Miller C D, Miller W L
Department of Biochemistry, North Carolina State University, Raleigh 27695-7622, USA.
Endocrinology. 1996 Aug;137(8):3437-46. doi: 10.1210/endo.137.8.8754772.
Direct transcriptional inhibition of the gene that encodes ovine FSH beta-subunit (oFSH beta) by 17 beta-estradiol (E2) has been previously demonstrated by our laboratory. To determine which cis-acting elements in the 5'-flanking region of this gene may be involved in E2 regulation, DNA constructs containing deletions of the 5'-end of the oFSH beta gene were fused to a luciferase reporter and tested in transient transfection assays. These oFSH beta-luciferase constructs and the human E2 receptor expression vector (HEO) were transfected into primary cultures of ovine pituitary cells and subsequently tested with E2. Expression of the largest oFSH beta-luciferase construct (-4741 to +759 of oFSH beta) was inhibited 50% by 20 nM E2. Repression was dependent upon cotransfection of estrogen receptor (HEO) and was E2 dose dependent, with an apparent ED50 similar to that of the positive control consensus estrogen-responsive element construct, ERETk-LUC (ED50 = 50 pM). Deletion studies indicated that sequences between- 105 and -84 bp are necessary for this repression. In addition, a synthetic nucleotide containing oFSH beta sequences from - 105 to -72 could direct E2-dependent repression of a heterologous thymidine kinase promoter that drives luciferase expression. Additional experiments showed that no tissue-specific elements were required for either basal expression or E2-directed transcriptional repression. Although there are no consensus DNA response elements for the estrogen receptor between -105 and +759 of the oFSH beta gene, cotransfection of a mutant E2 receptor lacking the DNA-binding domain (HE-11) failed to mediate E2-dependent inhibition. Gel retardation studies, using the oligonucleotide-containing oFSH beta sequences from -105 to -72, indicated no evidence of direct binding of the estrogen receptor to DNA from -105 to -72. The studies presented here indicate that transcriptional repression of the oFSH beta gene by E2 may be directed in vivo by 5'-flanking sequences between -105 and -72 of the oFSH beta gene. Furthermore, the data suggest that inhibition is mediated via E2 receptor-protein interactions with basal transcription factors that may bind to the -105/-72 DNA directly.
我们实验室先前已证明，17β-雌二醇（E2）可直接转录抑制编码绵羊促卵泡激素β亚基（oFSHβ）的基因。为了确定该基因5'侧翼区域中的哪些顺式作用元件可能参与E2调控，将含有oFSHβ基因5'端缺失的DNA构建体与荧光素酶报告基因融合，并在瞬时转染实验中进行测试。将这些oFSHβ-荧光素酶构建体和人E2受体表达载体（HEO）转染到绵羊垂体细胞的原代培养物中，随后用E2进行测试。20 nM E2可使最大的oFSHβ-荧光素酶构建体（oFSHβ的-4741至+759）的表达抑制50%。抑制作用依赖于雌激素受体（HEO）的共转染，且呈E2剂量依赖性，其表观ED50与阳性对照共有雌激素反应元件构建体ERETk-LUC的相似（ED50 = 50 pM）。缺失研究表明，-105至-84 bp之间的序列对于这种抑制作用是必需的。此外，一个包含oFSHβ基因-105至-72序列的合成核苷酸可指导驱动荧光素酶表达的异源胸苷激酶启动子的E2依赖性抑制。额外的实验表明，基础表达或E2介导的转录抑制均不需要组织特异性元件。尽管在oFSHβ基因的-105至+759之间没有雌激素受体的共有DNA反应元件，但共转染缺乏DNA结合结构域的突变E2受体（HE-11）未能介导E2依赖性抑制。使用包含oFSHβ基因-105至-72序列的寡核苷酸进行的凝胶阻滞研究表明，没有证据表明雌激素受体与-105至-72的DNA直接结合。此处呈现的研究表明，E2对oFSHβ基因的转录抑制可能在体内由oFSHβ基因-105至-72之间的5'侧翼序列介导。此外，数据表明抑制作用是通过E2受体与可能直接结合到-105 / -72 DNA的基础转录因子之间的蛋白质相互作用介导的。
