HIF-1/COMPASS调控缺氧诱导Brg1和Brm表达上调的机制研究

Pulmonary vasoconstriction mediated by endothelin, and inflammation associated with the up-regulation of cell adhesion molecules (CAMs) play an important role in the development of hypoxia pulmonary hypertension. Our previous studies found that Brg1 and Brm were upregulated by hypoxia in rat pulmonary vascular and endothelial cells. Brg1 and Brm were able to enhance the activation of CAMs and ET-1 by hypoxia, and play an important role in HPH. But the mechanism of Brg1/Brm transactivation in response to hypoxia in endothelial cells leading up to HPH is not fully appreciated. Bioinformatics analysis found that the Brg1 and Brm promoter regions contain HRE. Our preliminary experiment found that when knocked down the expression of endogenous Wdr5、Ash2、Set1 using siRNA, the induction of endogenous Brg1 and Brm messages was limited in endothelial cell. So we speculated that the nuclear transcription factor HIF-1, histone H3K4 methylation mediated by histone methylation transferase COMPASS complex, as well as their crosstalk influence hypoxia-induced Brg1/Brm transactivation. This study intends to adopt the pulmonary artery endothelial cells and rat HPH model as the research object, using plasmid overexpressing, RNAi interference technology, combining with methods such as reporter assays, CHIP, Re-CHIP to clarify the mechanism of Brg1/Brm transactivation. This study will further clarify the molecular mechanism of HPH. And provide theoretical and experimental evidence for HPH prevention and cure.

我们最近研究发现，Brg1和Brm介导的表观遗传调控在缺氧性肺动脉高压(HPH)的发生发展中具有重要作用。缺氧可诱导大鼠肺血管及内皮细胞Brg1和Brm表达增多，介导粘附分子(CAMs)和ET-1转录激活，参与肺血管炎症反应及肺血管重构，最终导致HPH。但是，缺氧时Brg1和Brm表达上调的转录调控机制目前尚不清楚。我们经生物信息学分析发现Brg1和Brm启动子区域含有缺氧反应元件(HRE)；预实验发现，干扰组蛋白甲基转移酶复合物(COMPASS)Wdr5、Ash2和Set1亚基可显著抑制Brg1表达，提示HIF-1和COMPASS可能分别或相互作用，共同调控缺氧诱导Brg1和Brm表达上调。因此，本研究拟从HPH动物和细胞水平，研究缺氧时HIF-1/COMPASS介导Brg1和Brm表达上调的转录调控机制，深入阐明HPH发生的分子机制，为临床HPH防治提供理论和实验依据。

低氧性肺动脉高压(HPH) 是高原心脏病和肺源性心脏病发病的中心环节，以持续的肺血管收缩和肺血管结构改建为基本特征。我们前期研究发现，低氧可诱导内皮细胞中染色质重构蛋白Brg1和Brm表达上调，在CAMs和ET-1的转录调控及HPH形成中发挥着重要的表观遗传调控作用。 但低氧如何诱导Brg1和Brm表达增多，其机制目前尚不明确。本研究重点探讨了HIF-1α及组蛋白甲基转移酶COMPASS在低氧诱导内皮细胞Brg1和Brm 表达上调中的作用机制。在内皮细胞（HUVEC）中，过表达HIF-1α可显著增强低氧诱导的Brm和Brg1的启动子活性，Brm的mRNA及蛋白表达显著上调； Brg1的mRNA表达也显著升高。采用siRNA干扰HIF-1α的表达后，Brm的启动子活性、mRNA及蛋白表达显著受抑；Brg1的mRNA表达也下调。CHIP实验发现低氧可显著增加内皮细胞中HIF-1α与Brm和Brg1启动子的结合，并初步明确了HIF-1α与Brm启动子的结合位点在-130-24之间；HIF-1α与Brg1启动子的结合位点在-284-96之间。siRNA干扰内皮细胞中的组蛋白甲基转移酶复合物亚基Wdr5和Ash2l后，Brm的启动子活性、mRNA和蛋白表达均显著降低，Brg1的mRNA表达也显著降低。过表达Wdr5和Ash2l后Brm启动子活性、mRNA和蛋白表达显著升高，Brg1的mRNA表达也显著上调，Ash2l过表达后Brg1的启动子活性也有所升高。ChIP实验结果显示低氧可显著增加Brm和Brg1启动子上HIF-1α结合区域的组蛋白H3K4三甲基化水平。将内皮细胞进行低氧培养后进行IP实验探讨HIF-1α与Wdr5之间的相互作用关系，结果显示，HIF-1α与Wdr5之间无明确的相互作用关系，但ChIP实验发现，干扰HIF-1α后，Wdr5和Ash2与Brg1和Brm启动子的结合显著受到抑制。本项目研究表明，低氧可促进HIF-1α与Brm和Brg1启动子结合，直接调控Brm和Brg1的转录和表达，HIF-1α也可通过影响Wdr5和Ash2的招募来介导组蛋白H3K4三甲基化修饰来共同调控Brm和Brg1的转录和表达。本研究为深入研究HPH发生的分子机制提供了新的思路和线索，为临床HPH 防治提供了理论和实验依据。