同型半胱氨酸经组蛋白和DNA甲基化相互作用调控ERO1α促内质网应激的分子机制

Endoplasmic reticulum stress (ERS) play an important role in homocysteine (Hcy) induced atherosclerosis (As). ERO1α is a key regulator of protein folding in the endoplasmic reticulum. Our previous studies showed that coexisting hypermethylation with hypomethylation of different genes under the same condition, which suggests a more complex regulation mechanism. The histone methylation coordinating with the DNA methylation regulates gene transcription function, but the significance of histone and DNA methylation regulating of ERO1α in homocysteine-induced ER stress has not been elucidated. Therefore, after established HHcy model, we will detect ERO1α with immunofluorescence and verify the crucial role of ERO1α in atherosclerosis by siRNA.Then we will analyze key genes of ERS by transfecting interference vectors of ERO1α and mapping them , explore the role of ERO1α regulating its target genes using constitutively active- and dominant negative-plasmids of ERO1α. Finally, we will measure the methylation status of H3K9 and ERO1α by MeDIP-qPCR, confirm the coordination of histone and DNA methylation regulating ERO1α when methylation homeostasis disturbed by silencing LSD1 and DNA methylation inhibitor 5-azacytidine. We hope that these studies would provide important insights into the effect and mechanism of hyperhomocysteinemia, thus identifying novel targets for therapeutic interventions of atherosclerosis.

内质网应激(ERS)是同型半胱氨酸(Hcy)致动脉粥样硬化(As)的重要机制，ERO1α是内质网上调控蛋白质折叠的关键基因，前期研究发现同条件下不同基因高低甲基化并存，提示存在更深层次机制，组蛋白甲基化具有协同DNA甲基化调控基因转录的作用，但Hcy是否经组蛋白和DNA甲基化相互作用介导ERO1α调控ERS机制尚未阐明，因此拟复制HHcy模型，免疫荧光分析ERO1α的变化，在细胞中使其沉默予以验证，明确其在As中的作用；转染ERO1α干扰载体分析ERS关键基因的变化并定位，将ERO1α持续失活或激活突变体与启动子报告质粒共转染，揭示ERO1α对靶基因的调控作用；运用MeDIP-qPCR检测组蛋白和ERO1αDNA甲基化的水平，沉默LSD1以逆转组蛋白甲基化及5-氮杂胞苷阻断DNA甲基化，探讨各自平衡模式打破后，组蛋白和DNA甲基化相互作用对靶基因表达的调控机制，为As靶向治疗提供理论依据。

内质网应激(ERS)是同型半胱氨酸(Hcy)致动脉粥样硬化(As)的重要机制，ERO1α是内质网上调控蛋白质折叠的关键基因，前期研究发现同条件下不同基因高低甲基化并存，提示存在更深层次机制，组蛋白甲基化具有协同DNA 甲基化调控基因转录的作用，但Hcy 是经组蛋白和DNA 甲基化相互作用介导ERO1α 调控ERS 机制尚未阐明。本研究探讨了同型半胱氨酸(Hcy)对ERO1α表达的影响，证实Hcy诱导的动脉粥样硬化是通过ERO1α调控的内质网应激而引起的。高蛋氨酸饮食喂养APOE-/-小鼠主动脉内质网应激因子表达上调、H3K4me2水平下降。原代巨噬细胞过表达ERO1α促进巨噬细胞凋亡，而敲减ERO1α后巨噬细胞凋亡减轻。这些数据表明，ERO1α在高同型半胱氨酸介导动脉粥样硬化斑块不稳定中发挥重要作用，组蛋白甲基化可能是Hcy致动脉粥样硬化的一个新的调控靶点。