Sterling K, Bresnick E
Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical Center, Worcester 01655, USA.
Mol Pharmacol. 1996 Feb;49(2):329-37.
The rat CYP1A1 negative regulatory element (NRE) contains AP-1 and Oct-1 motifs at -808 to -788 bp. The CYP1A1 sequence from -813 to -779 bp and an identical sequence bearing a point mutation in the octamer motif were synthesized. Gel mobility shift assays showed the formation of two complexes with the wild-type CYP1A1 sequence and nuclear extracts from H4IIE and HepG2 hepatoma cells and from rat liver. The formation of the major complex was significantly reduced with the mutant octamer-containing oligomer and was specifically competed by an Oct-1 oligodeoxyribonucleotide. The addition of Oct-1 antibody caused a supershift of the major complex. The presence of the wild-type sequence, but not the mutant octamer sequence, caused a 3-fold decrease in SV40 enhancerless promoter activity in transfected HepG2 cells. Co-transfection of an Oct-1 expression vector with rat CYP1A1 NRE octamer-containing, promoter/reporter gene constructs specifically further decreased promoter activity of the wild-type octamer-containing constructs in HepG2 cells. The results indicate that Oct-1 binds to the rat CYP1A1 promoter NRE and is a negative regulator of rat CYP1A1 expression.
大鼠细胞色素P450 1A1（CYP1A1）负调控元件（NRE）在-808至-788碱基对处含有激活蛋白-1（AP-1）和八聚体转录因子1（Oct-1）基序。合成了从-813至-779碱基对的CYP1A1序列以及在八聚体基序处带有一个点突变的相同序列。凝胶迁移率变动分析显示，野生型CYP1A1序列与来自H4IIE和HepG2肝癌细胞以及大鼠肝脏的核提取物形成了两种复合物。含突变八聚体的寡聚物使主要复合物的形成显著减少，并且被Oct-1寡脱氧核糖核苷酸特异性竞争。加入Oct-1抗体导致主要复合物出现超迁移。野生型序列而非突变八聚体序列的存在，使转染的HepG2细胞中无SV40增强子的启动子活性降低了3倍。将Oct-1表达载体与含大鼠CYP1A1 NRE八聚体的启动子/报告基因构建体共转染，在HepG2细胞中特异性地进一步降低了含野生型八聚体构建体的启动子活性。结果表明，Oct-1与大鼠CYP1A1启动子NRE结合，并且是大鼠CYP1A1表达的负调控因子。
