Pützer B M, Gnauck J, Kirch H C, Brockmann D, Esche H
Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Germany.
J Gen Virol. 1997 Apr;78 ( Pt 4):879-91. doi: 10.1099/0022-1317-78-4-879.
Transcription of the E1A gene of the highly oncogenic adenovirus 12 (Ad12) initiates at two start sites (TS1 and TS2). We have previously shown that the E2F and ATF motifs distal of TS1 co-operatively participate in E1A autostimulation from the TS1 promoter region. Here we report the identification of a second E2F-like target region (E2DFII) immediately upstream of the E1A-stimulating factor 1 binding site (ESF-1), important for 13S-mediated autoactivation from TS2. Reporter constructs lacking distinct TS2 cis-acting elements were analysed for their levels of CAT expression in the absence and presence of the E1A 13S protein in transient expression assays. In the absence of 13S, full promoter activity was observed only for a construct containing all elements (the E2F-like motif, and E-Box and the TATA element). Promoter activation increased significantly in Ad12 E1A-co-transfected cells. Induction by the 13S protein was also detected for the construct containing a non-functional ESF-1 sequence. Our results indicate that the E2F-like motif is responsible for activation medicated by the 13S protein from TS2, while ESF-1-or TATA-binding protein activity were not involved. Additionally, the TATA sequence appeared to be dispensable for transactivation. Gel-shift experiments using the E2F-like promoter element as a probe indicated the binding of an E2F-5 or E2F-5-like transcription factor to this region. We conclude that transcription through the TS1 as well as the TS2 promoter region is stimulated by the Ad 12 13S protein. Moreover, transfection of the construct including both TS1 and TS2 indicates an E2F-site-mediated synergism between both regions with respect ot E1A-induced transactivation.
高致癌性腺病毒12（Ad12）的E1A基因转录起始于两个起始位点（TS1和TS2）。我们先前已经表明，TS1远端的E2F和ATF基序协同参与来自TS1启动子区域的E1A自激活。在此，我们报告了在E1A刺激因子1结合位点（ESF-1）紧邻上游鉴定出第二个E2F样靶区域（E2DFII），这对于从TS2进行的13S介导的自激活很重要。在瞬时表达试验中，分析了缺乏不同TS2顺式作用元件的报告基因构建体在有无E1A 13S蛋白情况下的CAT表达水平。在没有13S的情况下，仅对于包含所有元件（E2F样基序、E盒和TATA元件）的构建体观察到完全的启动子活性。在Ad12 E1A共转染的细胞中，启动子激活显著增加。对于包含无功能ESF-1序列的构建体，也检测到了13S蛋白的诱导作用。我们的结果表明，E2F样基序负责13S蛋白从TS2介导的激活，而ESF-1或TATA结合蛋白活性未参与其中。此外，TATA序列对于反式激活似乎是可有可无的。使用E2F样启动子元件作为探针的凝胶迁移实验表明，一种E2F-5或E2F-5样转录因子与该区域结合。我们得出结论，Ad 12 13S蛋白刺激通过TS1以及TS2启动子区域的转录。此外，包含TS1和TS2的构建体的转染表明，在E1A诱导的反式激活方面，两个区域之间存在E2F位点介导的协同作用。
