Specific Repression of Prolactin Gene Expression

催乳素基因表达的特异性抑制

• 批准号: 6846243
• 负责人: RICHARD N DAY
• 金额: $ 25.9万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-11 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6846243
• 关键词: cell nucleus; fluorescence resonance energy transfer; gene expression; gene induction /repression; genetic promoter element; genetic transcription; immunoprecipitation; polymerase chain reaction; prolactin; protein protein interaction; protein structure function; site directed mutagenesis; transcription factor

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.

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，以及如何被疾病破坏。需要PRL维持正常的生殖过程，其调节表达对于控制体内平衡至关重要。在垂体中，催乳素（PRL）基因的转录仅限于植物营养谱系的细胞，使其成为定义指定细胞选择性基因转录的机制的绝佳模型。需要特异性的同源域蛋白pit，需要建立植物营养细胞谱系，并且对于调节PRL基因的调节转录也是必要但不足的。 PIT-1在垂体细胞类型的起源中的作用是通过影响PIT-1的活性的患者明确的，这些突变会影响垂体激素缺乏症（CPHD），这种疾病的特征是这些细胞缺乏这些细胞产生的激素。通过与其他转录因子和CO调节蛋白的平衡相互作用，通过PIT-1对选择性基因表达的控制。 PIT-1与共激活因子复合物的关联包括介导激素刺激的PRL基因转录的CBP。 PIT 1与包括N-COR和SMRT在内的核共抑制蛋白的关联与核共抑制蛋白的关联相反。我们自己和其他人的最新工作表明，转录因子和共调节蛋白定位于细胞核内的特定位点。 我们已经开发了创新的成像方法，可以直接可视化活核核中的合作蛋白相互作用。我们的观察结果表明，由PIT-L形成的蛋白质相互作用表面的破坏及其核蛋白伴侣会严重影响这些蛋白质复合物在细胞核中组装的地方，并最终对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，该突变在活细胞中观察时会极大地影响合作蛋白相互作用。 通过体外分析和活细胞成像的组合，我们将解决以下问题：1。蛋白质相互作用表面如何指导与激活PRL转录的其他转录因子的合作相互作用？ 2。如何调节PIT-1和共抑制蛋白的功能相互作用？ 3。是否参与直接控制垂体细胞内源性靶基因的转录复合物？如果我们要理解疾病过程和设计治疗策略，则必须定义特定基因调节复合物如何组装在细胞核中，以及它们的定位如何影响组合代码指定特定基因表达的组合。我们的分析将提供重要的组织原理，这些主管是垂体细胞特异性基因表达的基础，并为理解控制真核基因表达所涉及的机制而言是基础。