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特定的基因表达程序，以及这是如何被疾病破坏的。PRL是维持正常生殖过程所必需的，它的调节表达对动态平衡的控制至关重要。在脑垂体中，催乳素(PRL)基因的转录仅限于躯体催乳细胞系的细胞，使其成为确定特定细胞选择性基因转录机制的极佳模型。垂体特异的同源结构域蛋白Pit-1是建立体乳细胞谱系所必需的，也是调节PRL基因转录所必需的，但不是充分的。Pit-1在垂体细胞类型发生中的作用是清楚的，因为患者携带影响Pit-1活性的突变，他们患上了复合性垂体激素缺乏症(CPHD)，这种疾病的特征是缺乏这些细胞产生的激素。Pit-1通过与其他转录因子和共调控蛋白的平衡相互作用来控制选择性基因的表达。Pit-1与共激活复合体的结合包括介导激素刺激的PRL基因转录的CBP。这种激活被Pit 1与包括N-COR和SMRT在内的核共抑制蛋白的关联所抵消。我们和其他人最近的工作揭示了转录因子和协同调节蛋白定位于细胞核内的特定位置。我们已经开发了创新的成像方法来直接可视化活细胞核中的协作蛋白质相互作用。我们的观察表明，Pit-L及其核蛋白伙伴形成的蛋白质相互作用面的破坏可以严重影响这些蛋白质复合体在核中的组装位置，最终影响PRL转录的调节。我们通过观察与CPHD相关的Pit-1蛋白突变来展示这些相互作用表面的重要性，当在活细胞中观察时，这种突变极大地影响了合作蛋白的相互作用。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指导与其他激活PRL转录的转录因子的协同作用？2.Pit-1和共抑制蛋白的功能相互作用是如何调节的？3.Pit-1是否参与了直接控制脑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，重要的是定义特定的基因调控复合体如何在细胞核内组装，以及它们的位置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，这些原则是垂体细胞特异性基因表达的基础，并为理解真核基因表达调控机制奠定了基础。

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.