Rust W, Stedronsky K, Tillmann G, Morley S, Walther N, Ivell R
Institute for Hormone and Fertility Research, University of Hamburg, Germany.
J Mol Endocrinol. 1998 Oct;21(2):189-200. doi: 10.1677/jme.0.0210189.
The bovine gene for the steroidogenic acute regulatory protein (StAR) was cloned and sequenced, including 2 kb of the upstream control region of the gene. The gene comprises seven exons arranged similarly to those of the human and mouse gene sequences. The sequence analysis identified three cis elements corresponding to the binding motif for the transcription factor SF-1/Ad4BP, at - 100, - 240 and - 1190 from the transcription start site. Electrophoretic mobility shift analysis (EMSA) using nuclear proteins from bovine corpus luteum and bovine adrenal as well as in vitro transcribed/translated SF-1/Ad4BP consistently showed that only the site at -1190 bound the transcription factor significantly. Very weak binding was detectable also at the - 240 site, but none at the -100 site. Heterologous transfection of StAR promoter deletion-reporter constructs into Hela cells cotransfected with an expression vector for bovine SF-1/Ad4BP, showed that this transcription factor can specifically act on the bovine StAR gene promoter, but preferentially in regions corresponding to the two proximal SF-1/Ad4BP elements at - 100 and - 240, though with only low relative effect. Furthermore, additional cotransfection of a construct expressing a constitutive protein kinase A catalytic subunit to mimic the effects of cAMP stimulation, led to a small SF-1/Ad4BP-dependent increase in reporter activity mediated only by the same proximal sites. Since the bovine StAR gene promoter does not appear to have a functional cAMP responsive element (CRE), either this effect is mediated in this system directly by SF-1/Ad4BP, or by other factors interacting with this transcription factor, but which do not involve CRE-mediated gene activation. Taken together, the results show that there is a discrepancy between the results of the EMSA experiments and those using transfection of promoter-reporter constructs, which needs to be resolved before a clear understanding of SF-1/Ad4BP-mediated regulation of the StAR gene is attained.
克隆并测序了牛的类固醇生成急性调节蛋白（StAR）基因，包括该基因上游2 kb的控制区域。该基因由七个外显子组成，其排列方式与人类和小鼠的基因序列相似。序列分析确定了三个顺式元件，它们对应于转录因子SF-1/Ad4BP的结合基序，位于转录起始位点上游-100、-240和-1190处。使用来自牛黄体和牛肾上腺的核蛋白以及体外转录/翻译的SF-1/Ad4BP进行的电泳迁移率变动分析（EMSA）始终表明，只有-1190处的位点能与转录因子发生显著结合。在-240位点也可检测到非常微弱的结合，但在-100位点则没有。将StAR启动子缺失报告基因构建体异源转染到与牛SF-1/Ad4BP表达载体共转染的Hela细胞中，结果表明该转录因子可特异性作用于牛StAR基因启动子，但优先作用于对应于-100和-240处两个近端SF-1/Ad4BP元件的区域，不过相对效应较低。此外，额外共转染一个表达组成型蛋白激酶A催化亚基的构建体以模拟cAMP刺激的效应，导致仅由相同近端位点介导的报告基因活性出现小幅度的SF-1/Ad4BP依赖性增加。由于牛StAR基因启动子似乎没有功能性的cAMP反应元件（CRE），因此这种效应在该系统中要么直接由SF-1/Ad4BP介导，要么由与该转录因子相互作用的其他因子介导，但不涉及CRE介导的基因激活。综上所述，结果表明EMSA实验结果与使用启动子报告基因构建体转染的实验结果之间存在差异，在清楚了解SF-1/Ad4BP介导的StAR基因调控之前，这一差异需要得到解决。
