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: 3245651
• 负责人: MARGARET A SHUPNIK
• 金额: $ 8.13万
• 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/3245651
• 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）是垂体糖蛋白激素，在脑垂体中发挥着关键作用， 甲状腺激素的产生，从而在正常的新陈代谢。 TSH包括 所有糖蛋白激素共有的α亚基， TSH β亚基，其处于更严格的生理调节下。 我们以前已经证明，下丘脑肽促甲状腺激素- 促甲状腺激素释放激素（TRH）对大鼠促甲状腺激素β（TSH β）基因转录的影响 垂体细胞培养物，以及垂体细胞的5 '侧翼区域的部分。 基因赋予TRH刺激的反应，以报告基因在瞬时 表达测定。 我们的第一个具体目标是确定具体的 赋予TRH对荧光素酶反应性的TSH β基因元件 GH 3和培养的垂体细胞中的构建体。 Bal 31 手指， 将进行基因的连接子扫描和点突变， 确定相关DNA元件的精确定位。 瞬态 表达实验将确定TRH敏感基因区域 与钙和激酶C反应共定位，钙的作用 对正常垂体TSH β基因转录的影响 细胞将被测量。 第二个具体目标是确定和 分离的垂体DNA结合蛋白，其赋予TRH刺激， TSH β基因。 核蛋白与TSH β基因的结合将是 通过DNA酶I或核酸外切酶III足迹法研究评估。 具体 确定的基因元件和转录因子之间的结合将是 通过凝胶迁移率变动分析和Southwestern印迹分析测量。 Scatchard将计算DNA-蛋白质相互作用的亲和力 分析和竞争和平衡约束研究。 因为 被称为Pit-1/GHF-1的转录因子被认为在TRH中起作用 刺激催乳素、DNA结合和转染实验， 将进行Pit-1表达载体和TSH β基因。 这些 研究将确定Pit-1是否与TSH β基因结合，并评估其 在促甲状腺激素释放激素刺激促甲状腺激素β的潜在作用。 转录因子 将从表达载体中克隆参与TRH应答的 通过筛选具有特异性TSH β基因的DNA结合蛋白的文库 增强子寡核苷酸。 这些潜在的生物学作用 将通过共转染实验使用克隆的 因子和TRH受体cDNA。 或者，蛋白质因子可以是 通过DNA增强子寡核苷酸亲和层析分离 列. 这些方法的结合将直接定义DNA 和实现TRH转录应答所必需的蛋白质，以及 将提供重要的洞察机制，膜作用 激素在基因水平上发挥作用。