Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter - Basal transcription is mediated by Sp1 and Sp3
Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter - Basal transcription is mediated by Sp1 and Sp3
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DOI：
10.1074/jbc.m101814200
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发表时间：
2001-09-07
影响因子：
4.8
通讯作者：
Eling, TE
中科院分区：
文献类型：
--
作者：
Baek, SJ;Horowitz, JM;Eling, TE
非甾体抗炎药激活基因（NAG-1）是已知的与抗肿瘤活性，属于转化生长因子β超家族。在本研究中，我们克隆了启动子区（-3500至+41），并研究了人NAG-1基因基础表达的转录调控机制。几个潜在的转录因子结合位点在这个区域被确定。基于来自巢式缺失的克隆的结果，在-133和+41碱基对之间的构建体包含三个Sp1结合位点（Sp1-A、Sp1-B和Sp1-C），其赋予NAG-1表达的基础转录特异性活性。当Spi-C位点突变（GG至TT）时，在HCT-116细胞中观察到启动子活性降低60-80%。凝胶位移，共转染，染色质免疫沉淀试验表明，SP转录因子结合到Spl-binding网站和反式激活NAG-1的表达。此外，鸡卵清蛋白上游启动子转录因子I可以与Sp1和Sp3蛋白的C-末端区域相互作用，并通过SpI和Sp3转录因子诱导NAG-1启动子活性。这些结果确定了人NAG-1基础启动子的关键调控区域。此外，结果表明，NAG-1基因的表达水平将取决于Sp蛋白的可用性和辅因子，如鸡卵清蛋白上游启动子转录因子1。
Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-beta superfamily. In the present study, we cloned the promoter region (-3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between -133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C, which confer basal transcription specific activity of NAG-1 expression. When the Spi-C site was mutated (GG to TT), a 60-80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, cotransfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Spl-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter transcription factor I can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through SpI and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1.