Rein T, Schreck R, Willenbrink W, Neubert W J, Zorbas H, Bäuerle P A
Institut für Biochemie der Ludwig-Maximilians-Universität, Munich, Germany.
J Inflamm. 1995;45(4):269-82.
The promoter of the interferon regulatory factor-1 (IRF-1) gene contains at position -47 to -38 an evolutionary conserved binding sequence for the inducible transcription factor NF-kappa B. This site is highly homologous to a transcriptionally active site from the MHC class I enhancer. In this study, we show by in vitro assays using purified NF-kappa B that the kappa B motif in the IRF-1 promoter binds the factor specifically and with high affinity, comparable to various other cis-acting kappa B elements. Two copies of the IRF-1 kappa B site fused to the heterologous c-fos promoter conferred induction of a chloramphenicol acetyl transferase (CAT) reported gene in response to stimulation of L929 fibroblasts with various NF-kappa B inducers, such as tumor necrosis factor alpha (TNF alpha) or phorbol 12-myristate 13-acetate (PMA). Mutation of the binding site completely abolished transcriptional inducibility of the heterologous promoter. Surprisingly, the same IRF-1 kappa B motif in context of the homologous IRF-1 promoter was transcriptionally inactive in CAT assays. The very weak induction of the IRF-1 promoter in response to TNF treatment or infection of fibroblasts with Newcastle disease virus (NDV) was barely affected by point mutation of the kappa B site or loss of the site by truncation of the promoter. Analysis of the occupational state of the chromosomal IRF-1 kappa B site by in vivo foot-printing revealed that no footprint was induced over the kappa B motif in the IRF-1 promoter after PMA treatment of L929 fibroblast cells, despite the simultaneous induction of IRF-1 mRNA and NF-kappa B binding activity. Constitutive footprints were detected at a CCAAT and GC-rich region in the promoter. This is the first example of a high-affinity NF-kappa B binding site within a promoter which may not participate in transcriptional regulation under conditions activating NF-kappa B DNA binding and gene expression.
干扰素调节因子-1（IRF-1）基因的启动子在-47至-38位含有一个进化保守的诱导型转录因子NF-κB结合序列。该位点与MHC I类增强子的一个转录活性位点高度同源。在本研究中，我们通过使用纯化的NF-κB进行体外试验表明，IRF-1启动子中的κB基序特异性且高亲和力地结合该因子，与其他各种顺式作用κB元件相当。与异源c-fos启动子融合的两个IRF-1 κB位点拷贝，在使用各种NF-κB诱导剂（如肿瘤坏死因子α（TNFα）或佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA））刺激L929成纤维细胞后，可诱导氯霉素乙酰转移酶（CAT）报告基因的表达。结合位点的突变完全消除了异源启动子的转录诱导性。令人惊讶的是，在CAT试验中，同源IRF-1启动子背景下的相同IRF-1 κB基序在转录上是无活性的。IRF-1启动子对TNF处理或新城疫病毒（NDV）感染成纤维细胞的非常微弱的诱导，几乎不受κB位点的点突变或启动子截短导致该位点缺失的影响。通过体内足迹分析染色体IRF-1 κB位点的占据状态发现，尽管同时诱导了IRF-1 mRNA和NF-κB结合活性，但在用PMA处理L929成纤维细胞后，IRF-1启动子中的κB基序上没有诱导出足迹。在启动子的一个CCAAT和富含GC的区域检测到组成型足迹。这是启动子内一个高亲和力NF-κB结合位点的首个例子，在激活NF-κB DNA结合和基因表达的条件下，该位点可能不参与转录调控。
