REGULATION OF THE ERYTHROPOIETIN GENE

红细胞量的生理调节依赖于促红细胞生成素（Epo）基因对缺氧反应的转录增强。 Epo基因表达的研究在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作出反应的机制中是有用的。 缺氧引起的Epo基因转录的上调由至少两种已知的DNA结合转录因子HIF-1和HNF-4介导，它们在一个关键的约50 bp的3'增强子中与同源反应元件结合。 低氧激活HIF-1依赖于选择性保护其α亚基免受泛素依赖性蛋白水解。 HNF-4是一种孤儿核受体，在肾脏和肝脏中组成性表达，并与HIF-1合作产生最大的缺氧诱导。 在这次更新申请中，我们将继续研究缺氧诱导Epo基因表达显著增加的机制。 具体目标1关注缺氧对HIF-1的激活。 定点诱变将确定磷酸化和/或甲硫氨酸氧化是否介导HIF-1 α的氧依赖性降解。 我们还将研究与ARNT或热休克蛋白90的组装是否有助于HIF-1 α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。 我们还将评估是否这种核受体在介导促红细胞生成素基因表达的功能是由一个假定的天然配体的影响。 作为对这些Epo基因转录研究的补充，我们将在特定目标3中深入研究Epo表达是否也在mRNA稳定性水平上受到调控。 Epo mRNA的3'非翻译区将通过使用标记的Epo基因的转染实验、沿着凝胶迁移实验和无细胞RNA稳定性测定来研究。 具体目标4侧重于水蚤中血红蛋白在暴露于缺氧时显著上调的分子基础。 这些研究应提供独特的和高度相关的信息，使生物体适应缺氧的氧传感，信号转导和基因调控的共同机制。

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.