ESTROGEN RECEPTOR INTERACTION WITH DNA

雌激素受体是发育过程中的重要组成部分， 分化和生长。它被认为在 一些乳腺癌的发展。对其作用机制的阐明 雌激素对转录的调节对于理解 这些癌症的病因和有效治疗方法的设计。一个 雌激素调节转录的第一步是结合 一种称为雌激素反应性的特定DNA序列的受体 元素(ERE)。在这里达成了一项共识，这是一个完美的 回文序列。这一共识已经被证明是可行的。 特定的雌激素反应性。两人之间的相互作用 带有ERE的激素激活受体影响RNA的活性 聚合酶II通过一种未知的机制。 这个项目的目标是确定 激素激活的雌激素受体识别含有 偏离共识的ERE和受体的机制 激活RNA聚合酶II。有证据表明 成分，即侧翼序列和蛋白质，除了 雌激素受体是不完善的ERE发挥功能所必需的 活着。这一假设将得到检验，即这些组件允许 不完善的ERE通过增加结合亲和力而在体内发挥作用 ERE的雌激素受体。不完美的前部和侧翼 大鼠黄体生成素B基因上游区序列分析 将在对HeLa的瞬时转基因实验中进行分析 细胞或凝胶迁移率变化分析。 受体与ERE的结合似乎不够充分。 用于信号转导。有证据表明类固醇 受体似乎竞争共同的限制性转录因子 它们不是基本转录启动机制的组成部分。 这一假设将被检验，即特定相互作用的蛋白质 通过激素激活-雌激素受体帮助受体在 影响RNA聚合酶11活性。这些雌激素受体结合 蛋白质将通过凝胶迁移率变化分析或 标记N-末端结构域的分离表位的免疫沉淀 雌激素受体或通过使用雌激素受体亲和柱。 这些蛋白质的功能将在任一种情况下进行分析 “转录干扰”实验或类固醇受体依赖 体外转录。

The estrogen receptor is an important component in development, differentiation and growth. It is thought to play a role in the development of some breast cancers. Elucidation of the mechanism of estrogen regulation of transcription is important in understanding the cause of these cancers and in the design of effective treatments. An initial step in estrogen regulation of transcription is the binding of the receptor to a specific DNA sequence called an estrogen responsive element (ERE). A consensus ERE has been derived which is a perfect palindromic sequence. This consensus ERE has been shown to confer specific estrogen responsiveness. The interaction of the hormone-activated receptor with the ERE influences the activity of RNA polymerase II by an unknown mechanism. The objectives of this project are to determine the mechanism by which hormone activated-estrogen receptor recognizes EREs which contain deviations from the consensus ERE and the mechanism by which the receptor activates RNA polymerase II. There is evidence which suggests that components, that is, flanking sequences and proteins in addition to the estrogen receptor are required for imperfect EREs to be functional in vivo. The hypothesis will be tested that these components allow imperfect EREs to function in vivo by increasing the binding affinity of the estrogen receptor for the ERE. The imperfect ERE and flanking sequences from the upstream region of the rat luteinizing hormone B gene will be analyzed in either transient transfection experiments into HeLa cells or gel mobility shift assays. The binding of the receptor to the ERE does not appear to be sufficient for signal transduction. There is evidence which suggests that steroid receptors appear to compete for common limiting transcription factors which are not components of the basal transcription initiation machinery. The hypothesis will be tested that proteins which specifically interact with the hormone activated-estrogen receptor aid the receptor in influencing RNA polymerase 11 activity. These estrogen receptor-binding proteins will be identified by either gel mobility shift assays or immunoprecipitation with an isolated epitope tagged N-terminal domain of the estrogen receptor or by use of an estrogen receptor affinity column. The function of these proteins will be analyzed in either "transcriptional interference" experiments or steroid receptor-dependent in vitro transcription.