Shu Jin, Wang Xiao-Hua, Zhou Lan-Bo, Jiang Chun-Ming, Yang Wei-Xia, Jin Rui, Wang Lu-Lu, Zhou Guo-Ping
Department of Pediatrics, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
2013 Clinical Class 7, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Mol Med Rep. 2016 Sep;14(3):2815-22. doi: 10.3892/mmr.2016.5565. Epub 2016 Jul 27.
The transcription factor, interferon regulatory factor 5 (IRF5), is important in the induction of type I interferon, proinflammatory cytokines and chemokines, and is involved in autoimmune diseases and tumourigenesis. However, the mechanisms underlying the transcriptional regulation of wild‑type IRF5 remain to be fully elucidated. The present study was primarily designed to clarify whether specificity protein 1 (Sp1) was involved in the regulation of IRF5. Initially, the IRF5 promoter region was cloned and its promoter activity was examined using Hela and HEK 293 cells. Deletion analyses revealed that the region spanning ‑179 to +62 was the minimal promoter of IRF5. Bioinformatics analyses showed that this region contained three putative Sp1 binding sites, and mutational analyses revealed that all the Sp1 sites contributed to transcriptional activity. Secondly, the overexpression of Sp1 was found to increase the activity of the IRF5 promoter and the mRNA level of IRF5, determined using reporter gene assays and polymerase chain reaction analysis, respectively. By contrast, treatment with mithramycin and Sp1 small interfering RNA significantly reduced the activity of the IRF5 promoter and the mRNA level of IRF5. Finally, the results of an electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated that Sp1 bound to the promoter region of IRF5 in vitro and in vivo. These results suggested that the Sp1 transcription factor is the primary determinant for activating the basal transcription of the IRF5.
转录因子干扰素调节因子5（IRF5）在I型干扰素、促炎细胞因子和趋化因子的诱导中起重要作用，并参与自身免疫性疾病和肿瘤发生。然而，野生型IRF5转录调控的潜在机制仍有待充分阐明。本研究主要旨在阐明特异性蛋白1（Sp1）是否参与IRF5的调控。首先，克隆了IRF5启动子区域，并使用Hela和HEK 293细胞检测其启动子活性。缺失分析表明，跨度为-179至+62的区域是IRF5的最小启动子。生物信息学分析表明，该区域包含三个假定的Sp1结合位点，突变分析表明所有Sp1位点均对转录活性有贡献。其次，发现Sp1的过表达增加了IRF5启动子的活性和IRF5的mRNA水平，分别使用报告基因测定和聚合酶链反应分析确定。相比之下，用放线菌素D和Sp1小干扰RNA处理显著降低了IRF5启动子的活性和IRF5的mRNA水平。最后，电泳迁移率变动分析和染色质免疫沉淀分析的结果表明，Sp1在体外和体内均与IRF5的启动子区域结合。这些结果表明，Sp1转录因子是激活IRF5基础转录的主要决定因素。
