An estrogen receptor binding site within the human galanin gene
An estrogen receptor binding site within the human galanin gene
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DOI：
10.1210/en.138.11.4649
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发表时间：
1997-11-01
期刊：
影响因子：
4.8
通讯作者：
Hyde, JF
中科院分区：
文献类型：
--
作者：
Howard, G;Peng, LH;Hyde, JF
在啮齿类动物中，垂体前叶（AP）中甘丙肽基因表达的调节受到雌激素的积极影响，在人类中尚未确定。本研究的目的是通过鉴定人甘丙肽启动子内可能作为雌激素反应元件（ERE）发挥作用的任何推定的雌激素受体（ER）结合序列来研究雌激素诱导甘丙肽的机制。在甘丙肽6 '侧翼序列中鉴定出两个区域，gERE 1和gERE 2，其与先前在卵黄蛋白原基因（vERE）中定义的完整13个碱基的ERE共有序列相似。在迁移率变动分析中测试两个序列结合从大鼠AP组织或MtTW-10垂体瘤分离的核蛋白的能力，只有-527（gERE 1）处的远端序列产生ERE特异性DNA/蛋白复合物，其特征在于迁移率和与vERE的交叉竞争。垂体组织和肿瘤提取物之间的DNA/蛋白质复合物的凝胶迁移率模式是可比的。然而，DNA/蛋白质亲和力估计表明，垂体蛋白质对gERE 1的亲和力高于vERE序列。电泳迁移率改变提供了人ER（hER）确实识别人甘丙肽基因中的gERE 1序列的证据。此外，在超移位实验中，hER特异性抗体识别gERE 1/蛋白复合物，在大鼠GH（3）垂体细胞系的瞬时转染中检测到gERE 1的增强子活性，导致在雌激素存在下由异源胸苷激酶启动子驱动的表达诱导增加4倍。ER调节gERE 1增强子的证据通过以下证明：1）使用特异性ER拮抗剂ICI 164，384抑制增强;和2）通过gERE 1的增强是来自vERE元件的增强的一半，并且可以反映两个序列之间ER复合物的亲和力或组成的差异。证明了在人甘丙肽基因内存在功能性ERE序列，其可能在AP中作为雌激素作用的调节元件发挥作用。
Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the meChanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 6'-flanking sequence with similarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors.Only the distal sequence at -527 (gERE1) yielded an ERE-specific DNA/protein complex distinguished by mobility and cross-competition with vERE. The gel mobility pattern of the DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift. assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift, experiments.Enhancer activity by gERE1 was detected in transient transfections of the rat GH(3) pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter inthe presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition of enhancement using the specific ER antagonist ICI 164,384; and 2) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of the ER complex between the two sequences.These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.