HORMONAL REGULATION OF RAT PROLACTIN GENE EXPRESSION

大鼠催乳素基因(PRL)提供了一个有用的模型系统来检测 基因调控的几个方面：1)激活和抑制 PRL的表达受两种正向作用因子的调节 (EQ。雌激素受体)和负性作用因子(E.Q。糖皮质激素 受体)；2)细胞特异性表达；PRL仅在乳汁中表达 脑垂体前叶细胞；3)远距离活动；两种激素 而细胞特异性表达是通过两个不同的 PRL内1500个碱基的转录调控元件 基恩的5‘侧翼DNA序列。我们目前对PRL的大部分理解 基因调控来自瞬时转基因研究或体外实验。 蛋白质-DNA结合研究。然而，这些研究并没有考虑到 考虑到细胞中存在的催乳素基因结构的复杂性 原子核。在体内，催乳素DNA不是简单的一系列核苷酸序列 容易与特定蛋白质介体相互作用的物质 抄写。在体内，PRL DNA被超级卷曲并组织成一个 核小体结合的染色质结构。这个项目的重点是 了解染色质结构对类固醇作用机制的影响 激素对催乳素基因表达的调节。我们开发了一种独特的 扩增的微染色体系统，包含转录的 PRL的监管要素。PRL-微染色体以一种方式作出反应 在转录水平上与内源性PRL基因相似 其高度有序的染色质结构的调节和变化。这个 将使用PRL微染色体系统在核苷酸水平上进行检测 类固醇对PRL染色质结合蛋白的影响 调节转录的荷尔蒙和细胞特异性因子。这些 研究将确定PRL染色质的特定区域，其中蛋白质 与PRL DNA结合并可能在不同的时间相互作用 PRL表达的不同阶段。我们打算分离这些蛋白质， 表征它们的染色质结合特性，并通过使用 分离的PRL-微染色体体外转录检测 模板，决定了这些蛋白质在调节 染色质相关PRL的转录。最后，互动 结合在PRL中两个不同调控区的蛋白质之间 染色质及其在PRL表达中的作用将使用一种新的 核连接试验和体内交联物研究。

The rat prolactin gene (PRL) provides an useful model system to examine a number of aspects of gene regulation: 1) Activation and repression of expression; expression of PRL is regulated by both positive acting factors (e.q. estrogen receptor) and negative acting factors (e.q. glucocorticoid receptor); 2) Cell-specific expression; PRL is only expressed in lactotroph cells of the anterior pituitary; and 3) Action at a distance; both hormone and cell-specific expression is mediated through two distinct transcriptional regulatory elements separated by 1500 bp within the PRL gene's 5' flanking DNA sequences. Much of our current understanding of PRL gene regulation has come from transient transfection studies or in vitro protein-DNA binding studies. However, these studies do not take into account the complexities of the PRL gene structure as it exist in the cell nucleus. In vivo, PRL DNA is not simply a series of nucleotide sequences that are readily available for interaction with specific protein mediators of transcription. In vivo, PRL DNA is supercoiled and organized into a nucleosome-bound chromatin structure. This project focuses on understanding the effect of chromatin structure on the mechanism of steroid hormone regulation of PRL gene expression. We have developed a unique amplified minichromosome system that contains the transcriptional regulatory elements of PRL. The PRL-minichromosome responds in a manner similar to the endogenous PRL gene with respect to transcriptional regulation and alterations in its highly ordered chromatin structure. The PRLminichromosome system will be used to examine at the nucleotide level changes in protein binding to the PRL chromatin in response to steroid hormones and cell-specific factors that regulate transcription. These studies will identify specific regions of the PRL chromatin where proteins bind to the PRL DNA and possibly interact with each other during various stages of PRL expression. We intend to isolate these proteins, characterize their chromatin binding properties, and through the use of an in vitro transcription assay using isolated PRL-minichromosomes as templates, determine the role of these proteins in regulating the transcription of chromatin-associated PRL. Finally, the interaction between proteins bound to the two distinct regulatory regions in PRL chromatin and their role in PRL expression will be examined using a novel nuclear ligation assay and in vivo crosslinking studies.