TRANSCRIPTION FACTOR INTERACTIONS AT THE GH PROMOTER

GH 启动子处的转录因子相互作用

• 批准号: 6177957
• 负责人: Fred J SCHAUFELE
• 金额: $ 22.08万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177957
• 关键词: chimeric proteins; developmental genetics; enhancer binding protein; fluorescence resonance energy transfer; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; green fluorescent proteins; hormone regulation /control mechanism; laboratory mouse; pituitary gland; protein kinase A; protein kinase C; protein localization; protein protein interaction; protein structure function; site directed mutagenesis; somatotropin; transcription factor; western blottings

Growth hormone (GH) is a powerful regulator of linear growth and metabolic homeostasis that acts at almost all organ systems. Exogenous GH therapy is costly but effective at counteracting the profound metabolic effects associated with idiopathic GH-deficiency and, seemingly, the normal age-related decline in GH gene transcription. GH synthesis is restricted to the somatotroph cells of the anterior pituitary gland where the transcription of the GH gene is tightly regulated in distinct physiologic, age-related and circadian patterns by systemic and hypothalamic hormones, many of which operate via protein kinase systems. Our long-term goal is to identify the molecular mechanisms of hormone- regulated, somatotroph-specific GH gene expression as a first step in the design of cost-effective therapies targeting GH transcription. The transcription factor C/EBPalpha cooperates with the pituitary-specific transcription factor Pit-1 and the coactivator CBP to control protein kinase A and C-regulated GH gene transcription. Cooperative activation likely arises from mutual or complementary, kinase-regulated actions of C/EBPalpha, Pit-1 and CBP at specific basal factor and/or structural targets. C/EBPalpha, Pit-1 and CBP each interact with, and colocalize to a nuclear substructure with, the basal transcription factor TBP that participates in activation by those transcription factors and cofactor. The molecular basis for cooperative action will be assessed by examining C/EBPalpha, CBP, Pit-1, and TBP activities required for kinase-regulated GH promoter activation by, nuclear localization of, and physical interactions between, those factors. We have already developed a series of innovative in vivo cellular assays which will allow us to determine the: 1. structures in C/EBPalpha and CBP affecting kinase-regulated cooperative activation of the GH promoter, 2. structures in TBP required for kinase-regulated C/EBPalpha, CBP and Pit-1 GH promoter activation, 3. nuclear positioning of C/EBPalpha, CBP, Pit-1, TBP and TFIIB and the spatiotemporal relationships of interactions between those factors under the same cellular conditions used in the functional assays. 4. the role of C/EBPalpha in the embryonic onset of GH gene transcription in C/EBPalpha knockout mice which die shortly after birth.

生长激素(GH)是一种强大的线性生长和代谢稳态调节剂，作用于几乎所有的器官系统。外源性生长激素治疗费用昂贵，但在抵消与特发性生长激素缺乏症相关的深刻代谢影响以及似乎与年龄相关的正常生长激素基因转录下降方面有效。生长激素的合成仅限于垂体前叶的促生长激素细胞，生长激素基因的转录在不同的生理、年龄相关和昼夜节律模式下受到全身和下丘脑激素的严格调控，其中许多激素通过蛋白激酶系统发挥作用。我们的长期目标是确定激素调节的生长激素特异性生长激素基因表达的分子机制，作为设计针对生长激素转录的成本效益疗法的第一步。转录因子C/EBPalpha与垂体特异性转录因子Pit-1和共激活因子CBP协同作用，控制蛋白激酶A和C调节的生长激素基因转录。协同激活可能源于C/EBPalpha、Pit-1和CBP在特定的基础因子和/或结构靶点上相互或互补的、激酶调节的作用。C/EBPalpha、Pit-1和CBP分别与参与这些转录因子和辅因子激活的基础转录因子TBP相互作用并与其共定位到核亚结构。协同作用的分子基础将通过检测C/EBPalpha、CBP、Pit-1和TBP活性来评估，这些活性是通过这些因子的核定位和物理相互作用激活激酶调节的生长激素启动子所需的。我们已经开发了一系列创新的体内细胞分析方法，这些方法将使我们能够确定：1.影响激酶调控的GH启动子协同激活的C/EBPalpha和CBP中的结构；2.在激酶调控的C/EBPalpha、CBP和Pit-1 GH启动子激活所需的TBP中的结构；3.C/EBPalpha、CBP、Pit-1、TBP和TFIIB的核定位以及在功能分析中使用的相同细胞条件下这些因素之间相互作用的时空关系。4.在出生后不久死亡的C/EBPalpha基因敲除小鼠中，C/EBPalpha在GH基因转录的胚胎启动中的作用。