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

ACTN4 与功能性 SNP rs9277336 结合控制肺动脉中的基因组结构和内皮病理表型

• 批准号: 10750069
• 负责人: Anna Kirillova
• 金额: $ 5.27万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-26 至 2027-03-25
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750069
• 关键词: ATF6 gene; Alleles; Angiogenesis Inhibition; Antigen-Presenting Cells; Architecture; Behavior; Binding; Biological Assay; Blood Vessels; Cardiovascular system; Cell Extracts; Cell physiology; Cells; Chromatin; Clinical; DNA; Data; Diagnosis; Diagnostic; Disease; Distal; Endothelial Cells; Endothelium; Enhancers; Exhibits; Exposure to; Gene Targeting; Genes; Genetic; Genome; Genotype; Hematological Disease; Histocompatibility Antigens Class II; Human; Immune; Inflammatory; Link; Linkage Disequilibrium; Lung; Lung diseases; Maps; Mediating; Mediator; Mutation; Nuclear; Nucleotides; Oligonucleotides; Pathogenicity; Pathologic; Patients; Peripheral Blood Mononuclear Cell; Persons; Phenotype; Physicians; Population; Populations at Risk; Process; Property; Protein Isoforms; Regulation; Risk; Role; SNP genotyping; Scientist; Severities; Severity of illness; Single Nucleotide Polymorphism; Specificity; Testing; Therapeutic; Training; Untranslated RNA; Variant; Vascular remodeling; angiogenesis; career; cell motility; cell type; chromatin immunoprecipitation; clinical translation; cohort; diagnostic tool; endothelial dysfunction; experimental study; gain of function; genome wide association study; induced pluripotent stem cell; insight; loss of function; migration; mortality; novel diagnostics; physical model; prognostic tool; pulmonary arterial hypertension; pulmonary artery endothelial cell; pulmonary vasoconstriction; transcription factor; vasoconstriction

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 why a significant proportion of persons are at risk for more severe PAH. Such insight would have substantial impact on better PAH diagnostics and therapeutics, thus preparing me for a long career as a physician-scientist.

项目总结