Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression.
Nuclear transcription factor Oct-1 binds to the 5'-upstream region of CYP1A1 and negatively regulates its expression.
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DOI：
10.1016/1357-2725(95)00122-0
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发表时间：
1996-02
期刊：
影响因子：
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通讯作者：
Rashid Brat;James A. Weaver;Kenneth M. Sterling;Edward Bresnick
中科院分区：
文献类型：
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作者：
Rashid Brat;James A. Weaver;Kenneth M. Sterling;Edward Bresnick
细胞色素P450依赖性单加氧酶是一个广泛的超家族，催化许多内源性和外源性物质的生物转化。这些血红素蛋白之一，细胞色素P4501 A1，是最密切相关的多环芳烃，如苯并[a]芘，这可能在环境致癌作用的生物活化。在细胞色素P4501 A1基因（CYP 1A 1）的5′-上游区域，距转录位点-843 ~-746碱基对处存在一个负调控元件（NRE）。本研究的目的是确定反式作用蛋白质与顺式元件的任何相互作用。使用大鼠肝细胞核作为反式作用蛋白的来源，并通过聚合酶链反应技术从含有CYPIA 1的质粒中制备生物素化的NRE携带片段（−782至−843 bp）。凝胶迁移率变动分析用于证明该NRE片段和核蛋白之间的相互作用。证明了与CYP 1A 1 5′-上游区域含八聚体基序的特异性结合;这被用作从大鼠肝脏部分纯化转录因子Oct-1的步骤。也使用了常规的层析程序和DNA识别位点阿米蒂层析。用pMCoLUC+和Oct-1表达载体转染HepG 2人肝癌细胞，pMCoLUC+含有作为由CYP 1A 1启动子（包括NRE）驱动的报告基因的hhciferase基因。双转染细胞中的荧光素酶活性/mg蛋白显著低于仅含有pMCoLUC+的细胞。核转录因子Oct-1与大鼠CYP 1A 1的NRE的一部分相互作用，抑制该基因的表达。这些发现可能有助于解释哺乳动物系统中CYP 1A 1基础表达水平低的原因。
The cytochrome P450-dependent monooxygenases, which represent an extended superfamily, catalyze the biotransformation of many endogenous and exogenous substances. One of these hemoproteins, cytochrome P4501A1, is most closely associated with the bioactivation of polycyclic aromatic hydrocarbons such as benzo[alpyrene, which may play a role in environmental carcinogenesis. A negative regulatory element (NRE) has been localized in the 5′-upstream region of the cytochrome P4501A1 gene (CYPlA1) at -843 to -746 base pairs from the site of transcription. The purpose of this research was to define any interactions of trans-acting proteins with this cis element. Rat liver nuclei were used as the source of trans-acting proteins and a biotinylated NRE-bearing fragment (−782 to −843 bp) from a plasmid which contained the CYPIA 1 was prepared by the polymerase chain reaction technique. Gel mobility shift assays were used to demonstrate interactions between this NRE fragment and nuclear proteins. The specific binding to an octamer-containing motif in the 5′-upstream region of CYP1A1 was demonstrated; this was used as a step in the partial purification from rat liver of the transcription factor, Oct-1. Conventional chromatographic procedures and DNA recognition site amity chromatography were also used. HepG2 human hepatoma cells were transfected with both pMCoLUC+which contains the hhciferase gene as a reporter gene driven by the CYP1A1 promoter (including the NRE), and an Oct-1 expression vector. Luciferase activity/mg protein in the doubly-transfected cells was significantly lower than in cells containing only pMCoLUC+. A nuclear transcription factor Oct-1 interacts with a portion of the NRE of the rat CYP1A1, suppressing the expression of this gene. These findings may help to explain the low level of basal expression of CYP1A1 in mammalian systems.