Quek Sue Ing, Chan Woon Khiong
Department of Biological Sciences, National University of Singapore, Singapore, Republic of Singapore 117543.
J Mol Endocrinol. 2009 Sep;43(3):121-30. doi: 10.1677/JME-09-0029. Epub 2009 May 28.
The cytochrome P450scc (cholesterol side-chain cleavage enzyme) encoded by CYP11A1 catalyzes the first step in steroidogenesis by converting cholesterol to pregnenolone, and thus, controls the synthesis rate of steroid hormones. In mammals, steroidogenic factor 1 (SF1) has been implicated in the cAMP-mediated transcriptional activation of CYP11A1 promoter. In zebrafish, Ff1b has been established as the homolog of SF1. To assess the dependency of cyp11a1 expression on Ff1b, the putative promoter of zebrafish cyp11a1, spanning 1.7 kb, was isolated and bioinformatic analysis revealed two conserved FF1 response elements (FREs) that potentially bind Ff1b. Transfection studies in cell lines of different lineages confirmed that this promoter fragment contained the necessary regulatory elements required for its basal transcription. Truncation and mutagenesis studies performed in Y1 adrenocortical cells revealed that only the proximal FRE was essential for transcriptional activation. Electrophoretic mobility shift assay, however, indicated that Ff1b bound to both FREs, while their in vivo occupancy was confirmed using a chromatin immunoprecipitation assay. Lastly, the cyp11a1 promoter was able to direct EGFP expression specifically to the interrenal gland and genital ridge when transiently expressed in microinjected zebrafish embryos, and the promoter activity is potentiated by ff1b overexpression as measured from luciferase reporter activity in zebrafish embryos.
由CYP11A1编码的细胞色素P450scc（胆固醇侧链裂解酶）通过将胆固醇转化为孕烯醇酮来催化类固醇生成的第一步，因此，它控制着类固醇激素的合成速率。在哺乳动物中，类固醇生成因子1（SF1）参与了cAMP介导的CYP11A1启动子的转录激活。在斑马鱼中，Ff1b已被确定为SF1的同源物。为了评估cyp11a1表达对Ff1b的依赖性，分离了斑马鱼cyp11a1的推定启动子，其跨度为1.7 kb，生物信息学分析揭示了两个保守的FF1反应元件（FREs），它们可能与Ff1b结合。在不同谱系的细胞系中进行的转染研究证实，该启动子片段包含其基础转录所需的必要调控元件。在Y1肾上腺皮质细胞中进行的截短和诱变研究表明，只有近端FRE对转录激活至关重要。然而，电泳迁移率变动分析表明，Ff1b与两个FREs都结合，而使用染色质免疫沉淀分析证实了它们在体内的占有率。最后，当在显微注射的斑马鱼胚胎中瞬时表达时，cyp11a1启动子能够将EGFP表达特异性地导向间肾腺和生殖嵴，并且从斑马鱼胚胎中的荧光素酶报告基因活性测量可知，ff1b的过表达增强了启动子活性。
