Petit F G, Métivier R, Valotaire Y, Pakdel F
Equipe d'Endocrinologie Moléculaire de la Reproduction, Université de Rennes I, France.
Eur J Biochem. 1999 Jan;259(1-2):385-95. doi: 10.1046/j.1432-1327.1999.00072.x.
In all oviparous, liver represents one of the main E2-target tissues where estrogen receptor (ER) constitutes the key mediator of estrogen action. The rainbow trout estrogen receptor (rtER) gene expression is markedly up-regulated by estrogens and the sequences responsible for this autoregulation have been located in a 0.2 kb upstream transcription start site within - 40/- 248 enhancer region. Absence of interference with steroid hormone receptors and tissue-specific factors and a conserved basal transcriptional machinery between yeast and higher eukaryotes, make yeast a simple assay system that will enable determination of important cis-acting regulatory sequences within rtER gene promoter and identification of transcription factors implicated in the regulation of this gene. Deletion analysis allowed to show a synergistic effect between an imperfect estrogen-responsive element (ERE) and a consensus half-ERE to achieve a high hormone-dependent transcriptional activation of the rtER gene promoter in the presence of stably expressed rtER. As in mammalian cells, here we observed a positive regulation of the rtER gene promoter by the chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) through enhancing autoregulation. Using a point mutation COUP-TFI mutant unable to bind DNA demonstrates that enhancement of rtER gene autoregulation requires the interaction of COUP-TFI to the DNA. Moreover, this enhancement of transcriptional activation by COUP-TFI requires specifically the AF-1 transactivation function of ER and can be observed in the presence of E2 or 4-hydroxytamoxifen but not ICI 164384. Thus, this paper describes the reconstitution of a hormone-responsive transcription unit in yeast in which the regulation of rtER gene promoter could be enhanced by the participation of cis-elements and/or trans-acting factors, such as ER itself or COUP-TF.
在所有卵生动物中，肝脏是主要的雌激素作用靶组织之一，雌激素受体（ER）是雌激素作用的关键介质。雌激素能显著上调虹鳟雌激素受体（rtER）基因的表达，负责这种自身调节的序列位于转录起始位点上游0.2 kb处的-40/-248增强子区域内。酵母与高等真核生物之间不存在对类固醇激素受体和组织特异性因子的干扰，且具有保守的基础转录机制，这使得酵母成为一种简单的检测系统，能够确定rtER基因启动子内重要的顺式作用调控序列，并鉴定参与该基因调控的转录因子。缺失分析表明，在稳定表达rtER的情况下，不完全雌激素反应元件（ERE）和共有半ERE之间存在协同效应，可实现rtER基因启动子的高激素依赖性转录激活。与哺乳动物细胞一样，在这里我们观察到鸡卵清蛋白上游启动子转录因子I（COUP-TFI）通过增强自身调节对rtER基因启动子有正向调节作用。使用无法结合DNA的点突变COUP-TFI突变体表明，rtER基因自身调节的增强需要COUP-TFI与DNA相互作用。此外，COUP-TFI对转录激活的这种增强作用特别需要ER的AF-1反式激活功能，并且在存在E2或4-羟基他莫昔芬但不存在ICI 164384的情况下可以观察到。因此，本文描述了酵母中激素反应性转录单元的重建，其中rtER基因启动子的调控可通过顺式元件和/或反式作用因子（如ER本身或COUP-TF）的参与而增强。
