Specific Repression of Prolactin Gene Expression

生物医学研究面临的一个主要挑战是确定涉及基因调控蛋白的相互作用网络如何在活细胞内的自然环境中受到控制，并了解疾病过程如何影响这些活动。催乳素（PRL）基因在垂体前叶催乳细胞中的表达是一个已被证实的模型系统，以定义有助于控制细胞类型特异性基因调控的分子机制。在垂体细胞核中，同源结构域（HD）转录因子Pit-1协调控制PRL基因表达的调节蛋白网络的活动。该提案的广泛目标是使用核结构和染色质重塑中的新兴概念来确定Pit-1如何协调特定基因增强子和启动子处的多蛋白复合物的活性。Pit-1通过与其他辅调节蛋白（包括CCAAT/增强子结合蛋白α（C/EBP 1））的相互作用来调节PRL转录。C/EBP 1又与着丝粒异染色质区域的异染色质结合蛋白1 α（HP 11）结合，Pit-1可以从致密染色质区域募集C/EBP 1。Pit-1中的致病突变破坏了这种蛋白质相互作用的网络，这些结果对许多与HD蛋白质突变相关的人类疾病具有广泛的意义。该提案中的研究使用生物化学分析和活细胞成像的组合来测试Pit-1与C/EBP 1-HP 11复合物相互作用的假设，以重塑密集包装的染色质，允许垂体特异性转录因子进入靶基因。第一个目的是确定C/EBP 1和HP 11在垂体细胞异染色质区域的相互作用，然后确定Pit-1在调节蛋白质相互作用网络中的作用。第二个目的是确定这些相互作用如何控制局部染色质重塑。第三个目标将利用新开发的转基因小鼠模型，允许明确识别活的催乳素或促生长素细胞，以映射正常成熟小鼠垂体细胞中的Pit-1相互作用的网络。 相关性：垂体前叶激素PRL具有许多不同的生理作用，并且不能调节PRL合成导致男性和女性的生殖障碍，并且可以导致催乳素瘤，最常见的颅内原发性肿瘤。如果我们要了解疾病的过程和设计治疗策略，重要的是要确定特定的基因调控复合物是如何组装在完整的细胞核。发现核结构如何控制基因表达将是理解基因组如何工作的基石。

DESCRIPTION (provided by applicant): A major challenge facing biomedical research is to determine how the network of interactions involving gene regulatory proteins is controlled in the context of the natural environment inside living cells, and to understand how disease processes affect these activities. The expression of the prolactin (PRL) gene in anterior pituitary lactotrope cells is a proven model system to define the molecular mechanisms that contribute to the control of cell type-specific gene regulation. In the pituitary cell nucleus, the homeodomain (HD) transcription factor Pit-1 orchestrates the activities of a network of regulatory proteins that control PRL gene expression. The broad objective of this proposal is to use emerging concepts in nuclear architecture and chromatin remodeling to determine how Pit-1 coordinates the activities of multi-protein complexes at specific gene enhancers and promoters. Pit-1 regulates PRL transcription through its interactions with other coregulatory proteins including the CCAAT/enhancer-binding protein alpha (C/EBP1). C/EBP1 in turn associates with the heterochromatin binding protein 1 alpha (HP11) in regions of centromeric heterochromatin, and Pit-1 can recruit C/EBP1 from the regions of compact chromatin. Disease-causing mutations in Pit-1 disrupt this network of protein interactions, and these results have broad implications for many human diseases linked to mutations in the HD proteins. The studies in this proposal use the combination of biochemical analysis and live-cell imaging to test the hypothesis that Pit-1 interactions with the C/EBP1-HP11 complex function to remodel densely packaged chromatin, allowing the access of pituitary-specific transcription factors to target genes. The first aim is to define the interactions of C/EBP1 and HP11 in regions of heterochromatin in pituitary cells, and then to determine the role of Pit-1 in regulating this network of protein interactions. The second aim is to determine how these interactions function to control local chromatin remodeling. The third aim will take advantage of newly developed transgenic mouse models that allow the unambiguous identification of living lactotrope or somatotropes cells to map the network of Pit-1 interactions in the normal mature mouse pituitary cells. Relevance: The anterior pituitary hormone PRL has many diverse physiological roles, and the failure to regulate PRL synthesis leads to reproductive disturbances in both men and women, and can lead to prolactinomas, the most common intracranial primary tumor. If we are to understand disease processes and design therapeutic strategies, it is important to define how specific gene regulatory complexes are assembled in the intact cell nucleus. Discovering how nuclear architecture controls gene expression will be the cornerstone for understanding how genomes work.