Potter J J, Mezey E, Cornelius P, Crabb D W, Yang V W
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Arch Biochem Biophys. 1992 Jun;295(2):360-8. doi: 10.1016/0003-9861(92)90529-6.
The rat class I alcohol dehydrogenase (ADH) gene is primarily expressed in the liver. We previously showed that the liver-enriched transcription factor, the CCAAT/enhancer binding protein (C/EBP), binds to the proximal promoter of the rat class I ADH gene between positions -11 and -22 relative to the start site of transcription. We now demonstrate that another transcription factor, the liver activator protein (LAP), also interacts with the same region of the promoter based on the following observations: (1) LAP synthesized by in vitro transcription and translation of cloned cDNA sequence forms complexes with an oligonucleotide containing the C/EBP-binding sequence within the ADH promoter as determined by the electrophoretic mobility shift assay (EMSA), (2) purified LAP interacts with the proximal ADH promoter when analyzed by the DNase I protection assay, and (3) an ADH promoter-reporter gene construct containing the C/EBP-binding site is transactivated by an eukaryotic expression vector containing the LAP sequence. EMSA of an oligonucleotide containing the first 22 base pairs (between positions -1 and -22) of the ADH promoter with rat liver nuclear extracts (RLNE) resulted in the formation of two major complexes. Complex 1 was competed away by a heterologous oligonucleotide containing a C/EBP-binding site within the promoter of the adipocyte 422 (aP2) gene, while complex 2 was not. Additional competition experiments with the ADH or 422 (aP2) oligonucleotide using either RLNE or extracts from 3T3-L1 adipocytes demonstrated that complex 1 contains either C/EBP or LAP, while complex 2 contains a DNA-binding protein that binds to a novel sequence 5'-TGGCCCAGTT-3' between positions -1 and -10 of the ADH promoter. Ultraviolet cross-linking between RLNE and a labeled oligonucleotide containing the above sequence indicates that this protein, designated EDBP (for enhancer-site downstream binding protein), has an estimated molecular weight of 47 kDa, which is larger than that reported for either C/EBP (42 kDa) or LAP (36 kDa).
大鼠I类乙醇脱氢酶（ADH）基因主要在肝脏中表达。我们之前表明，肝脏富集转录因子CCAAT/增强子结合蛋白（C/EBP）与大鼠I类ADH基因启动子近端结合，该区域相对于转录起始位点位于-11至-22位之间。我们现在证明，另一种转录因子肝脏激活蛋白（LAP）也与启动子的同一区域相互作用，基于以下观察结果：（1）通过对克隆的cDNA序列进行体外转录和翻译合成的LAP与包含ADH启动子内C/EBP结合序列的寡核苷酸形成复合物，这通过电泳迁移率变动分析（EMSA）得以确定；（2）通过DNase I保护分析可知，纯化的LAP与ADH启动子近端相互作用；（3）含有C/EBP结合位点的ADH启动子-报告基因构建体被含有LAP序列的真核表达载体反式激活。用大鼠肝核提取物（RLNE）对包含ADH启动子前22个碱基对（-1至-22位之间）的寡核苷酸进行EMSA，结果形成了两个主要复合物。复合物1被含有脂肪细胞422（aP2）基因启动子内C/EBP结合位点的异源寡核苷酸竞争掉，而复合物2则没有。使用RLNE或3T3-L1脂肪细胞提取物对ADH或422（aP2）寡核苷酸进行的额外竞争实验表明，复合物1包含C/EBP或LAP，而复合物2包含一种与ADH启动子-1至-10位之间的新序列5'-TGGCCCAGTT-3'结合的DNA结合蛋白。RLNE与含有上述序列的标记寡核苷酸之间的紫外线交联表明，这种被命名为EDBP（增强子位点下游结合蛋白）的蛋白质估计分子量为47 kDa，大于报道的C/EBP（42 kDa）或LAP（36 kDa）的分子量。
