Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins.
Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins.
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C/EBP 相关蛋白对 CYP2B1 和 CYP2B2 基因的转录调节。
DOI：
10.1016/0006-2952(95)02190-6
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发表时间：
1996
影响因子：
5.8
通讯作者：
Shaw,PM
中科院分区：
文献类型：
--
作者：
Luc,PV;Adesnik,M;Ganguly,S;Shaw,PM
细胞色素P450(Cyp)2B1和2B2是由两个密切相关的基因--细胞色素P450(CyP)2B1和2B2编码的，它们在成年大鼠肝脏中低水平表达，但在动物体内被药物苯巴比妥(PB)或其他结构无关的疏水化合物显著诱导。人们对控制这些基因的基础转录和诱导转录的分子机制知之甚少。我们已经确定了两个肝脏特异的DNase I与CYP2B1和CYP2B2(CYP2B)基因相关的超敏位点。其中一个位点位于−2.2和−2.3kb之间的5‘侧翼区，对DNA ase I裂解具有更强的抵抗力；另一个超敏位点则相反，它位于−0.0 5和−0.15kb之间的近端启动子区域。DNase1足迹分析显示，在近端超敏部位附近的启动子区域有三个显著的足迹区域和一个微弱的足迹区域。利用竞争对手寡核苷酸，我们确定了位于−42和−66之间的一个足迹区域(FT2)可能代表CCAATT增强子结合蛋白(C/EBP)家族成员的结合位点。事实上，细菌表达了结合在这个位点的重组C/eBPα，并形成了与肝核提取液观察到的相同的足迹模式。体外转录分析表明，FT2位点对启动子活性的贡献很大，因为它的突变使转录减少了80%。通过足迹分析确定的另外两个位置(FT1和FT3)也需要在体外转录实验中维持CYP2B2启动子结构的高基础转录。瞬时转染实验证实了C/eBPα可以激活1.4kb的CYP2B启动子的预期，FT2位点的突变会削弱外源C/eBPα的基础转录和反式激活。
Cytochrome P450 (CYP) 2B1 and 2B2 are encoded by two closely related genes, CYP2B1 and CYP2B2, that are expressed at low levels in adult rat liver but are induced markedly by the administration of the drug phenobarbital (PB) or other structurally unrelated hydrophobic compounds to animals. Very little is understood about the molecular mechanisms that control both basal and induced transcription of these genes. We have identified two liver specific DNase I hypersensitive sites associated with the CYP2B1 and CYP2B2 (CYP2B) genes. One site, which maps to a region in the 5′-flanking region between −2.2 and −2.3 kb, became more resistant to DNase I cleavage in nuclei from PB-treated rats; the converse was true of the other hypersensitive site, which maps to the proximal promoter region between −0.05 and −0.15 kb. DNase 1 footprint analysis revealed three prominent and one weak footprinted regions in the promoter region in the vicinity of the proximal hypersensitive site. Using competitor oligonucleotides, we determined that one footprinted region (FT2), between −42 and −66 bp, is likely to represent a binding site for CCAATT enhancer binding protein (C/EBP) family members. Indeed, bacterial expressed recombinant C/EBPα bound at this site and formed a footprint pattern identical to the pattern observed with liver nuclear extract. In vitro transcription assays demonstrated that the FT2 site contributed strongly to promoter activity, since its mutation reduced transcription by 80%. Two other sites identified by footprint analysis (FT1 and FT3) are also required to maintain high basal transcription of CYP2B2 promoter constructs in an in vitro transcription assay. Transient transfection experiments confirmed the expectation that C/EBPα could activate the 1.4 kb CYP2B promoter constructs, with mutation of the FT2 site impairing both basal transcription and transactivation by exogenous C/EBPα.