Genetic and hypoxic control of a lncRNA axis orchestrates endothelial reprogramming in pulmonary hypertension

lncRNA轴的遗传和缺氧控制协调肺动脉高压中的内皮重编程

• 批准号: 10622021
• 负责人: Stephen Y Chan
• 金额: $ 73.74万
• 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: 2023-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622021
• 关键词: Alleles; American; Automobile Driving; Binding; Biology; Blood specimen; CRISPR/Cas technology; Caring; Cells; ChIP-seq; DNA; Data; Development; Disease; Endothelial Cells; Endothelium; Ensure; Epigenetic Process; Genes; Genetic; Genetic Carriers; Genomics; Genotype; Hematological Disease; Histone-Lysine N-Methyltransferase; Histones; Human; Hypoxia; Individual; Introns; Knock-out; Link; Lung; Lung diseases; Lysine; Medical; Meta-Analysis; Metabolic; Metabolic dysfunction; Metabolism; Methylation; Modeling; Molecular; Mus; Pathogenicity; Pathway interactions; Patients; Phenocopy; Phenotype; Positioning Attribute; Proteins; Publishing; Pulmonary Hypertension; Quantitative Trait Loci; RNA; Rattus; Regulation; Risk; Rodent; Role; Severities; Single Nucleotide Polymorphism; Slice; Small Interfering RNA; Specificity; Structure of parenchyma of lung; Transcript; Untranslated RNA; Validation; Vascular Diseases; Vascular remodeling; Work; adeno-associated viral vector; cadherin 5; chromatin remodeling; cohort; epigenetic therapy; functional genomics; genome editing; histone methylation; humanized mouse; in vivo; induced pluripotent stem cell; inhibitor; insight; knock-down; lung hypoxia; molecular targeted therapies; nanoparticle; promoter; pulmonary vascular remodeling; response; transcription factor

Background and Hypothesis: Pulmonary hypertension (PH) is a deadly disease, where Group 1 PAH and Group 3 PH are driven by hypoxia, HIF-2, and non-coding RNAs. We found that the lncRNA KMT2E-AS1 is up-regulated in Groups 1/3 PH and is induced by HIF-2. This lncRNA gene neighbors KMT2E, a gene controlling histone 3 lysine 4 trimethylation (H3K4me3) and chromatin remodeling. In pulmonary endothelial cells (ECs), KMT2E-AS1 stabilizes KMT2E to increase H3K4me3, thus driving HIF-2-specific metabolic and pathogenic alterations. The G-allele of single nucleotide variant (SNV) rs73184087 within KMT2E is associated with risk of developing Group 1 PAH (in discovery/validation cohorts and a meta-analysis of 2,181 PAH vs. 10,060 controls). rs73184087 also displays more avid allele (G)-specific association with HIF-2 leading to induction of this lncRNA-KMT2E pair. A mouse deficient in the conserved lncRNA sequence is protected against Groups 1/3 PH; this is phenocopied by inhibition of histone methylation in PAH rats. We postulate that the KMT2E-AS1/KMT2E axis is a central lynchpin in pathogenic reprogramming in ECs, promoting PH. Aim 1) Define the allele-specific role of the KMT2E SNV rs73184087 in controlling HIF-2-dependent EC lncRNA-KMT2E expression and PH pathophenotypes. Using ECs derived from genome-edited inducible pluripotent stem cells (iPSC) as well as primary lung ECs carrying rs73184087 A and G alleles, we will determine if (G) increases lncRNA-KMT2E by more HIF-2 binding and drives more severe EC phenotypes. We will also pursue expression quantitative trait loci (eQTL) analysis in blood samples from PAH patients (discovery/validation cohorts) and PAH lung tissues carrying A and G alleles of rs73184087. Aim 2) Define the role of this lncRNA-KMT2E axis and H3K4me3 in promoting PH in vivo. We will quantify Groups 1/3 PH severity in rodents after EC-specific knockdown of this lncRNA vs. lncRNA+KMT2E and after AAV-driven EC- specific expression of lncRNA vs. lncRNA+KMTE2. We will also determine if MM-589, a specific H3K4me3 inhibitor, reverses PAH in rats. Thus, we aim to determine if lncRNA+KMT2E together are necessary and sufficient to drive Group 1/3 PH and if PAH is dependent upon H3K4me3 activity, thus offering a new epigenetic PH therapy. Aim 3) Define the causative role of the G allele of rs73184087 on pulmonary vascular remodeling and PH in vivo. Culturing human precision cut lung slices, we will determine if the rs73184087 G allele drives vascular remodeling via regulation of the lncRNA-KMT2E axis and H3K4me3. We have also inserted the human rs73184087 G vs. A allele in mice and will use these “humanized” mice to study these alleles in vivo. With these 2 unique platforms, we will determine if the G allele drives HIF-2-specific EC phenotypes and PH. Significance: We plan to shift paradigms of lncRNA biology in PH, via defining the links of hypoxia to epigenetics and metabolism and by introducing new epigenetic therapies. By establishing the causative role of rs73184087 in PH, we are poised to leverage functional genomics to gain mechanistic insight in PH specifically for humans.

背景和假设：肺动脉高压（pH）是一种致命疾病，其中1组PAH和 3组pH由缺氧，HIF-2和非编码RNA驱动。我们发现lncrna kmt2e-as1是 以1/3 pH为单位上调，并由HIF-2诱导。这个lncRNA基因邻居kmt2e，一个基因 控制Hisstone 3赖氨酸4三甲基化（H3K4ME3）和染色质重塑。在肺部内皮细胞中 （ECS），KMT2E-AS1稳定KMT2E以增加H3K4Me3，从而驱动HIF-2特异性代谢和 致病性改变。 KMT2E中的单个核苷酸变体（SNV）RS73184087的G-优质相关 有开发1组PAH的风险（在发现/验证队列中，荟萃分析为2,181 pah vs。 10,060个控件）。 RS73184087还显示了与HIF-2的特定狂热等位基因（G） 诱导该lncRNA-KMT2E对。缺乏配置的lncRNA序列缺乏的小鼠受到保护 组1/3 pH；这是通过抑制PAH大鼠组蛋白甲基化来表达的。我们假设 KMT2E-AS1/KMT2E轴是EC中的致病重编程中的中央林奇宾，促进了pH。目的 1）定义KMT2E SNV RS73184087在控制HIF-2依赖性EC中的特定等位基因特异性作用 lncRNA-KMT2E表达和pH病理表达。使用源自基因组编辑的诱导的EC 多能干细胞（IPSC）以及携带RS73184087 A和G等位基因的原发性肺ECS，我们将确定 如果（g）通过更多的HIF-2结合增加LNCRNA-KMT2E并驱动更严重的EC表型。我们也会 在PAH患者的血液样本中追求表达定量性状基因座（EQTL）分析 （发现/验证队列）和含有rs73184087的A和G等位基因的PAH肺组织。目标2）定义 该lncRNA-KMT2E轴和H3K4me3在促进体内pH值中的作用。我们将量化组1/3 pH EC特异性敲低的LNCRNA与lncRNA+KMT2E和AAV驱动的EC-之后的啮齿动物的严重程度 lncRNA与lncRNA+kmte2的特异性表达。我们还将确定MM-589是否是特定的H3K4ME3 抑制剂，逆转大鼠的PAH。这，我们旨在确定lncrna+kmt2e是否需要在一起，并且 足以驱动组1/3 pH，如果PAH取决于H3K4ME3活性，从而提供了一种新的表观遗传学 pH疗法。目标3）定义RS73184087 G等位基因在肺血管上的病因作用 重塑和体内pH。培养人类精确切割肺切片，我们将确定RS73184087 g是否 等位基因通过调节LNCRNA-KMT2E轴和H3K4me3的调节来驱动血管重塑。我们也插入了 人类RS73184087 g与小鼠中的等位基因相比，将使用这些“人性化”小鼠在体内研究这些等位基因。 使用这两个独特的平台，我们将确定G等位基因是否驱动HIF-2特异性EC表型和pH。 意义：我们计划通过定义缺氧与表观遗传学的联系来改变pH中lncRNA生物学的范例 和新陈代谢，并引入新的表观遗传疗法。通过建立RS73184087的因果关系 在pH中，我们被毒化以利用功能基因组学来获得专门针对人类的pH的机械洞察力。