DNA羟甲基化酶TET2调控Th17细胞分化及其在系统性红斑狼疮发病中的作用

Active demethylation induced by DNA hydroxymethylation plays important roles in various developmental processes, such as embryonic development and stem cell differentiation, as well as in human brain and myeloid malignancies. However, the specific role of DNA hydroxymethylation in the activation and differentiation of immune cells remains poorly understood. This project will investigate the role and mechanism of DNA hydroxymethylation regulating the aberrant Th17 differentiation in the pathogenesis of systemic lupus erythematosus (SLE). Our previous studies showed that 5-hydroxymethylcytosine (5hmC) content and DNA hydroxymethylase TET2 expression, are significantly up-regulated in CD4+ T cells from patients with SLE. Moreover, we found that DNA hydroxymethylation level in the promoter region of RORγt gene, a key gene determining differentiation of Th17 cell, was increased significantly accroding to DNA hydroxymethylation chip assay. Based on these observations, we propose that the increased DNA hydroxymethylation induced by TET2 leads to the aberrant Th17 differentiation, thus contributing to the pathogenesis of SLE. Our project will test this hypothesis by investigating the regulation of TET2 on the DNA hydroxymethylation and methylation levels in the promoter region of RORγt gene and the effect of interfering TET2 on Th17 differentiation and pathogenesis of SLE. These studies will help demonstrate a novel mechanism in the pathogenesis of SLE and identify new, effective target for SLE therapy.

DNA羟甲基化修饰介导的主动去甲基化过程在胚胎发育、干细胞分化、脑组织及造血系统肿瘤发生中发挥重要作用，然而其在免疫细胞活化与分化过程中的作用仍是未知。本项目将从Th17细胞分化入手，探讨DNA羟甲基化修饰在调控SLE患者外周血Th17细胞异常分化中的作用及机制。我们的前期预实验显示，SLE患者CD4+T细胞基因组5-羟甲基化胞嘧啶含量增加，羟甲基化修饰酶TET2表达显著上调。利用芯片筛选发现决定Th17细胞分化的关键基因RORγt启动子羟甲基化水平显著升高。因此，我们提出TET2调控的DNA羟甲基化修饰水平升高导致Th17细胞异常分化，从而在SLE发生、发展中起重要作用的全新假说。本项目拟通过TET2对RORγt基因启动子甲基化修饰状态的调控，干预TET2对Th17细胞分化及SLE发病的影响来证实这一假说。该研究将有助于进一步揭示SLE的发病机制，并为寻找治疗靶点提供新的思路。

系统性红斑狼疮（SLE）患者外周血中Th17细胞明显增加，Th17细胞通过分泌大量炎性细胞因子如IL-17A促进SLE发病。然而，SLE患者Th17细胞异常增加的分子机制目前尚未阐明。通过本项目研究，我们发现SLE患者CD4+T细胞和体外诱导分化的Th17细胞中TET2和Th17细胞相关基因IL-17A表达均显著升高，并且二者呈显著正相关。我们利用基因过表达或干扰实验发现TET2对IL-17A基因表达及Th17细胞分化具有正调控作用。进一步我们发现与原始的CD4+T细胞相比，Th17细胞中IL17A基因启动子区DNA羟甲基化水平升高，TET2蛋白结合增加，甲基化水平降低。与正常人CD4+T细胞相比，SLE患者CD4+T细胞中IL17A基因启动子区DNA羟甲基化水平升高，TET2蛋白结合增加，甲基化水平降低。过表达或干扰TET2表达可调控IL17A基因启动子区TET2结合、DNA羟甲基化和甲基化水平。此外，我们利用hMeDIP-seq高通量测序的方法筛选了SLE患者CD4+T细胞中全基因组范围内差异羟甲基化修饰基因，发现许多羟甲基化修饰改变基因的表达水平亦发生相应改变，提示DNA羟甲基化修饰通过调控基因参与SLE发病。进一步我们发现转录因子CTCF通过招募TET2到靶基因SOCS1启动子区，过表达或干扰CTCF可调控SOCS1基因启动子区羟甲基化和甲基化水平。以上重要研究发现揭示了TET2通过调控IL17A基因启动子区羟甲基化和甲基化修饰，影响Th17细胞分化及IL17A基因表达，TET2过度表达在SLE发病中发挥重要作用。本项目研究成果为阐明SLE发病的表观遗传学机制奠定了重要基础，为SLE治疗提供了新靶点。