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.

项目概要 背景：肺动脉高压（PAH）的特点是肺血管收缩和 血管重塑。全基因组关联研究（GWAS）定义了单个基因之间的关联 核苷酸多态性 (SNP) 和 PAH。例如，非编码 SNP rs2856830，接近 HLA- DPA1 与 PAH 风险和生存率增加有关。然而，这种“标签”SNP 可能不会导致疾病 关联，因为它们通常与邻近的真正“功能性”SNP 存在连锁不平衡 (LD) (fSNP) 驱动疾病。我们发现 SNP rs2856830 与 fSNP rs9277336 处于 LD 状态，fSNP rs9277336 具有增强子 活性并结合具有等位基因特异性的转录因子 ACTN4。 rs9277336 (G) 等位基因相关 随着 PAH 严重程度的增加。 fSNP rs9277336 通过 ACTN4 的等位基因特异性结合调节 HLA-DPA1 与远端基因靶标 ATF6B 接触。因此，我假设 rs9277336 (G) 等位基因 通过减少 ACTN4 的等位基因特异性结合来控制内皮功能障碍和 PAH，从而 通过近端和远端染色质相互作用破坏 HLA-DPA1 和 ATF6B 的调节。这 将通过以下具体目标来检验假设： (1) 确定 SNP rs9277336 (G) 等位基因是否显示 与 ACTN4 的结合减少。将寡核苷酸暴露于肺动脉内皮细胞 (PAEC) 核 在离体细胞提取物中，rs9277336 (G) 等位基因表现出比 (A) 更低的与 ACTN4 的结合。在这里，我将执行 更明确的实验以确定 ACTN4 是否显示出与 rs9277336 (G) 与 (A) 的等位基因特异性结合 完整细胞中的等位基因，主要使用同基因诱导多能干细胞衍生的内皮细胞 (iPSC-EC) 携带单个 SNP 核苷酸编辑。 (2)确定ACTN4与SNP rs9277336的结合是否调节HLA- DPA1 表达。我们发现 ACTN4 功能的丧失和获得可以相互调节 HLA- 的表达。 PAEC 中的 DPA1。 ACTN4 或 HLA-DPA1 缺陷介导血管生成和 PAEC 迁移。我会评估 减少的 ACTN4 与 rs9277336 (G) 等位基因的结合是否调节并依赖于 HLA-DPA1 水平 iPSC-EC 中的控制功能，从而证明 (G) 等位基因的致病作用。 (3)判断是否为ATF6B 控制 PAH 的病理内皮功能。根据现有的 Hi-C 染色质接触图，我们发现 有证据表明 ACTN4 与 rs9277336 结合可能通过长程相互作用调节 ATF6B。 ATF6B 是一种同工型 ATF6，一种已知的 PAH 介质。我将执行染色质确认捕获 (3C) 以确定 rs9277336 是否 物理接触 ATF6B 基因。我还将评估 ATF6B 功能丧失和获得是否会扰乱 PAEC 功能，从而建立了 rs9277336 与 ATF6B 远端相互作用并介导病理学的范例 内皮功能，以及对 HLA-DPA1 的影响。意义：由于风险 (G) 等位基因显着 在人群中富集 (MAF=0.852)，该 SNP 致病活性的证据将定义为什么显着 有比例的人面临更严重的 PAH 风险。这种洞察力将对更好地发挥作用产生重大影响 PAH 诊断和治疗，从而为我作为一名医师科学家的长期职业生涯做好准备。