Functional fine-mapping of the 9p21.3 cardiovascular disease locus implicates the lncRNA MIR31HG as an effector transcript in endothelial cells

BACKGROUND

The chromosome 9p21.3 locus is among the strongest genetic risk loci for cardiovascular disease (CVD), yet the regulatory mechanisms underlying this association remain poorly understood. Previous vascular disease studies focused predominantly on the role of this locus in smooth muscle cell (SMC) dysfunction, whereas its potential role in endothelial cells (ECs) remains less well characterized. Here, we sought to identify candidate regulatory variants at 9p21.3, their distal target genes, and effector transcripts that contribute to CVD-related phenotypes in ECs.

METHODS

Promoter capture Hi-C was used to map chromatin interactions at 9p21.3 in primary human ascending aortic SMCs (n=6), supplementing the previously obtained ECs (n=16) dataset. To support the regulatory relevance of these interactions, expression quantitative trait locus analysis was performed in 136 vascular biopsies. Variant effects were prioritized in silico using AlphaGenome. The regulatory activity of the prioritized region was validated using CRISPR activation.

RESULTS

Chromatin interaction mapping identified a complex regulatory architecture at the 9p21.3 locus involving 19 putative regulatory elements and 15 genes across ECs and SMCs. MIR31HG showed the most recurrent distal promoter connectivity with the 9p21.3 region. Consistent with the chromatin interaction map, expression of MIR31HG transcripts was associated with genetic variation at the 9p21.3 locus. In ECs, the lncRNA MIR31HG, but not miR-31, was responsive to hemodynamic flow. Depletion of MIR31HG in ECs was associated with endothelial activation. Epigenetic tracks and deep learning predictions highlighted an active enhancer element at 9p21.3, which was subsequently validated by CRISPR activation and increased MIR31HG expression, confirming its capacity to regulate MIR31HG over a large genomic distance of approximately 538 kb.

CONCLUSIONS

Altogether, these data implicate lncRNA MIR31HG as a likely effector of the 9p21.3 CVD locus, linking the cis-regulatory element and heritable genetic variation in the respective region to endothelial activation through long-range enhancer regulation.

Clinical Perspective

What Is New?

• Promoter capture Hi-C in primary human vascular cells identified recurrent long-range interactions between the 9p21.3 cardiovascular disease locus and the MIR31HG promoter, extending the regulatory landscape beyond the nearest genes.

• Expression of MIR31HG transcripts was associated with 9p21.3 genetic variation in vascular tissue; the lncRNA MIR31HG was responsive to hemodynamic flow in endothelial cells and influenced the endothelial activation transcriptional programs upon depletion.

• Integrated sequence-based, epigenomic, genetic, and CRISPR activation analyses nominated the 9p21.3 enhancer as a regulator of MIR31HG in endothelial cells.

What Are the Clinical Implications?

• The findings suggest that the cardiovascular disease associations of 9p21.3 may involve stronger than previously anticipated endothelial regulatory mechanisms in addition to characterized effects in other vascular cell types.

• lncRNA MIR31HG provides a molecular link between noncoding genetic variation at 9p21.3 and endothelial cell-state regulation relevant to vascular disease.