Specific Repression of Prolactin Gene Expression

描述（由申请人提供）：生物医学研究面临的一个主要挑战是确定在活细胞内的自然环境中，涉及基因调控蛋白的相互作用网络是如何被控制的，以及了解疾病过程是如何影响这些活动的。泌乳素（PRL）基因在垂体前乳细胞中的表达是一个被证实的模型系统，用于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。本提案的主要目的是利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动。Pit-1通过与包括CCAAT/增强子结合蛋白α (C/EBP1)在内的其他协同调节蛋白的相互作用来调节PRL的转录。C/EBP1反过来与着丝性异染色质区域的异染色质结合蛋白1 α (HP11)结合，而Pit-1可以从致密染色质区域招募C/EBP1。Pit-1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蛋白突变相关的人类疾病具有广泛的意义。本研究采用生化分析和活细胞成像相结合的方法来验证Pit-1与C/EBP1-HP11复合体功能相互作用以重塑密集包装的染色质，从而允许垂体特异性转录因子进入靶基因的假设。第一个目的是确定C/EBP1和HP11在垂体细胞异染色质区域的相互作用，然后确定Pit-1在调节这一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，该模型允许明确识别活的嗜乳性或嗜体性细胞，以绘制正常成熟小鼠垂体细胞中Pit-1相互作用的网络。

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.