CD4+ T细胞组蛋白修饰异常对CREMα基因表达的调控及其在系统性红斑狼疮发病中的作用

In recent years, accumulating evidence has demonstrated that increased CREMα in CD4+ T cells plays an essential role in the pathogenesis of systemic lupus erythematosus (SLE). However, the molecular mechanisms that result in CREMα over expression remain poorly understood. In our previous work, we identified markedly increased histone H3 lysine 4 trimethylation (H3K4me3) enrichment at the CREMα promoter of SLE CD4+ T cells relative to controls. In addition, a striking increase was observed in Set1 (a H3K4 methyltransferase) binding. All these results suggested that aberrant histone modification was involved in over expression of CREMα in SLE CD4+ T cells. Therefore, we plan to further compare the levels of H3K4me3 and H3K4 methyltransferases at the CREMα promoter between normal and SLE CD4+ T cells; And to knock down H3K4 methyltransferases in CD4+ T cells from SLE patients, subsequently to examine the effects on CREMα expression, on the levels of H3K4me3, histone acetylation and DNA methylation at the CREMα promoter, and on the productions of IL-2 and IL-17A. By these ways, we would reveal the causation and detailed mechanism of CREMα increase in SLE CD4+ T cells, and could help provide a novel way and target for effective SLE therapy.

近年来的研究表明CD4+ T细胞CREMα表达升高在系统性红斑狼疮（SLE）的发病中起到至关重要的作用，然而CREMα过度表达的分子机制尚未完全阐明。我们前期实验发现SLE患者CD4+ T细胞CREMα启动子区H3K4me3水平显著高于正常对照，且该区H3K4甲基化酶Set1水平亦显著升高，提示CREMα在SLE CD4+ T细胞中过表达可能与异常的组蛋白修饰相关。因此，我们拟进一步证实正常对照和SLE患者CD4+ T细胞CREMα启动子区H3K4me3和H3K4甲基化酶的水平是否存在差异；在SLE CD4+ T细胞中抑制H3K4甲基化酶的表达，检测对CREMα转录水平、CREMα启动子区H3K4me3、组蛋白乙酰化、DNA甲基化水平以及IL-2、IL-17A水平的影响，以此探讨CREMα在SLE患者CD4+ T细胞中表达升高的原因及具体机制，并有望为SLE的治疗提供新的途径和有效靶点。

近年来，越来越多的研究表明系统性红斑狼疮（SLE）患者T细胞的cAMP反应元件调控因子α（CREMα）较正常升高，其可导致 IL-2 减少和IL-17A 增加，这在SLE的发病机理中起到重要作用。然而，在SLE中调控CREMα表达的机制仍不清楚。通过染色质免疫沉淀（ChIP）微阵列，我们发现SLE CD4+ T细胞的CREMα启动子区H3K4me3水平是正常对照的2.48倍。随后通过ChIP结合实时定量PCR，我们证实了SLE患者CD4+ T细胞CREMα启动子区H3K4me3水平显著升高（0.541 ± 0.188比0.842 ± 0.227，P<0.001）。此外，我们还发现SLE患者CD4+ T细胞CREMα启动子区Set1（一种H3K4甲基化酶）水平显著升高（0.398 ± 0.120比0.650 ± 0.189，P<0.001），而MLL1（也是一种H3K4甲基化酶）水平则与对照组无明显差异（0.162 ± 0.060比0.187 ± 0.082，P=0.278）。在SLE CD4+ T细胞中采用siRNA下调Set1后，CREMα启动子区Set1（1比0.424 ± 0.059，P=0.003）和H3K4me3（1比0.343 ± 0.062，P=0.003）水平均下降，同时DNA甲基化（1比2.501 ± 0.232，P=0.004）和DNA甲基转移酶3a（DNMT3a）（1比1.896 ± 0.135，P=0.007）水平都明显升高。上述改变抑制了CREMα的表达（1比0.313 ± 0.043，P=0.001），导致IL-2增多（1比3.013 ± 0.237，P=0.005），而IL-17A减少（1比0.493 ± 0.072，P=0.007）。随后，我们进一步证实了SLE患者CD4+ T细胞CREMα启动子区的DNMT3a减少（0.770 ± 0.144比0.664 ± 0.095，P=0.009）。我们的研究首次证实SLE患者CD4+ T细胞CREMα启动子区Set1水平显著升高，而过表达的Set1又导致此区域H3K4me3水平升高。升高的H3K4me3排斥DNMT3a，结果抑制了此区域的DNA甲基化。上述因素促使CREMα增多，结果致使IL-2减少和IL-17A增加，最终引起SLE的发病。