Project Summary Background: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and vascular remodeling. Genome-wide association studies (GWAS) have defined associations between single nucleotide polymorphisms (SNPs) and PAH. For example, non-coding SNP rs2856830, in proximity to HLA- DPA1, is linked to increased PAH risk and survival. Yet, such “tag” SNPs may not be responsible for disease association, because they are often in linkage disequilibrium (LD) with neighboring true “functional” SNPs (fSNPs) that drive disease. We showed that SNP rs2856830 is in LD with fSNP rs9277336, which has enhancer activity and binds a transcription factor, ACTN4, with allele-specificity. The rs9277336 (G) allele is associated with increased PAH severity. fSNP rs9277336 regulates HLA-DPA1 via allele-specific binding of ACTN4 and comes into contact with a distal gene target, ATF6B. Thus, I hypothesize that the rs9277336 (G) allele controls endothelial dysfunction and PAH through reduced allele-specific binding of ACTN4, thus disrupting the regulation of HLA-DPA1 and ATF6B via proximal and distal chromatin interactions. This hypothesis will be tested with the following Specific Aims: (1) Determine if SNP rs9277336 (G) allele displays reduced binding to ACTN4. With oligonucleotides exposed to pulmonary artery endothelial cell (PAEC) nuclear cell extracts ex vivo, the rs9277336 (G) allele exhibited lower binding to ACTN4 than (A). Here, I will perform the more definitive experiment to determine if ACTN4 shows such allele-specific binding to the rs9277336 (G) vs (A) allele in intact cells, primarily using isogenic inducible pluripotent stem cell-derived endothelial cells (iPSC-EC) that carry single SNP nucleotide edits. (2) Determine if ACTN4 binding to SNP rs9277336 regulates HLA- DPA1 expression. We found that loss- and gain-of-function of ACTN4 reciprocally regulated expression of HLA- DPA1 in PAECs. ACTN4 or HLA-DPA1 deficiency mediated angiogenesis and PAEC migration. I will assess whether reduced ACTN4 binding to the rs9277336 (G) allele regulates and depends upon HLA-DPA1 levels to control function in iPSC-ECs, thus proving the pathogenic action of the (G) allele. (3) Determine if ATF6B controls pathologic endothelial function in PAH. Based on existing Hi-C chromatin contact maps, we found evidence that ACTN4 binding to rs9277336 may regulate ATF6B via long-range interaction. ATF6B is an isoform of ATF6, a known mediator of PAH. I will perform chromatin confirmation capture (3C) to determine if rs9277336 physically contacts the ATF6B gene. I will also assess if ATF6B loss- and gain-of-function disrupts PAEC function, thus establishing a paradigm by which rs9277336 distally interacts with ATF6B and mediates pathologic endothelial function, in addition to its effects on HLA-DPA1. Significance: Since the risk (G) allele is significantly enriched in the population (MAF=0.852), proof of this SNP’s pathogenic activity would define wh...