Ohmori Y, Hamilton T A
Research Institute, Cleveland Clinic Foundation, Ohio 44195.
J Biol Chem. 1993 Mar 25;268(9):6677-88.
The transcriptional regulation of the murine IP-10 gene in lipopolysaccharide (LPS) or interferon gamma (IFN gamma)-treated macrophages was investigated by analysis of regions of the gene that flank the transcription start site. A series of sequence fragments were placed 5' to the chloramphenicol acetyltransferase (CAT) reporter gene and ability to mediate transcription of CAT in response to IFN gamma or LPS treatment was studied following transient transfection in the macrophage-like cell line RAW 264.7. Analysis of larger constructs identified a potential negative regulatory site for IFN gamma response in the region between nucleotide positions -2002 and -930 and a positive regulator for LPS response in the region between bases -930 and -676. A 227-base fragment spanning positions -228 to -2 was the minimal sequence able to mediate LPS- and IFN gamma-dependent transcription of CAT. Deletion of 24 bases, which included a highly conserved IFN stimulus response element (ISRE) from the -228 construct, abolished response to IFN gamma. A 33-base fragment containing the IP-10 ISRE was able to confer both IFN gamma and LPS sensitivity upon a heterologous promoter. The ability of LPS to stimulate CAT via the ISRE was apparently mediated by intermediate expression of endogenous IFN alpha/beta. Elimination of bases -204 to -102 abolished sensitivity to LPS. This region contains two kappa B binding sites. Site-directed mutagenesis of key nucleotides in the ISRE and the two kappa B sites demonstrated that optimal response to IFN gamma required both the ISRE and one of the two kappa B sites, whereas optimal response to LPS required either both kappa B sites or one kappa B site and the ISRE. IFN gamma or LPS treatment induced sequence-specific binding activity for the ISRE and the two kappa B sites. These results indicate that the 230 nucleotides upstream from the transcription start site are important for transcriptional control of the IP-10 gene in response to IFN gamma and LPS. The three defined regulatory elements function in distinct fashion for each of the two stimuli; optimal response to either IFN gamma or LPS requires cooperation between at least two sites.
通过分析小鼠IP - 10基因转录起始位点侧翼区域，研究了脂多糖（LPS）或干扰素γ（IFNγ）处理的巨噬细胞中该基因的转录调控。将一系列序列片段置于氯霉素乙酰转移酶（CAT）报告基因的5'端，并在巨噬细胞样细胞系RAW 264.7中进行瞬时转染后，研究其在IFNγ或LPS处理下介导CAT转录的能力。对更大构建体的分析确定了在核苷酸位置 - 2002至 - 930之间的区域中存在一个潜在的IFNγ反应负调控位点，以及在碱基 - 9至 - 676之间的区域中存在一个LPS反应正调控位点。一个跨越位置 - 228至 - 2的227个碱基的片段是能够介导CAT的LPS和IFNγ依赖性转录的最小序列。从 - 228构建体中缺失24个碱基（其中包括一个高度保守的IFN刺激反应元件（ISRE）），消除了对IFNγ的反应。一个包含IP - 10 ISRE的33个碱基的片段能够赋予异源启动子对IFNγ和LPS的敏感性。LPS通过ISRE刺激CAT的能力显然是由内源性IFNα/β的中间表达介导。消除 - 204至 - 102碱基消除了对LPS的敏感性。该区域包含两个κB结合位点。对ISRE和两个κB位点中的关键核苷酸进行定点诱变表明，对IFNγ的最佳反应需要ISRE和两个κB位点之一，而对LPS的最佳反应需要两个κB位点或一个κB位点和ISRE。IFNγ或LPS处理诱导了ISRE和两个κB位点的序列特异性结合活性。这些结果表明，转录起始位点上游的230个核苷酸对于IP - 10基因响应IFNγ和LPS的转录控制很重要。对于这两种刺激中的每一种，三个定义的调控元件以不同方式起作用；对IFNγ或LPS的最佳反应需要至少两个位点之间协同作用。
