Shuler F D, Chu W W, Wang S, Evans M I
Department of Biochemistry, School of Medicine,. Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown 26506-9142, USA.
DNA Cell Biol. 1998 Aug;17(8):689-97. doi: 10.1089/dna.1998.17.689.
During periods of egg laying in the chicken, when circulating levels of estrogen are increased, the liver-specific estrogen-dependent very low density apolipoprotein II (apoVLDLII) gene is expressed. This expression takes place primarily at the level of transcription, driven by two estrogen response elements that reside in the promoter. In transient transfection assays, expression is increased fourfold when the first intron is added to the promoter construct, indicating that 75% of the regulation comes from intron A. Using in vitro DNase I footprinting, six protein-binding sites were revealed throughout the first intron. The functional significance of these binding sites was evaluated by mutation and transient transfection. Two of the protein-binding regions were shown to increase transcription. Site-specific mutations introduced at either the +66 to +86 or +112 to +129 sites disrupted trans-factor binding and reduced the estrogen-dependent expression by 45% and 34%, respectively. A plasmid containing both mutations resulted in a 43% decrease in expression, indicating that the contributions of these regions are not additive. Competition with known sequences in electrophoretic mobility shift assays suggested that the +66 to +86 site binds a chicken member of the nuclear receptor transcription factor family.
在母鸡产蛋期间，当循环中的雌激素水平升高时，肝脏特异性雌激素依赖性极低密度载脂蛋白II（apoVLDLII）基因会表达。这种表达主要发生在转录水平，由启动子中的两个雌激素反应元件驱动。在瞬时转染实验中，当将第一个内含子添加到启动子构建体中时，表达增加了四倍，这表明75%的调控来自内含子A。使用体外DNase I足迹法，在整个第一个内含子中发现了六个蛋白质结合位点。通过突变和瞬时转染评估了这些结合位点的功能意义。其中两个蛋白质结合区域显示出可增加转录。在+66至+86或+112至+129位点引入的位点特异性突变破坏了反式因子结合，分别使雌激素依赖性表达降低了45%和34%。含有这两种突变的质粒导致表达下降43%，这表明这些区域的作用不是累加的。在电泳迁移率变动分析中与已知序列的竞争表明，+66至+86位点结合核受体转录因子家族的一个鸡成员。
