Barbulescu K, Meyer zum Büschenfelde K H, Neurath M F
Department of Medicine, University of Mainz, Germany.
Eur J Immunol. 1997 May;27(5):1098-107. doi: 10.1002/eji.1830270509.
Interferon-gamma (IFN-gamma) is a key cytokine of T lymphocytes with major regulatory functions in the immune system. To determine and compare protein/DNA interactions at the native IFN-gamma locus in T cells, we analyzed the human IFN-gamma promoter by ligation-mediated polymerase chain reaction (LM-PCR) techniques. Accordingly, Jurkat T cells and primary CD45RA and CD45R0 CD4+ T cell subsets isolated from peripheral blood using immunomagnetic beads were cultured and analyzed by LM-PCR. Constitutive and inducible protein/DNA interactions of the IFN-gamma promoter in vivo were detected in all T cells tested. Interestingly, an inducible footprint between -183 and -196 was consistently observed in Jurkat T cells and CD45RA and CD45R0 T helper subsets upon stimulation with phorbol 12-myristate 13-acetate+phytohemagglutinin (PMA+PHA) that was highly sensitive to treatment with corticosteroids. This novel target site, denoted the C-site, was shown by several criteria, including cell distribution studies, stimulation experiments, supershift assays, and cross-competition electrophoretic mobility shift assays to bind the transcription factor AP-1. Mutation of the C-site that prevented AP-1 binding to this site was sufficient strikingly to reduce inducible promoter activity in primary CD45R0 T cells. In summary, the data demonstrate that IFN-gamma gene transcription in primary T cells is regulated in vivo at the level of constitutive and inducible protein/DNA interactions. We propose a model where basal transcription is maintained by binding of various transcription factors to the IFN-gamma promoter, whereas PMA+PHA-inducible IFN-gamma transcription in CD45R0 T cells is associated with binding of AP-1 to the C-site.
干扰素-γ（IFN-γ）是T淋巴细胞的一种关键细胞因子，在免疫系统中具有主要调节功能。为了确定并比较T细胞中天然IFN-γ基因座处的蛋白质/DNA相互作用，我们通过连接介导的聚合酶链反应（LM-PCR）技术分析了人IFN-γ启动子。据此，培养了Jurkat T细胞以及使用免疫磁珠从外周血中分离的原代CD45RA和CD45R0 CD4 + T细胞亚群，并通过LM-PCR进行分析。在所有测试的T细胞中均检测到了体内IFN-γ启动子的组成型和诱导型蛋白质/DNA相互作用。有趣的是，在用佛波醇12-肉豆蔻酸酯13-乙酸酯+植物血凝素（PMA + PHA）刺激后，在Jurkat T细胞以及CD45RA和CD45R0 T辅助亚群中始终观察到-183至-196之间的诱导型足迹，该足迹对皮质类固醇治疗高度敏感。通过包括细胞分布研究、刺激实验、超迁移分析和交叉竞争电泳迁移率变动分析在内的多项标准表明，这个新的靶位点（称为C位点）可结合转录因子AP-1。阻止AP-1结合该位点的C位点突变足以显著降低原代CD45R0 T细胞中的诱导型启动子活性。总之，数据表明原代T细胞中的IFN-γ基因转录在体内受组成型和诱导型蛋白质/DNA相互作用水平的调节。我们提出了一个模型，其中基础转录通过各种转录因子与IFN-γ启动子的结合来维持，而CD45R0 T细胞中PMA + PHA诱导的IFN-γ转录与AP-1与C位点的结合相关。
