HYPOXIA INDUCIBLE FACTOR 1 AND OXYGEN HOMEOSTASIS

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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.