Reddig P J, Kim Y J, Verma A K
Department of Human Oncology, University of Wisconsin-Madison Medical School 53792, USA.
Mol Carcinog. 1996 Oct;17(2):92-104. doi: 10.1002/(SICI)1098-2744(199610)17:2<92::AID-MC6>3.0.CO;2-V.
In a previous study, we narrowed the region of the human ornithine decarboxylase (ODC) promoter responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA) to nt -42 to +54 around the transcription initiation site (Kim YJ, Pan H, Verma AK, Mol Carcinog 10:169-179, 1994). Here we report defining the role of the TATA box in TPA-induced transcription from the -42/+54 ODC promoter fragment. A transversion mutation at the third position of the TATA box (TATAAGT-->TAAAAGT) reduced TPA responsiveness of the reporter construct -42/+54 ODC-Luc by 49%. Electrophoretic mobility shift assays (EMSAs) using HeLa cell nuclear protein extracts revealed no differences in the binding pattern between the natural -42/+54 ODC promoter element and the -42/+54 ODC promoter element containing the T-->A mutation. However, antibodies to the general transcription factor TFIIB disrupted the DNA-protein complexes normally formed with the -42/+54 ODC promoter element in EMSAs. A consensus TATA box oligonucleotide formed two bands, with the faster mobility band displaying enhanced binding with nuclear protein extracts from TPA treated cells. Furthermore, incubation of HeLa cell nuclear extracts with an oligonucleotide containing the ODC TATA box also caused formation of two specific bands in EMSA. Both bands exhibited augmented binding to nuclear proteins from TPA-treated cells. Introduction of the T-->A transversion mutation in the ODC TATA oligonucleotide eliminated binding of the faster migrating band formed with the natural ODC TATA oligonucleotide. These results indicate that TPA modulation of the general transcription machinery may play a role in the TPA-activated transcription of the human ODC promoter.
在之前的一项研究中，我们将人鸟氨酸脱羧酶（ODC）启动子对12-O-十四酰佛波醇-13-乙酸酯（TPA）产生应答的区域缩小至转录起始位点周围的-42至+54核苷酸处（Kim YJ、Pan H、Verma AK，《分子致癌作用》10:169 - 179，1994年）。在此，我们报告确定TATA盒在TPA诱导的来自-42/+54 ODC启动子片段的转录中的作用。TATA盒第三个位置的颠换突变（TATAAGT→TAAAAGT）使报告构建体-42/+54 ODC-Luc的TPA应答性降低了49%。使用HeLa细胞核蛋白提取物进行的电泳迁移率变动分析（EMSA）显示，天然的-42/+54 ODC启动子元件与含有T→A突变的-42/+54 ODC启动子元件之间的结合模式没有差异。然而，针对通用转录因子TFIIB的抗体破坏了在EMSA中通常与-42/+54 ODC启动子元件形成的DNA-蛋白质复合物。一个共有TATA盒寡核苷酸形成了两条带，迁移速度较快的带与来自TPA处理细胞的核蛋白提取物显示出增强的结合。此外，用含有ODC TATA盒的寡核苷酸孵育HeLa细胞核提取物也在EMSA中导致形成两条特异性带。两条带都显示出与来自TPA处理细胞的核蛋白的结合增强。在ODC TATA寡核苷酸中引入T→A颠换突变消除了与天然ODC TATA寡核苷酸形成的迁移速度较快的带的结合。这些结果表明，TPA对通用转录机制的调节可能在人ODC启动子的TPA激活转录中起作用。
