REGULATION OF THE ERYTHROPOIETIN GENE

促红细胞生成素基因的调节

• 批准号: 6380614
• 负责人: H. Franklin Bunn
• 金额: $ 57.54万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6380614
• 关键词: Branchiopoda; DNA binding protein; Malacostraca; erythropoiesis; erythropoietin; gene expression; gene induction /repression; genetic enhancer element; genetic regulation; genetic transcription; globin; hypoxia; messenger RNA; molecular site; oxidation; phosphorylation; posttranscriptional RNA processing; protein degradation; protein structure function; reporter genes; site directed mutagenesis; tissue /cell culture; transcription factor; transfection /expression vector; yeast two hybrid system

The physiologic regulation of the red cell mass depends upon enhanced transcription of the erythropoietin (Epo) gene in response to hypoxia. Studies of Epo gene expression have been useful in investigating the mechanism by which cells and tissues sense hypoxia and respond with biologically appropriate alterations in gene expression. The up-regulation of Epo gene transcription by hypoxia is mediated by at least two known DNA binding transcription factors, HIF-1 and HNF-4, which bind to cognate response elements in a critical approximately 50 bp 3' enhancer. The activation of HIF-1 by hypoxia depends upon the selective protection of its alpha subunit from ubiquitin- dependent proteolysis. HNF-4 is an orphan nuclear receptor which is constitutively expressed in kidney and liver, and cooperates with HIF-1 to give maximal hypoxic induction. In this renewal application we will continue our investigation of the mechanism by which hypoxia induces a marked increase in Epo gene expression. Specific Aim 1 focuses on the activation of HIF-1 by hypoxia. Site directed mutagenesis will determine whether phorphorylation and/or methionine oxidation mediates the oxygen- dependent degradation of HIF-1alpha. We will also investigate whether assembly with ARNT or with Heat shock protein 90 contributes to the stability and nuclear localization of HIF- 1alpha. Specific Aim 2 addresses the role of HNF-4 in mediating tissue-specific hypoxia induction of the Epo gene. We will employ transfection experiments to test the importance of HNF-4 in vivo. Several strategies will be used to ascertain the proteins that interact with HNF-4 on the Epo enhancer. We will also assess whether the function of this nuclear receptor in mediating Epo gene expression is affected by a putative natural ligand. As a complement to these studies of Epo gene transcription, we will, in Specific Aim 3, investigate in depth whether Epo expression is also regulated at the level of mRNA stability. The 3' untranslated region of Epo mRNA will be investigated by transfection experiments employing a marked Epo gene, along with gel shift experiments and cell free RNA stability assays. Specific Aim 4 focuses on the molecular basis of the marked up-regulation of hemoglobin in the water flea Daphnea, upon exposure to hypoxia. These studies should provide unique and highly relevant information about common mechanisms of oxygen sensing, signal transduction and gene regulation that enable organisms to adapt to hypoxia.

红细胞团的生理调节取决于促红细胞生成素 (Epo) 基因在缺氧时的转录增强。 Epo 基因表达的研究有助于研究细胞和组织感知缺氧并以生物学上适当的基因表达改变做出反应的机制。 缺氧对 Epo 基因转录的上调是由至少两种已知的 DNA 结合转录因子 HIF-1 和 HNF-4 介导的，它们与关键的大约 50 bp 3' 增强子中的同源反应元件结合。 缺氧对 HIF-1 的激活取决于选择性保护其 α 亚基免受泛素依赖性蛋白水解作用。 HNF-4是一种孤儿核受体，在肾脏和肝脏中组成型表达，与HIF-1配合产生最大程度的缺氧诱导。 在此更新应用中，我们将继续研究缺氧诱导 Epo 基因表达显着增加的机制。 具体目标 1 侧重于缺氧激活 HIF-1。 定点诱变将确定磷酸化和/或甲硫氨酸氧化是否介导 HIF-1α 的氧依赖性降解。 我们还将研究与 ARNT 或与热休克蛋白 90 的组装是否有助于 HIF-1α 的稳定性和核定位。 具体目标 2 阐述了 HNF-4 在介导 Epo 基因的组织特异性缺氧诱导中的作用。 我们将采用转染实验来测试HNF-4在体内的重要性。 将使用多种策略来确定与 Epo 增强子上的 HNF-4 相互作用的蛋白质。 我们还将评估该核受体介导 Epo 基因表达的功能是否受到假定的天然配体的影响。 作为对 Epo 基因转录研究的补充，我们将在具体目标 3 中深入研究 Epo 表达是否也在 mRNA 稳定性水平上受到调节。 Epo mRNA 的 3' 非翻译区将通过使用标记的 Epo 基因的转染实验以及凝胶迁移实验和无细胞 RNA 稳定性测定进行研究。 具体目标 4 重点关注水蚤水蚤中暴露于缺氧后血红蛋白显着上调的分子基础。 这些研究应该提供有关氧传感、信号转导和基因调控的常见机制的独特且高度相关的信息，使生物体能够适应缺氧。