Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.
Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.
复制标题
转基因小鼠中大鼠 CYP2B2 基因的 5 侧翼区域赋予苯巴比妥反应性。
DOI：
10.1016/s0378-1119(98)00612-x
复制
发表时间：
1999
期刊：
影响因子：
3.5
通讯作者：
Omiecinski,CJ
中科院分区：
文献类型：
--
作者：
Ramsden,R;Beck,NB;Sommer,KM;Omiecinski,CJ
苯巴比妥 (PB) 是一类药物的原型，可对许多基因（包括某些细胞色素 P-450）产生显着的转录激活作用。我们使用转基因小鼠方法和多基因报告基因来评估大鼠 CYP2B2 基因 2.5kb 5' 侧翼区域内特定缺失和位点特异性突变的功能后果。 PBRU 结构域的蛋白质-DNA 相互作用也得到了表征。使用转基因模型，我们证明CYP2B2基因的-2500和-1700bp之间的序列对于PB诱导至关重要；具有 1700 或 800bp 5' 侧翼 CYP2B2 序列的小鼠不具有 PB 反应性。 DNA亲和力富集技术以及免疫印迹和电迁移率分析用于确定核因子1（NF-1）与CYP2B2的PB反应单元（PBRU）中以-2200bp为中心的位点强烈相互作用。为了测试 NF-1 在 PB 激活中的功能贡献，我们在 PBRU NF-1 元件内引入了特定突变，并证明这些突变完全消除了结合相互作用。然而，将突变型 NF-1 序列整合到野生型 -2500/CYP2B2 转基因中的转基因小鼠保持了完全的 PB 反应性。这些结果表明，尽管体外 PBRU 内各自的 DNA-蛋白质相互作用具有亲和力，但 NF-1 相互作用并不是指导体内 PB 转录激活的重要因素。
Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific mutations within the 2.5kb 5′-flanking region of the rat CYP2B2 gene. Protein–DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between −2500 and −1700bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800bp of 5′-flanking CYP2B2 sequence are not PB responsive. DNA affinity enrichment techniques and immunoblotting and electromobility shift assays were used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at −2200bp in the PB responsive unit (PBRU) of CYP2B2. To test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type −2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA–protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.