Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2.
Nuclear factor-1 motif and redundant regulatory elements comprise phenobarbital-responsive enhancer in CYP2B1/2.
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核因子 1 基序和冗余调节元件构成 CYP2B1/2 中的苯巴比妥反应增强子。
DOI：
10.1089/dna.1998.17.461
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发表时间：
1998
影响因子：
3.1
通讯作者：
Kemper,B
中科院分区：
文献类型：
--
作者：
Liu,S;Park,Y;Rivera-Rivera,I;Li,H;Kemper,B
虽然苯巴比妥对药物代谢系统的诱导作用已被认识了约40年，但细胞色素P450基因表达增加的机制仍不清楚。CYP 2B 2中约-2.2Kb的163-bp片段已显示在原代大鼠肝细胞中介导苯巴比妥诱导（Trottier，et al. [1995] Gene 158：263-268），并且通过大鼠肝中的原位瞬时转染测定（Park，Y.，等人[1996]。271：23725-23728）。该片段的缺失突变表明，如果检测与CYP 2C 1近端启动子融合的缺失片段的单拷贝，则从-2258到-2170的88-bp延伸是在原位系统中能够介导苯巴比妥诱导的最小序列。如果存在三个拷贝的片段，5′和3′缺失定义了最小的37 bp核心片段，尽管这是苯巴比妥反应所必需的，但这是不够的，除非在两端存在额外的序列，这表明在两个侧翼区域存在冗余元件。88-bp片段内NF-1位点的位点特异性诱变和跨片段的接头扫描诱变表明，NF-1位点和该位点5′侧的区域有助于反应的幅度，但NF-1突变和任何接头扫描突变都不能消除对苯巴比妥的反应。NF-1位点3′区的突变导致基础表达升高，而对苯巴比妥诱导的表达无实质性影响。通过凝胶移位竞争研究和NF-1抗血清对蛋白质-DNA复合物的supershifts建立NF-1与37 bp核心片段的结合。在NF-1位点侧翼区域检测到额外的蛋白质-DNA复合物。这些研究表明，CYP 2B 2苯巴比妥反应增强子含有多个组成元件和苯巴比妥反应元件。从控制或苯巴比妥处理的animalin体外核蛋白结合到这个区域是非常相似的。检测到的唯一差异是一种复合物，该复合物被苯巴比妥处理大幅减少，并定位于NF-1位点的3′侧。
Although the induction of drug-metabolizing systems by phenobarbital has been recognized for about 40 years, the mechanism by which cytochrome P450 gene expression is increased is still not well understood. A 163-bp fragment at about -2.2 Kb inCYP2B2has been shown to mediate phenobarbital induction in primary rat hepatocytes (Trottier,et al. [1995] Gene 158:263-268) and by anin situtransient transfection assay in rat liver (Park, Y.,et al. [1996]. J. Biol. Chem. 271:23725-23728). Deletion mutations of this fragment indicated that the 88-bp stretch from -2258 to -2170 was the minimal sequence that could mediate phenobarbital induction in the in situ system if single copies of the deleted fragments fused to theCYP2C1proximal promoter were assayed. If three copies of the fragments were present, 5′ and 3′ deletions defined a minimal 37-bp core fragment, which, although necessary for phenobarbital responsiveness, was not sufficient unless additional sequence was present at either end, suggesting that redundant elements were present in the two flanking regions. Site-specific mutagenesis of an NF-1 site within the 88-bp fragment and linker scanning mutagenesis across the fragment indicated that the NF-1 site and a region to the 5′ side of the site contributed to the magnitude of the response, but neither the NF-1 mutations nor any of the linker scanning mutations eliminated the response to phenobarbital. Mutation in a region 3′ of the NF-1 site resulted in elevated basal expression without substantial effects on phenobarbital-induced expression. Binding of NF-1 to the 37-bp core fragment was established by gel-shift competition studies and by supershifts of the protein-DNA complexes by antisera to NF-1. Additional protein-DNA complexes were detected in the regions flanking the NF-1 site. These studies indicate that theCYP2B2phenobarbital-responsive enhancer contains multiple constitutive and phenobarbital-responsive elements. Binding of nuclear proteins from control or phenobarbital-treated animalsin vitroto this region was very similar. The only difference detected was a complex that was substantially reduced by phenobarbital treatment and mapped to the 3′ side of the NF-1 site.