TRH REGULATION OF THE RAT THYROTROPIN BETA GENE

大鼠促甲状腺素β基因的TRH调节

• 批准号: 2143551
• 负责人: MARGARET A SHUPNIK
• 金额: $ 13.55万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143551
• 关键词: DNA binding protein; DNA footprinting; calcium; chemical binding; clone cells; gel mobility shift assay; gene expression; genetic enhancer element; genetic library; genetic regulation; genetic regulatory element; genetic transcription; hormone regulation /control mechanism; hypothalamic pituitary axis; laboratory rat; molecular cloning; nuclear runoff assay; pituitary gland; protein kinase C; reporter genes; restriction mapping; site directed mutagenesis; southern blotting; thyrotropin; thyrotropin releasing hormone; tissue /cell culture; transcription factor; transfection; western blottings

Thyrotropin (TSH) pituitary glycoprotein hormone, plays a critical role in thyroid hormone production and thus in normal metabolism. TSH consists of an alpha subunit common to all glycoprotein hormones, and the unique TSHbeta subunit which is under more stringent physiological regulation. We have previously demonstrated that the hypothalamic peptide thyrotropin- releasing hormone (TRH) stimulates TSHbeta gene transcription in rat pituitary cell cultures, and portions of the 5'-flanking region of the gene confer TRH-stimulated responses to reporter genes in transient expression assays. Our first specific aim is to identify the specific TSHbeta gene elements which confer TRH responsiveness to luciferase constructs in GH3 and cultured pituitary cells. Bal 31 digestions, linker-scanner and point mutations of the gene will be performed, and the exact positioning of relevant DNA elements identified. Transient expression experiments will determine if TRH-sensitive gene regions colocalize with calcium and kinase C responses, and the effect of calcium on basal and TRH-stimulated TSHbeta gene transcription in normal pituitary cells will be measured. The second specific aim is to identify and isolate pituitary DNA-binding proteins which confer TRH stimulation to the TSHbeta gene. Binding of nuclear proteins to the TSHbeta gene will be assessed by DNAse I or Exonuclease III footprinting studies. Specific binding between defined gene elements and transcription factors will be measured by gel mobility shift assays and Southwestern blot analysis. Affinities of DNA-protein interactions will be calculated by Scatchard analysis and by competition and equilibrium binding studies. Because a transcription factor termed Pit-1/GHF-1 is thought to play a role in TRH stimulation of prolactin, DNA binding and transfection experiments with Pit-1 expression vector and the TSHbeta gene will be performed. These studies will determine if Pit-1 binds to the TSHbeta gene and assesses its potential role in TRH stimulation of TSHbeta. Transcription factors involved in the TRH response will be cloned from expression vector libraries by screening for DNA-binding proteins with specific TSHbeta gene enhancer oligonucleotides. The potential biological role of these proteins will be evaluated by cotransfection experiments using cloned factors and TRH receptor cDNA. Alternatively, protein factors can be isolated by chromatography on DNA enhancer oligonucleotide affinity columns. The combination of these approaches will directly define the DNA and proteins necessary to implement the TRH transcriptional response, and will provide important insight into the mechanism by which membrane-acting hormones exert effects at the gene level.

促甲状腺激素(TSH)是一种垂体糖蛋白激素，在 甲状腺激素的产生，从而处于正常的新陈代谢。促性腺激素包括 一种所有糖蛋白荷尔蒙共有的阿尔法亚单位，以及唯一的 TSHβ亚基处于更严格的生理调控之下。 我们之前已经证明了下丘脑肽促甲状腺激素- 释放激素(TRH)刺激大鼠促甲状腺激素释放激素(TSHβ)基因转录 培养的垂体细胞和部分脑垂体5‘侧翼区域 在瞬变过程中，基因赋予TRH对报告基因的刺激反应 表达分析。我们的第一个具体目标是确定特定的 TSHbeta基因元件赋予TRH对荧光素酶的反应性 在GH3和培养的垂体细胞中构建。BAL 31摘要， 将进行链接器扫描和基因的点突变，并 确定了相关DNA元素的准确位置。瞬变 表达实验将确定TRH敏感基因区域 与钙共定位和激酶C反应，以及钙的影响 正常垂体基础和TRH刺激下TSHbeta基因转录的研究 细胞将被测量。第二个具体目标是确定和 分离垂体DNA结合蛋白，使TRH刺激 TSHβ基因。核蛋白与TSHbeta基因的结合将是 通过DNase I或核酸外切酶III足迹研究进行评估。特定的 已定义的基因元件和转录因子之间的结合将是 通过凝胶迁移率变化分析和西南印迹分析进行检测。 Scatchard将计算DNA-蛋白质相互作用的亲和力 分析和通过竞争和均衡约束研究。因为一个 转录因子Pit-1/GHF-1被认为在TRH中发挥作用 对催乳素的刺激作用、DNA结合和转染实验 PIT-1表达载体和TSHbeta基因的构建。这些 研究将确定Pit-1是否与TSHbeta基因结合，并评估其 促肾上腺皮质激素释放激素刺激促甲状腺激素释放激素β的潜在作用。转录因子 参与TRH反应的基因将从表达载体中克隆 筛选具有特异性TSHbeta基因的DNA结合蛋白文库 增强子寡核苷酸。它们潜在的生物学作用 蛋白质将通过共转染实验进行评估，克隆的 因子与TRH受体基因的关系。或者，蛋白质因子可以是 DNA增强子寡核苷酸亲和力的层析分离 柱子。这些方法的组合将直接定义DNA 和实现TRH转录反应所需的蛋白质，以及 将为膜作用的机制提供重要的见解 激素在基因水平上发挥作用。