Yan D H, Hung M C
Department of Tumor Biology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Mol Cell Biol. 1991 Apr;11(4):1875-82. doi: 10.1128/mcb.11.4.1875-1882.1991.
We used chloramphenicol acetyltransferase (CAT) assays to identify and characterize cis-acting elements responsible for rat neu promoter function. Deletion of a region of the neu promoter (-504 to -312) resulted in a marked decrease in CAT activity, indicating that this promoter region corresponds to a positive cis-acting element. Using band shift assays and methylation interference analyses, we further identified a specific protein-binding sequence, AAGATAAAACC (-466 to -456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. Our results demonstrate that RVF is the major DNA-binding protein contributing to enhancer activity. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.
我们使用氯霉素乙酰转移酶（CAT）分析来鉴定和表征负责大鼠neu启动子功能的顺式作用元件。删除neu启动子的一个区域（-504至-312）导致CAT活性显著降低，表明该启动子区域对应一个正向顺式作用元件。通过凝胶迁移分析和甲基化干扰分析，我们进一步鉴定出一个特定的蛋白质结合序列AAGATAAAACC（-466至-456），它结合一种特定的反式作用因子，称为RVF（针对neu启动子上的EcoRV因子）。RVF结合位点是大鼠neu启动子最大转录活性所必需的。在人和小鼠neu启动子的相应区域也发现了相同的序列。此外，该序列可以以方向独立的方式增强由胸苷激酶基因最小启动子驱动的CAT活性，因此它表现为一种增强子。我们的结果表明，RVF是对增强子活性有贡献的主要DNA结合蛋白。此外，使用RVF结合位点作为探针的蛋白质印迹（DNA-蛋白质）分析表明，一种60 kDa的多肽是RVF的潜在候选物。
