Epigenome Analysis Reveals TBX5 as a Novel Transcription Factor Involved in the Activation of Rheumatoid Arthritis Synovial Fibroblasts
Epigenome Analysis Reveals TBX5 as a Novel Transcription Factor Involved in the Activation of Rheumatoid Arthritis Synovial Fibroblasts
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DOI：
10.4049/jimmunol.1400066
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发表时间：
2014-11-15
影响因子：
4.4
通讯作者：
Neidhart, Michel
中科院分区：
文献类型：
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作者：
Karouzakis, Emmanuel;Trenkmann, Michelle;Neidhart, Michel
在这项研究中，我们分析了人类启动子在类风湿关节炎滑膜成纤维细胞（RASF）的甲基化状态。RASF和骨关节炎滑膜成纤维细胞（OASF）之间的差异甲基化基因进行了鉴定，甲基化DNA免疫沉淀和杂交人类启动子平铺阵列。通过焦磷酸测序确认甲基化状态。差异甲基化基因的基因和蛋白质表达进行了评估，实时PCR，蛋白质印迹和免疫组化。染色质免疫沉淀用于测量基因启动子相关的组蛋白乙酰化和甲基化。用微阵列和荧光素酶分析鉴定转录因子特异性靶点。我们发现转录因子T-box转录因子5（TBX 5）在类风湿关节炎（RA）滑膜和RASF中的甲基化程度低于骨关节炎（OA）样本。在RASF和RA滑膜中TBX 5启动子的去甲基化伴随着比OASF和OA滑膜中更高的TBX 5表达。在RA滑膜中，TBX 5表达主要定位于滑膜衬里。此外，TBX 5基因座在RASF中富含激活染色质标记，如组蛋白4赖氨酸4三甲基化和组蛋白乙酰化。在我们的功能研究中，我们观察到790个基因在OASF中过表达TBX 5后差异表达2-6倍。对这些基因的生物信息学分析显示，趋化因子IL-8、CXCL 12和CCL 20是OASF中TBX 5的共同靶标。总之，我们的数据表明TBX 5是RASF中重要趋化因子的新诱导剂。因此，我们得出结论，RASF有助于通过TBX 5的表观遗传控制在RA的发病机制中操作的炎症过程。
In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5.