Specific Repression of Prolactin Gene Expression

描述（由申请人提供）：我们的最终目标是更好地了解细胞核中的蛋白质相互作用如何控制基因表达的特定程序，以及疾病如何破坏这种相互作用。PRL是维持正常生殖过程所必需的，其调节表达对控制体内平衡至关重要。在垂体中，催乳素（PRL）基因的转录仅限于生长激素乳细胞系的细胞，使其成为定义指定细胞选择性基因转录机制的极好模型。垂体特异性同源结构域蛋白Pit-1是建立体乳营养细胞谱系所必需的，并且对于PRL基因的调节转录也是必需的，但不是充分的。Pit-1在垂体细胞类型发生中的作用通过携带影响Pit-1活性的突变的患者变得清楚，这些患者发展为联合垂体激素缺乏症（CPHD），这是一种以缺乏这些细胞产生的激素为特征的疾病。Pit-1通过与其他转录因子和共调节蛋白的平衡相互作用来控制选择性基因表达。Pit-1与辅激活因子复合物的关联包括介导孕酮刺激的PRL基因转录的CBP。这种激活通过Pit 1与核辅阻遏蛋白（包括N-CoR和SMRT）的结合来平衡。我们和其他人最近的工作揭示了转录因子和共调节蛋白定位于细胞核内的特定位点。 我们已经开发出创新的成像方法来直接可视化活细胞核中的合作蛋白质相互作用。我们的观察结果表明，Pit-1和它的核蛋白伴侣形成的蛋白质相互作用表面的破坏可以严重影响这些蛋白质复合物在核中组装的位置，并最终影响PRL转录的调节。我们通过观察与CPHD相关的Pit-1蛋白突变显示了这些相互作用表面的重要性，该突变在活细胞中观察时显著影响了合作蛋白相互作用。 通过体外分析和活细胞成像的结合，我们将解决以下问题：1。Pit-1上的蛋白质相互作用表面如何发挥作用来指导与其他激活PRL转录的转录因子的合作相互作用？2. Pit-1和辅阻遏蛋白的功能相互作用是如何调节的？3. Pit-1是否参与直接控制垂体细胞内源性靶基因的转录复合体？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在细胞核内组装，以及它们的定位如何影响指定特定基因表达的组合密码。我们的分析将提供重要的组织原则，垂体细胞特异性基因表达的基础，并形成一个基础，了解参与真核基因表达的控制机制。

DESCRIPTION (provided by applicant): Our ultimate goal is to better understand how protein interactions in the cell nucleus control specific programs of gene expression, and how this is disrupted by disease. PRL is required to maintain normal reproductive processes, and its regulated expression is critical to the control of homeostasis. In the pituitary, transcription of the prolactin (PRL) gene is restricted to cells of the somatolactotroph lineage, making it an excellent model for defining the mechanisms that specify cell-selective gene transcription. The pituitary-specific homeodomain protein Pit-1, is required to establish the somatolactotroph cell lineage, and is also necessary, but not sufficient, for regulated transcription of the PRL gene. The role of Pit-1 in the genesis of pituitary cell-types is made clear by patients harboring mutations that affect the activity of Pit-1 who develop combined pituitary hormone deficiency (CPHD), a disease characterized by the lack of the hormones produced by these cells. The control of selective gene expression by Pit-1 occurs through balanced interactions with other transcription factors and co regulatory proteins. The association of Pit-1 with a co-activator complex includes the CBP that mediates hormone-stimulated PRL gene transcription. This activation is counter-balanced by the association of Pit 1 with the nuclear co-repressor proteins including N-CoR and SMRT. Recent work by ourselves and others has revealed that transcription factors and co-regulatory proteins localize to specific sites within the cell nucleus. We have developed innovative imaging approaches to directly visualize cooperative protein interactions in the living ceil nucleus. Our observations indicate that disruption of protein interaction surfaces formed by Pit-l, and its nuclear protein partners can critically affect where these protein complexes are assembled in the nucleus, and ultimately the regulation of PRL transcription. We showed the importance of these interaction surfaces by observation of a Pit-1 protein mutation associated with CPHD that dramatically affected cooperative protein interactions when viewed in living cells. Through the combination of in vitro analysis and live-cell imaging we will address the following questions: 1. How do protein interaction surfaces on Pit-1 function to guide the cooperative interactions with other transcription factors that activate PRL transcription? 2. How are the functional interactions of Pit-1 and co-repressor proteins regulated? 3. Is Pit-1 engaged in transcriptional complexes that directly control endogenous target genes in pituitary cells? If we are to understand disease processes and design therapeutic strategies, it is important to define how specific gene regulatory complexes are assembled within the nucleus and how their positioning influences the combinatorial code specifying the expression of particular genes. Our analysis will provide important organizational principals that underlie pituitary cell specific gene expression and form a basis for understanding the mechanisms involved in the control of eukaryotic gene expression in general.