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PROLACTIN GENE EXPRESSION IN A PITUITARY DERIVED CELL

垂体来源细胞中催乳素基因的表达

基本信息

批准号：
2471057
负责人：
Wesley George Gray
金额：
$ 1.27万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-09-30 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/2471057
关键词：
PROLACTIN GENE EXPRESSION PITUITARY DERIVED

项目摘要

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.

这项提案的长期目标是了解雌激素调节催乳素（PRL）基因表达的机制。 PRL的合成受许多因素的调节，包括雌激素。这些调节剂的结合位点位于不同的距离PRL prom-.在雌激素的情况下，反应元件与启动子之间相隔超过1800个碱基对 (bp)的DNA。PRL基因的排列提供了一个独特的情况来研究雌激素的长距离转录激活。由于雌激素受体（ER）和组织特异性垂体因子1（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物理接触，从而介导PRL基因的长距离转录激活。因此，我们希望研究特定的蛋白质-蛋白质相互作用在急诊室和一号坑之间首先，ER-Pit-1相互作用将是使用Pit-1-蛋白质亲和柱在蛋白质-蛋白质色谱柱中进行分析。相互作用测定第二，ER将通过化学交联与Pit-1交联，交联剂如戊二醛和双马来酰亚胺己烷，通过UV诱导进行光化学反应。交联复合物将是通过标准生物化学和分子生物学技术分析。我们将通过以下方式解决ER Pit-1相互作用的功能相关性：证明ER和Pit-1之间的假设相互作用发生在体内。我们将使用体内UV交联测定直接测量Pit-1与ER的结合和ER-Pit-1与DNA的结合。结果将提供有关ER和Pit-1 共同调节PRL基因。