Song C S, Jung M H, Kim S C, Hassan T, Roy A K, Chatterjee B
Department of Cellular and Structural Biology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7762, USA.
J Biol Chem. 1998 Aug 21;273(34):21856-66. doi: 10.1074/jbc.273.34.21856.
Dehydroepiandrosterone sulfotransferase (Std) catalyzes sulfonation of androgenic steroids and certain aromatic procarcinogens. In rats, this enzyme is selectively expressed in the liver, and its expression is strongly repressed by androgens. DNase I footprinting and electrophoretic mobility shift analyses revealed two hepatocyte nuclear factor-1 (HNF1), three CCAAT/enhancer-binding protein (C/EBP), and one consensus palindromic thyroid hormone response elements within the first 215 base pairs (bp) of the promoter sequence of rat Std. This promoter is normally inactive in fibroblast-derived NIH 3T3 cells. However, overexpression of HNF1 and C/EBP resulted in synergistic activation of the Std promoter in this cell type, indicating essential roles of these two trans-regulators in liver-selective expression of the rat Std gene. On the other hand, point mutations at any one of five cis elements proximal to the -215 bp region markedly reduced reporter gene expression, suggesting that all of these sites are important for overall promoter function. Androgenic repression of the Std gene in rat liver can be recapitulated in androgen receptor (AR)-negative HepG2 hepatoma cells after cotransfection with an AR expression plasmid. Functional assay of a nested set of 5'-deleted promoters mapped the negative androgen response region between positions -235 and -310. Antibody supershift and oligonucleotide competition identified three OCT-1 and two C/EBP elements between bp -231 and -292. An additional OCT-1 site was found to overlap with a C/EBP element at the -262/-252 position. Mutational inactivation of any one of five cis elements within the -231/-292 region abolished negative androgen response. However, none of these cis elements showed DNase I protection by recombinant AR in footprinting assay, suggesting the absence of a direct AR-DNA interaction. Thus, these studies on rat Std promoter function indicate that (i) HNF1 and C/EBP are responsible for liver specificity of the rat Std gene; (ii) androgenic repression of the gene requires the presence of all of the OCT-1 and C/EBP elements between positions -231 and -292; and (iii) AR may exert its negative regulatory effect indirectly through transcriptional interference of OCT-1 and C/EBP rather than through a direct DNA-AR interaction.
硫酸脱氢表雄酮磺基转移酶（Std）催化雄激素类固醇和某些芳香族前致癌物的磺化反应。在大鼠中，这种酶在肝脏中选择性表达，其表达受到雄激素的强烈抑制。DNA酶I足迹法和电泳迁移率变动分析显示，在大鼠Std启动子序列的前215个碱基对（bp）内有两个肝细胞核因子-1（HNF1）、三个CCAAT/增强子结合蛋白（C/EBP）和一个共有回文甲状腺激素反应元件。该启动子在成纤维细胞来源的NIH 3T3细胞中通常无活性。然而，HNF1和C/EBP的过表达导致该细胞类型中Std启动子的协同激活，表明这两种反式调节因子在大鼠Std基因的肝脏选择性表达中起重要作用。另一方面，-215 bp区域附近五个顺式元件中任何一个的点突变都显著降低了报告基因的表达，表明所有这些位点对整体启动子功能都很重要。在与雄激素受体（AR）表达质粒共转染后，在AR阴性的HepG2肝癌细胞中可以重现大鼠肝脏中Std基因的雄激素抑制作用。一组嵌套的5'缺失启动子的功能分析将负雄激素反应区域定位在-235和-310位之间。抗体超迁移和寡核苷酸竞争鉴定出在-231至-292 bp之间有三个OCT-1和两个C/EBP元件。在-262/-252位发现一个额外的OCT-1位点与一个C/EBP元件重叠。-231/-292区域内五个顺式元件中任何一个的突变失活都消除了负雄激素反应。然而，在足迹分析中，这些顺式元件中没有一个显示出重组AR对DNA酶I的保护作用，这表明不存在直接的AR-DNA相互作用。因此，这些对大鼠Std启动子功能的研究表明：（i）HNF1和C/EBP负责大鼠Std基因的肝脏特异性；（ii）该基因的雄激素抑制需要-231至-292位之间所有的OCT-1和C/EBP元件的存在；（iii）AR可能通过对OCT-1和C/EBP的转录干扰间接发挥其负调节作用，而不是通过直接的DNA-AR相互作用。
