PROLACTIN GENE EXPRESSION IN A PITUITARY DERIVED CELL

这一提议的长期目标是理解分子 雌激素调节催乳素基因表达的机制。 催乳素的合成受多种因素的调节，包括雌激素。 这些调节子的结合位点位于不同的位置 距离PRL舞会的距离-。就雌激素而言，雌激素 反应元件与启动子之间有1800多个碱基对的分离 (个碱基)的DNA。PRL基因的排列提供了一种独特的情况 研究雌激素对远距离转录的激活作用。 由于雌激素受体(ER)和组织特异性垂体腺 第一因子(Pit-1)是PRL表达所必需的，我们希望确定 这两种蛋白质是否在饮食中相互接触， 从而介导PRL基因的远程转录激活。 因此，我们想要研究特定的蛋白质-蛋白质相互作用 在急诊室和1号坑之间。首先，ER-Pit-1交互作用将是 用Pit-1-蛋白质亲和层析柱分析蛋白质-蛋白质 相互作用分析。第二，内质网将通过化学方式与Pit-1交联 交联剂，如戊二醛和双马来亚胺己烷以及 通过紫外光诱导的光化学。交联的络合物将是 通过标准的生化和分子生物学技术进行分析。我们 将通过以下方式解决ER Pit-1相互作用的功能相关性 论证了内质网和Pit-1之间的假设相互作用 在活体内发生。我们将使用体内紫外光交联试验来直接 检测Pit-1与ER的结合和ER-Pit-1与DNA的结合。结果将会是 提供有关ER和Pit-1的机制的信息 在调节PRL基因方面进行合作。

The long-range goal of this proposal is to understand the molecular mechanism by which estrogen regulates prolactin (PRL) gene expression. PRL synthesis is regulated by a number of factors, including estrogen. The binding sites for these regulators are positioned at varying distances from the PRL prom-. In the case of estrogen, the estrogen response element is separated from the promoter by over 1800 base pairs (bp) of DNA. The arrangement of the PRL gene provides a unique situation in which to study long-range transcriptional activation by estrogen. Since both the estrogen receptor (ER) and the tissue-specific pituitary factor one (Pit-1) are required for PRL expression, we wish to determine whether these two proteins are in diet physical contact with each other, thus mediating long-range transcriptional activation of the PRL gene. Thus, we would like to investigate specific protein-protein interactions between the ER and Pit-1. First, the ER-Pit-1 interaction will be analyzed using a Pit-1-protein affinity column in a protein-protein interaction assay. Second, ER will be crosslinked to Pit-1 by chemical crosslinking agents such as glutaraldehyde and bismaleimidohexane and photochemically by UV induction. The crosslinked complexes will be analyzed by standard biochemical and molecular biological techniques. We will address the functional relevance of the ER Pit-1 interaction by demonstrating that the hypothesized interaction between the ER and Pit-1 occurs in vivo. We will use an in vivo UV crosslinking assay to directly measure Pit-1 binding to ER and ER-Pit-1 binding to DNA. The results will provide information regarding the mechanism by which ER and Pit-1 cooperate in regulating the PRL gene.