetraspanin 14). Regulatory elements harboring lead variants were functionally interrogated using chromatin accessibility, 3-dimensional chromatin organization, and clustered regularly interspaced short palindromic repeats–mediated deletion approaches in endothelial cells (ECs). Subsequently, TSPAN14 function in ECs was assessed using transcriptomic profiling, Notch signaling activation assays, and EC functional assay using gene knockout (KO) cell lines. RESULTS: Fine-mapping identified 2 lead variants, rs17680741 and rs12260962, located within regulatory elements predicted to affect TSPAN14 expression in monocytes and ECs, respectively. Chromatin accessibility, organization, and enhancer deletion assays demonstrated EC-specific function for the rs12260962-harboring regulatory element in TSPAN14 expression regulation. Loss of TSPAN14 resulted in significant transcriptomic changes related to Notch signaling, heart morphogenesis, cell adhesion, and wound healing. Functionally, TSPAN14 KO ECs exhibited impaired cell-cell junction integrity, reduced repair capacity, and diminished mechanosensitive responses. CONCLUSIONS: Together, these data identify a regulatory element harboring CAD-associated variant at the 10q23 locus that modulates TSPAN14 expression and downstream Notch signaling in ECs, thereby linking genetic risk to endothelial dysfunction relevant to CAD pathogenesis.