Shupnik M A, Rosenzweig B A
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22903.
J Biol Chem. 1991 Sep 15;266(26):17084-91.
Previous work from this laboratory demonstrated that 17-beta estradiol (E2) can directly stimulate the transcription rate of the rat luteinizing hormone beta (LH beta) gene and that an upstream portion of the LH beta gene between -2.0 and -0.6 kilobases could confer an E2-stimulated response to a reporter gene in transient expression assays. To localize the LH beta estrogen response element (ERE) by biological function, portions of the 5'-flanking region of the LH beta gene or synthetic oligonucleotides were inserted in expression vectors next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene. Constructs were transfected into GH3 cells, and transfected cells were treated for 48 h with E2. E2 stimulation of activity (2-4-fold) occurred with constructs containing the 15-base pair palindromic sequence (GGACACCATCTGTCC), found at bases -1173 to -1159 relative to the transcriptional start site in the LH beta gene. A construct containing a synthetic oligonucleotide of this putative LH beta ERE was stimulated 1.7-3-fold by E2, while a construct containing two copies of the sequence was stimulated to a slightly higher level (2.5-4.0-fold). An oligonucleotide in which the palindrome was mutated failed to confer E2 stimulation, and mutation of the palindromic region within the upstream region of the LH beta gene also eliminated the E2 response. The anti-estrogen tamoxifen could not elicit a response, nor could dehydrotestosterone or dexamethasone; however, thyroid hormone treatment resulted in a 2-2.5-fold stimulation. The 15-base pair LH beta gene palindrome was found to bind estrogen receptor (ER) complex directly by gel retardation experiments. Labeled LH beta ERE DNA formed three complexes with proteins from immature rat uterine extract. Two of these were associated with ER complexes, as determined by the comigration of [3H] estradiol bound to ER with these complexes, and by the ability of anti-ER antibody to associate with these complexes. The affinity of the LH beta ERE for ER was calculated by Scatchard analysis to be 2.2-5.0 nM, an approximately 5-10-fold lower affinity than for the ERE in the vitellogenin A2 gene region. The mutated ERE, which had no biological activity, could not compete effectively for binding to ER. ER which was heat-transformed at 30 degrees C had a similar affinity (2-5 nM) for the ERE as ER occupied with E2 (2-4 nM), while ER occupied by estrone had a lower affinity (9 nM).(ABSTRACT TRUNCATED AT 400 WORDS)
该实验室先前的研究表明，17-β雌二醇（E2）可直接刺激大鼠促黄体生成素β（LHβ）基因的转录速率，并且在瞬时表达试验中，LHβ基因-2.0至-0.6千碱基之间的上游部分可赋予报告基因E2刺激反应。为了通过生物学功能定位LHβ雌激素反应元件（ERE），将LHβ基因5'-侧翼区的部分片段或合成寡核苷酸插入到与氯霉素乙酰转移酶基因融合的单纯疱疹病毒胸苷激酶启动子旁边的表达载体中。构建体被转染到GH3细胞中，转染后的细胞用E2处理48小时。含有15个碱基对回文序列（GGACACCATCTGTCC）的构建体出现了E2刺激活性（2至4倍），该序列位于LHβ基因转录起始位点相对-1173至-1159碱基处。含有该推定的LHβERE合成寡核苷酸的构建体被E2刺激了1.7至3倍，而含有该序列两个拷贝的构建体被刺激到略高的水平（2.5至4.0倍）。回文序列发生突变的寡核苷酸未能赋予E2刺激作用，并且LHβ基因上游区域内回文区域的突变也消除了E2反应。抗雌激素他莫昔芬不能引发反应，脱氢睾酮或地塞米松也不能；然而，甲状腺激素处理导致2至2.5倍的刺激。通过凝胶阻滞实验发现，15个碱基对的LHβ基因回文序列直接结合雌激素受体（ER）复合物。标记的LHβERE DNA与未成熟大鼠子宫提取物中的蛋白质形成三种复合物。其中两种与ER复合物相关，这是通过与这些复合物共迁移的与ER结合的[3H]雌二醇以及抗ER抗体与这些复合物结合的能力来确定的。通过Scatchard分析计算，LHβERE对ER的亲和力为2.2至5.0 nM，比对卵黄生成素A2基因区域中ERE的亲和力低约5至10倍。没有生物学活性的突变ERE不能有效地竞争与ER的结合。在30℃下热转化的ER对ERE的亲和力（2至5 nM）与被E2占据的ER（2至4 nM）相似，而被雌酮占据的ER亲和力较低（9 nM）。（摘要截短至400字）
