Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene
Cloning, novel promoter sequence, and estrogen regulation of a rat oxytocin receptor gene
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DOI：
10.1210/en.138.3.1151
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发表时间：
1997-03-01
期刊：
影响因子：
4.8
通讯作者：
Dorsa, DM
中科院分区：
文献类型：
--
作者：
Bale, TL;Dorsa, DM
已知催产素受体（OR）基因在体内的表达受雌二醇（E（2））调节。我们已经克隆和测序了大鼠OR基因的BI侧翼DNA的4个内切酶（kb），并鉴定了在该启动子的最初出版物中不存在的1260个核苷酸的内部片段和另外的2kb上游序列。这个新的内部区域位于两个大的tg核苷酸重复序列之间。使用基因组DNA进行PCR扩增证实该序列存在于大鼠基因组中。为了解释E（2）的转录作用，在这个新序列中，在翻译起始位点的5'端约4kb处鉴定出了一个在雌激素受体结合中具有活性的回文雌激素反应元件（ERE）。在MCF 7细胞中的转染实验中测试了E（2）增强该启动子转录的能力。E（2）仅弱诱导截短构建体的转录。在基础启动子的背景下，ERE的突变分析表明，它作为一个增强子的功能，两个碱基的突变消除了这种活动。在迁移率凝胶位移试验中进一步支持了这种反应的有效性，其中OR ERE结合子宫提取物中存在的雌激素受体。在MCF 7细胞中使用I-125-鸟氨酸催产素的受体结合研究显示，E（2）显著上调内源性OR。Western印迹分析证实了E（2）处理MCF 7细胞后OR蛋白的增加。这些研究已经确定了大鼠OR启动子的一个新区域，该区域含有一个上游回文ERE，该区域赋予OR基因转录的E（2）诱导。
Expression of the oxytocin receptor (OR) gene in vivo is known to be regulated by estradiol (E(2)). We have cloned and sequenced 4 kilobases (kb) of BI-flanking DNA of the rat OR gene and identified an internal segment of 1260 nucleotides that was absent in an initial publication of this promoter and an additional 2 kb of upstream sequence. This novel internal region is located between two large tg nucleotide repeats. PCR amplification using genomic DNA verified that this sequence is present in the rat genome. To explain transcriptional effects of E(2), a palindromic estrogen response element (ERE) that is active in estrogen receptor binding was identified within this new sequence, approximately 4 kb 5' of the translational start site. The ability of E(2) to enhance transcription of this promoter was tested in transfection experiments in MCF7 cells. E(2) only weakly induced transcription of a truncated construct. Mutational analysis of the ERE in the context of a basal promoter indicated that it functions as an enhancer, and that mutation of two bases eliminates this activity. Further support of the efficacy of this response was shown in mobility gel shift assays in which the OR ERE bound estrogen receptor present in uterine extracts. Receptor binding studies using I-125-ornithine vasotocin in MCF7 cells revealed that E(2) dramatically up-regulated endogenous ORs. Western blot analysis confirmed this increase in OR protein with E(2) treatment of MCF7 cells. These studies have identified a novel region of the rat OR promoter containing an upstream palindromic ERE that imparts E(2) inducibility of OR gene transcription.