Lee Y H, Alberta J A, Gonzalez F J, Waxman D J
Laboratory of Molecular Carcinogenesis, NCI, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 May 20;269(20):14681-9.
Hepatic cytochrome P450 cholesterol 7 alpha-hydroxylase, CYP7, is regulated in vivo at the protein and the mRNA level in response to multiple physiological factors, including liver cholesterol synthesis, bile acid feedback inhibition, and diurnal rhythm. In the present study we investigated whether the liver transcription factor DBP (albumin promoter D-site binding protein), which undergoes a striking diurnal rhythm in rat liver (DBP levels during evening/morning approximately 100:1), contributes to the diurnal regulation of CYP7 gene expression. DNase I footprinting analysis using bacterially expressed DBP and a cloned 5'-flanking DNA segment of the rat CYP7 gene revealed five distinct DBP-binding sites, designated A-E, distributed between nucleotides (nts) -41 and -295 relative to the CYP7 transcription start site. CYP7-directed gene transcription in HepG2 cells transfected with a 5'-CYP7 promoter-chloramphenicol acetyl-transferase reporter was activated up to 12-fold upon cotransfection of a DBP expression vector, whereas an HNF-1 alpha expression vector did not stimulate CYP7 gene activity. 5'-Deletion analyses and site-specific mutagenesis revealed that this stimulating effect of DBP can in part be ascribed to its functional interaction with DBP binding sites B (nts -115/-125), C (nts -172/-195), and D (nts -214/-230). C/EBP beta (LAP), another liver-enriched basic-leucine zipper transcription factor, bound to these same sites but effected a more modest increase in CYP7-directed gene transcription (up to 3-4-fold) when expressed in HepG2 cells. Competition for CYP7 promoter-binding sites between C/EBP, which undergoes < or = 2-fold diurnal change in rat liver, and the diurnally regulated DBP is proposed to determine the relative rates of basal versus diurnally regulated CYP7 gene transcription and thus may be a primary mechanism for setting the 3-6-fold amplitude that characterizes the circadian rhythm of liver CYP7 expression. Moreover, since DBP is first expressed in rat liver 3-4 weeks after birth, these findings may account for both the enhanced expression and the onset of the diurnal pattern of CYP7 enzyme levels at this stage of development.
肝脏细胞色素P450胆固醇7α-羟化酶（CYP7）在体内受多种生理因素调控，包括肝脏胆固醇合成、胆汁酸反馈抑制和昼夜节律，在蛋白质和mRNA水平均有响应。在本研究中，我们调查了大鼠肝脏中呈现显著昼夜节律的肝脏转录因子DBP（白蛋白启动子D位点结合蛋白，傍晚/早晨的DBP水平约为100:1）是否参与CYP7基因表达的昼夜调节。使用细菌表达的DBP和大鼠CYP7基因的克隆5'侧翼DNA片段进行的DNase I足迹分析显示，有五个不同的DBP结合位点，命名为A - E，分布在相对于CYP7转录起始位点的核苷酸（nts）-41至-295之间。用5'-CYP7启动子-氯霉素乙酰转移酶报告基因转染的HepG2细胞中，CYP7指导的基因转录在共转染DBP表达载体后被激活高达12倍，而HNF-1α表达载体则不刺激CYP7基因活性。5'缺失分析和位点特异性诱变表明，DBP的这种刺激作用部分可归因于其与DBP结合位点B（nts -115/-125）、C（nts -172/-195）和D（nts -214/-230）的功能相互作用。另一种肝脏富集的碱性亮氨酸拉链转录因子C/EBPβ（LAP）也结合到这些相同位点，但在HepG2细胞中表达时，对CYP7指导的基因转录的增加作用较小（高达3 - 4倍）。大鼠肝脏中昼夜变化≤2倍的C/EBP与昼夜调节的DBP之间对CYP7启动子结合位点的竞争，被认为决定了基础转录与昼夜调节的CYP7基因转录的相对速率，因此可能是设定肝脏CYP7表达昼夜节律特征的3 - 6倍幅度的主要机制。此外，由于DBP在大鼠出生后3 - 4周首次在肝脏中表达，这些发现可能解释了在发育的这个阶段CYP7酶水平的表达增强和昼夜模式的出现。
