# ACTN4 Binding to Functional SNP rs9277336 Controls the Genome Architecture and Endothelial Pathophenotypes in Pulmonary Arterial

> **NIH NIH F30** · UNIVERSITY OF PITTSBURGH AT PITTSBURGH · 2024 · $53,974

## Abstract

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...

## Key facts

- **NIH application ID:** 10938016
- **Project number:** 5F30HL170649-02
- **Recipient organization:** UNIVERSITY OF PITTSBURGH AT PITTSBURGH
- **Principal Investigator:** Anna Kirillova
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $53,974
- **Award type:** 5
- **Project period:** 2023-09-26 → 2027-03-25

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10938016

## Citation

> US National Institutes of Health, RePORTER application 10938016, ACTN4 Binding to Functional SNP rs9277336 Controls the Genome Architecture and Endothelial Pathophenotypes in Pulmonary Arterial (5F30HL170649-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10938016. Licensed CC0.

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