REGULATION OF THE ERYTHROPOIETIN GENE

红细胞质量的生理调节依赖于促红细胞生成素(EPO)基因在低氧条件下的转录增强。EPO基因表达的研究有助于研究细胞和组织感知缺氧并对基因表达的生物学适当变化做出反应的机制。低氧对EPO基因转录的上调是由至少两个已知的DNA结合转录因子HIF-1和HNF-4介导的，它们与关键的约50bp3‘增强子中的同源反应元件结合。缺氧对HIF-1的激活依赖于对其α亚基的选择性保护，使其免受泛素依赖的蛋白降解。HNF-4是一种孤儿核受体，在肾脏和肝脏中有结构性表达，并与HIF-1协同发挥最大的低氧诱导作用。在这一更新应用中，我们将继续研究低氧诱导EPO基因表达显着增加的机制。特定目的1主要研究缺氧对HIF-1的激活作用。定点突变将确定磷酸化和/或蛋氨酸氧化是否介导HIF-1α的氧依赖降解。我们还将研究与Arnt或热休克蛋白90组装是否有助于HIF-1α的稳定性和核定位。特定目的2说明HNF-4在介导EPO基因的组织特异性低氧诱导中的作用。我们将通过转基因实验来测试HNF-4在体内的重要性。将使用几种策略来确定与EPO增强子上的HNF-4相互作用的蛋白质。我们还将评估这种核受体在调节EPO基因表达方面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，我们将在特定的目标3中深入研究EPO的表达是否也在mRNA稳定的水平上受到调控。EPO mRNA的3‘非翻译区将通过使用标记的EPO基因的转基因实验、凝胶漂移实验和细胞游离RNA稳定性分析来研究。具体目标4集中在暴露在低氧环境中的水蚤水蚤体内血红蛋白显著上调的分子基础。这些研究应该提供关于氧感应、信号转导和基因调控的共同机制的独特和高度相关的信息，使生物体能够适应低氧。

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.