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.
|Gorr, Thomas A; Gassmann, Max; Wappner, Pablo (2006) Sensing and responding to hypoxia via HIF in model invertebrates. J Insect Physiol 52:349-64|
|Gorr, Thomas A; Rider, Cynthia V; Wang, Helen Y et al. (2006) A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter. Mol Cell Endocrinol 247:91-102|
|Gorr, Thomas A; Tomita, Takeshi; Wappner, Pablo et al. (2004) Regulation of Drosophila hypoxia-inducible factor (HIF) activity in SL2 cells: identification of a hypoxia-induced variant isoform of the HIFalpha homolog gene similar. J Biol Chem 279:36048-58|
|Gorr, Thomas A; Cahn, Joshua D; Yamagata, Hideo et al. (2004) Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxia-inducible factor-dependent. J Biol Chem 279:36038-47|
|Huang, L Eric; Pete, Erin A; Schau, Maureen et al. (2002) Leu-574 of HIF-1alpha is essential for the von Hippel-Lindau (VHL)-mediated degradation pathway. J Biol Chem 277:41750-5|
|Gu, J; Milligan, J; Huang, L E (2001) Molecular mechanism of hypoxia-inducible factor 1alpha -p300 interaction. A leucine-rich interface regulated by a single cysteine. J Biol Chem 276:3550-4|
|Horiguchi, H; Kayama, F; Oguma, E et al. (2000) Cadmium and platinum suppression of erythropoietin production in cell culture: clinical implications. Blood 96:3743-7|
|Huang, L E; Willmore, W G; Gu, J et al. (1999) Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. J Biol Chem 274:9038-44|
|Zhu, H; Qiu, H; Yoon, H W et al. (1999) Identification of a cytochrome b-type NAD(P)H oxidoreductase ubiquitously expressed in human cells. Proc Natl Acad Sci U S A 96:14742-7|
|Ebert, B L; Bunn, H F (1999) Regulation of the erythropoietin gene. Blood 94:1864-77|
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