ESTROGEN RECEPTOR INTERACTION WITH DNA

雌激素受体是发育的重要组成部分， 分化和生长。 它被认为在 一些乳腺癌的发展。 阐明了 雌激素对转录的调节对于理解 这些癌症的原因和设计有效的治疗方法。 一个 雌激素调节转录的第一步是结合 一种特定DNA序列的受体称为雌激素反应性受体， 元素（ERE）。 一个共识的ERE已经推导出来，这是一个完美的 回文序列 这种共识ERE已被证明赋予 特异性雌激素反应性。 的相互作用 ERK激活受体影响RNA活性 聚合酶II通过未知的机制。 本项目的目标是确定 激素激活的雌激素受体识别含有 偏离共识ERE和受体的机制， 激活RNA聚合酶II。 有证据表明， 也就是说，侧翼序列和蛋白质除了 雌激素受体是不完全雌激素受体发挥功能所必需的， vivo. 将检验这些组件允许的假设 不完善的EREs在体内发挥作用，通过增加的结合亲和力， 雌激素受体。 不完美的ERE和侧翼 来自大鼠促黄体激素B基因上游区的序列 将在瞬时转染HeLa细胞的实验中进行分析 细胞或凝胶迁移率变化测定。 受体与ERE的结合似乎不充分 用于信号传导。 有证据表明类固醇 受体似乎竞争共同的限制性转录因子 其不是基础转录起始机制的组分。 这一假设将被验证，特异性相互作用的蛋白质 与激素激活的雌激素受体一起， 影响RNA聚合酶11的活性。 这些雌激素受体结合 蛋白质将通过凝胶迁移率变动分析或 免疫沉淀与分离的表位标记的N-末端结构域的 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以下两种方法中进行分析： “转录干扰”实验或类固醇受体依赖性 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.