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

背景和假设：肺动脉高压(PH)是一种致命的疾病，其中组1 PAH和 第3组PH由低氧、缺氧诱导因子-2和非编码RNA驱动。我们发现，lncRNA KMT2E-AS1是 在1/3PH组上调，并由HIF-2诱导。这个lncRNA基因与KMT2E基因相邻 控制组蛋白3赖氨酸4三甲基化(H3K4me3)和染色质重塑。在肺内皮细胞中 (ECs)，KMT2E-AS1稳定KMT2E增加H3K4me3，从而驱动HIF-2特异性代谢和 病原性改变。KMT2E中单核苷酸变异rs73184087的G等位基因与 有发生第1组多环芳烃的风险(在发现/验证队列中，以及对2,181个多环芳烃与 10,060个对照)。Rs73184087还显示出与HIF-2更狂热的等位基因(G)特异性关联，从而导致 诱导该lncRNA-KMT2E对。缺乏保守的lncRNA序列的小鼠可免受 1/3PH组；这是通过抑制PAH大鼠的组蛋白甲基化而出现的。我们假设 KMT2E-AS1/KMT2E轴是内皮细胞致病重编程的中心关键，促进了PH的发生。目标 1)确定KMT2E SNV rs73184087在控制HIF-2依赖的EC中的等位基因特异性作用 LncRNA-KMT2E表达与PH病理表型的关系使用来自基因组编辑的诱导物的ECs 多能干细胞(IPSC)以及携带rs73184087A和G等位基因的原代肺内皮细胞，我们将确定 IF(G)通过更多的HIF-2结合增加LncRNA-KMT2E，并驱动更严重的EC表型。我们还将 PAH患者外周血中EQTL分析 (发现/验证队列)和携带rs73184087的A和G等位基因的PAH肺组织。目标2)定义 这个LncRNA-KMT2E轴和H3K4me3在体内促进PH中的作用。我们将量化1/3 PH组 在EC特异性敲除该lncRNA与lncRNA KMT2E和AAV驱动的EC-KMT2E后，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-589，一种特定的H3K4me3 抑制剂，逆转大鼠的PAH。因此，我们的目标是确定lncRNA KMT2E是否一起是必要的和 足以驱动1/3组PH，如果PAH依赖于H3K4me3活性，因此提供了新的表观遗传学 PH值疗法。目的3)明确rs73184087的G等位基因对肺血管的致病作用 活体重塑和PH。培养人肺精密切片，我们将测定rs73184087G 等位基因通过调节LncRNA-KMT2E轴和H3K4me3来驱动血管重塑。我们还插入了 人类rs73184087G和A等位基因在小鼠中的对比，并将使用这些“人性化”的小鼠在体内研究这些等位基因。 有了这两个独特的平台，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：我们计划通过定义低氧与表观遗传学的联系，改变PH中lncRNA生物学的范式 通过引入新的表观遗传疗法。通过确定rs73184087的致病作用 在PH中，我们准备利用功能基因组学来获得专门针对人类的PH的机械性洞察。

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.