HYPOXIA INDUCIBLE FACTOR 1 AND OXYGEN HOMEOSTASIS

描述（改编自研究者摘要）：人类和所有 其他哺乳动物需要氧气来进行各种基本的代谢， 流程. 全身、局部和细胞内稳态反应 是由缺氧引起的，在这种状态下，氧气需求超过 供应许多低氧适应性反应涉及基因的改变， 发生在转录水平的表达。 HIF-1是一种 转录因子是转录所必需的 促红细胞生成素基因增强子介导的缺氧性活化 细胞在多种组织培养细胞中检测到HIF-1活性 经受缺氧。 HIF-1也参与了 糖酵解酶基因的转录调控 缺氧细胞，这表明该因子在 对细胞缺氧的转录反应。这项建议会 探索HIF-1活性的分子机制， 调节以响应氧张力的变化，并建立 缺氧诱导因子-1在氧稳态中作用 表达和体内表达缺陷的后果。 研究人员通过蛋白质微测序和cDNA克隆， HIF-1是一个异二聚体的碱性螺旋-环-螺旋PAS转录 HIF-1 α和HIF-1 β亚基都被诱导 在低氧细胞中稳态RNA和蛋白质水平， HIF-1的RNA、蛋白质和DNA结合活性迅速衰减， 缺氧后的细胞 GAL 4/HIF-1 α融合基因产物将被 在培养的人类细胞中表达，以确定 HIF-1 α RNA和蛋白质的稳定性，并证明存在 HIF-1 α蛋白的反式激活结构域。 的 研究者将分离人HIF-1 α基因，并建立 其结构和亚染色体位置。研究者将 证明缺氧诱导HIF-1 α基因转录 并确定顺式作用DNA序列和反式作用因子 介导这种反应。 缺氧诱导因子-1在系统性和 还将建立细胞生理学。 第一，表情 将在小鼠中分析HIF-1 RNA和蛋白质的功能， 解剖位置、环境氧分压和缺氧持续时间。 第二，在基因组中对于无效等位基因是纯合的小鼠， HIF-1 α基因座将通过同源重组产生， 胚胎干细胞这些实验很可能提供 深入了解氧稳态的分子机制， 作为理解病理生理学的基本基础 涉及缺氧的过程，如肿瘤进展，脑 血管意外和心肌缺血/梗塞。

DESCRIPTION (Adapted from Investigator's Abstract): Humans and all other mammals require oxygen for a variety of essential metabolic processes. Systemic, local and intracellular homeostatic responses are elicited by hypoxia, the state in which oxygen demand exceeds supply. Many adaptive responses to hypoxia involved changes in gene expression that occur at the level of transcription. HIF-1 is a transcription factor shown to be necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. HIF-1 activity is detected in a variety of tissue culture cells subjected to hypoxia. HIF-1 has also been implicated in the coordinate transcriptional regulation of genes encoding glycolytic enzymes in hypoxic cells, suggesting that this factor plays a key role in transcriptional responses to cellular hypoxia. This proposal will explore the molecular mechanisms by which HIF-1 activity is modulated in response to changes in oxygen tension and establish the role of HIF-1 in oxygen homeostasis by analyzing its normal expression and the consequences of deficient expression in vivo. The investigator has shown by protein microsequencing and cDNA cloning that HIF-1 is a heterodimeric basic helic-loop-helix PAS transcription factor, that both HIF-1alpha and HIF-1beta subunits are induced at the level of steady-state RNA and protein in hypoxic cells, and that HIF-1 RNA, protein, and DNA-binding activity rapidly decay in post-hypoxic cells. GAL4/HIF-1alpha fusion gene products will be expressed in cultured human cells to identify determinants of HIF-1alpha RNA and protein stability and to demonstrate the presence of a transactivation domain in the HIF-1alpha protein. The investigator will isolate the human HIF-1alpha gene, and establish its structure and subchromosomal location. The investigator will demonstrate that HIF-1alpha gene transcription is induced by hypoxia and determine the cis-acting DNA sequences and trans-acting factor that mediate this response. The role of HIF-1 in systemic and cellular physiology will also be established. First, expression of HIF-1 RNAs and proteins will be analyzed in mice as a function of anatomic location, ambient oxygen tension, and duration of hypoxia. Second, mice that are homozygous for a null allele at the HIF-1alpha locus will be generated by homologous recombination in embryonic stem cells. These experiments are likely to provide insights into molecular mechanisms of oxygen homeostasis that will serve as a basic foundation for understanding pathophysiologic processes that involve hypoxia, such as tumor progression, cerebral vascular accidents, and myocardial ischemia/infarction.