Halvorson L M, Kaiser U B, Chin W W
Division of Genetics, Department of Medicine, Brigham and Women's Hospital and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1996 Mar 22;271(12):6645-50. doi: 10.1074/jbc.271.12.6645.
The orphan nuclear receptor, steroidogenic factor-1 (SF-1), is expressed in the pituitary and in the gonadotrope precursor cell line, alphaT3-1, where it is believed to enhance expression of the common gonadotropin alpha-subunit gene through transactivation of the gonadotrope-specific element (GSE). Sequence analysis of the rat luteinizing hormone beta-subunit (LH beta) gene promoter revealed the presence of a consensus GSE at -127 to -119 (TGACCTTGT). We have demonstrated the ability of SF-1 to bind specifically to this putative GSE sequence by electrophoretic mobility shift assay, utilizing both alphaT3-1 nuclear extracts and in vitro translated SF-1. In addition, mutation of the putative LHbeta-GSE (TGAAATTGT) eliminated specific DNA binding. To examine the ability of SF-1 to enhance LHbeta promoter activity, CV-1 cells, which lack endogenous SF-1, were cotransfected with an SF-1-containing expression vector and an LHbeta-luciferase reporter construct. When cotransfected with -209/+5 of the LHbeta promoter, SF-1 increased luciferase activity by 56-fold. SF-1 responsiveness was markedly diminished with loss of the putative GSE region in deletion constructs and in the presence of a two base pair mutation, analogous to the mutation which eliminated DNA binding. Finally, the LHbeta-GSE was able to confer SF-1 responsiveness on a heterologous minimal growth hormone promoter, GH50 (57-fold). We conclude that SF-1 both binds to and transactivates the rat LHbeta promoter. These data suggest that SF-1 may participate in the expression of the LHbeta gene by the gonadotrope.
孤儿核受体类固醇生成因子1（SF-1）在垂体和促性腺激素前体细胞系αT3-1中表达，据信它通过激活促性腺激素特异性元件（GSE）来增强共同促性腺激素α亚基基因的表达。大鼠促黄体生成素β亚基（LHβ）基因启动子的序列分析显示，在-127至-119（TGACCTTGT）处存在一个共有GSE。我们利用αT3-1核提取物和体外翻译的SF-1，通过电泳迁移率变动分析证明了SF-1能够特异性结合这个假定的GSE序列。此外，假定的LHβ-GSE（TGAAATTGT）的突变消除了特异性DNA结合。为了检测SF-1增强LHβ启动子活性的能力，将缺乏内源性SF-1的CV-1细胞与含SF-1的表达载体和LHβ-荧光素酶报告构建体共转染。当与LHβ启动子的-209/+5共转染时，SF-1使荧光素酶活性增加了56倍。在缺失构建体中假定的GSE区域缺失以及存在一个两碱基对突变（类似于消除DNA结合的突变）时，SF-1的反应性显著降低。最后，LHβ-GSE能够赋予异源最小生长激素启动子GH50 SF-1反应性（57倍）。我们得出结论，SF-1既能结合大鼠LHβ启动子，又能激活它。这些数据表明，SF-1可能参与促性腺激素细胞中LHβ基因的表达。
