REGULATION OF THE ERYTHROPOIETIN GENE

促红细胞生成素基因的调节

基本信息

批准号：
6646459
负责人：
H. Franklin Bunn
金额：
$ 60.23万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-01 至 2005-08-31
项目状态：
已结题

项目摘要

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.

红细胞质量的生理调节取决于对缺氧的促红细胞生成素（EPO）基因的转录增强。 EPO基因表达的研究对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反应的机制很有用。缺氧对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，HIF-1和HNF-4与大约50 bp 3'增强子的关键响应元件结合。缺氧对HIF-1的激活取决于其α亚基免受泛素依赖性蛋白水解的选择性保护。 HNF-4是一种孤儿核受体，在肾脏和肝脏中组成型表达，并与HIF-1合作以产生最大的低氧诱导。在此续签应用中，我们将继续研究缺氧引起EPO基因表达显着增加的机制。特定的目标1专注于缺氧对HIF-1的激活。定位的诱变将确定追形性和/或蛋氨酸氧化是否介导HIF-1Alpha的氧依赖性降解。我们还将调查使用ARNT或热休克蛋白90的组装有助于HIF-1Alpha的稳定性和核定位。具体目标2解决了HNF-4在介导EPO基因介导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EPO增强剂上与HNF-4相互作用的蛋白质。我们还将评估该核受体在介导EPO基因表达中的功能是否受假定的天然配体的影响。作为对EPO基因转录的这些研究的补充，我们将在特定的AIM 3中深入研究EPO表达是否也受到mRNA稳定性水平的调节。 EPO mRNA的3'未翻译区域将通过采用明显的EPO基因的转染实验以及凝胶移位实验和无细胞RNA稳定性测定法进行研究。特定的目标4集中在暴露于缺氧后，水跳蚤的分子基础上的分子基础。这些研究应提供有关氧气传感，信号转导和基因调节的常见机制的独特且高度相关的信息，使生物能够适应缺氧。

项目成果

期刊论文数量（24）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells.

鉴定人类细胞中普遍表达的细胞色素 b 型 NAD(P)H 氧化还原酶。

DOI：
10.1073/pnas.96.26.14742
发表时间：
1999
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Zhu,H;Qiu,H;Yoon,HW;Huang,S;Bunn,HF
通讯作者：
Bunn,HF

Cis elements that regulate the erythropoietin gene.

调节促红细胞生成素基因的顺式元件。