PHYSIOLOGY OF THYROID HORMONE DEPENDENT GENE EXPRESSION

甲状腺激素是人类正常生长、大脑正常发育所必需的 发展和功能以及构成生热作用。多数 活性甲状腺激素3，5，3‘三碘甲腺原氨酸(T3) 在2%-3%的美国人口中， 甲状腺功能减退症，是由甲状腺素(T4)引起的。 一次脱碘。这个反应是由硒酶催化的， Dio1和2的1型和2型脱碘酶(d1和d2)产物 基因分别表达。T4和T3的失活，特别是在大脑中 和胎盘单位，由另一个催化的5脱碘发生 硒酶，3型脱碘酶(D3)以及双功能 D1。D1和d3都已被克隆，并且都受到调控 T3通过直接机制在mRNA水平上呈阳性反应 不需要新的蛋白质合成。这些关键的酶是 内部反馈回路的基本部分，用于维持T3 动态平衡。它们可以独立于下丘脑发挥作用- 脑垂体轴。本项目将分析dio1和dio3的调控。 作为受T3直接和正向调控的基因模型。特定的 目的：探讨大黄连的作用调节机制。 人dio1基因中的TRE2，并确定其作用机制 在特定细胞类型中，T3对dio1表达的负性调节。 这些研究将使用同源基因的瞬时表达分析。 含和不含甲状腺共表达启动子-报告基因的构建 激素受体(TR)或其他转录因子。特定目标 Ii将描述TRR-TRE互动的性质及其如何 利用人工合成和体内基因组足迹研究T_3的影响 内源基因的连接介导的聚合酶链式反应。特定目标III将 关注甲状腺调节D3表达的机制 激素，包括克隆5‘侧翼区，研究 甲状腺病变不同脑区D3表达的变化 应用原位杂交和原位杂交技术检测碘营养状况和碘缺乏 免疫组织化学、细胞培养系统和体内研究 基因组足迹。通过这些技术，我们将获得新的 关于T3如何直接调节基因转录的知识 编码催化甲状腺中必要步骤的酶 激素的激活和失活。

Thyroid hormone is essential for normal human growth, normal brain development and function and for constituitive thermogenesis. Most of the active thyroid hormone, 3,5, 3' triiodothyronine (T3), in intact individuals and all of this in the 2-3% of the U.S. population with hypothyroidism, is generated from thyroxine (T4) by 5' monodeiodination. This reaction is catalyzed by the selenoenzymes, Types 1 and 2 deiodinase (D1 and D2) products of the dio1 and 2 genes respectively. Inactivation of T4 and T3 especially in the brain and feto-placental unit, occurs by 5 deiodination catalyzed by another selenoenzyme, Type 3 deiodinase (D3) as well as by the bifunctional D1. Both D1 and D3 have been cloned and both are regulated positively by T3 at the mRNA level through direct mechanisms which do not require new protein synthesis. These critical enzymes are essential parts of an internal feedback loop acting to maintain T3 homeostasis. They can function independently of the hypothalamic- pituitary axis. This project will analyze the regulation of dio1 and 3 as models of genes directly and positively regulated by T3. Specific Aim I will explore the mechanism for regulation of the action of TRE2 in the human dio1 gene and determine the mechanism for negative regulation of dio1 expression by T3 in specific cell types. These studies will employ transient expression assays of homologous promoter-reporter constructs with and without co-expressed thyroid hormone receptor (TR) or other transcription factors. Specific Aim II will delineate the nature of the TR-TRE interaction and how it is affected by T3 using in vivo genomic footprinting of synthetic and endogenous genes by ligation-mediated PCR. Specific Aim III will focus on the mechanism for regulation of D3 expression by thyroid hormone including cloning of the 5' flanking region, studies of changes in D3 expression in various brain regions with altered thyroid status and iodine deficiency using in situ hybridization and immunohistochemistry, studies in cell culture systems, and in vivo genomic footprinting. By these techniques we will gain new knowledge of how T3 directly regulates the transcription of the genes encoding the enzymes which catalyze essential steps in thyroid hormone activation and inactivation.