Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.
Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix transcription factor that regulates hypoxia-inducible genes including the human erythropoietin (EPO) gene. In this study, we report structural features of the HIF-1alpha subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1alpha and HIF-1beta (ARNT; aryl hydrocarbon receptor nuclear ... translocator) subunits were coimmunoprecipitated from nuclear extracts, indicating that these proteins heterodimerize in the absence of DNA. In vitro-translated HIF-1alpha and HIF-1beta generated a HIF-1/DNA complex with similar electrophoretic mobility and sequence specificity as HIF-1 present in nuclear extracts from hypoxic cells. Compared to 826-amino acid, full-length HIF-1alpha, amino acids 1-166 mediated heterodimerization with HIF-1beta (ARNT), but amino acids 1-390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1alpha eliminated DNA binding without affecting heterodimerization. In cotransfection assays, forced expression of recombinant HIF-1alpha and HIF-1beta (ARNT) activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites. Deletion of the carboxy terminus of HIF-1alpha (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription. Overexpression of a HIF-1alpha construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.
Mesh Terms:
Base Sequence, Cell Nucleus, Cloning, Molecular, DNA, DNA-Binding Proteins, Helix-Loop-Helix Motifs, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Molecular Sequence Data, Nuclear Proteins, Precipitin Tests, Protein Binding, Protein Biosynthesis, Protein Conformation, Recombinant Proteins, Sequence Deletion, Subcellular Fractions, Transcription Factors, Transcription, Genetic, Transcriptional Activation
Base Sequence, Cell Nucleus, Cloning, Molecular, DNA, DNA-Binding Proteins, Helix-Loop-Helix Motifs, Humans, Hypoxia-Inducible Factor 1, Hypoxia-Inducible Factor 1, alpha Subunit, Molecular Sequence Data, Nuclear Proteins, Precipitin Tests, Protein Binding, Protein Biosynthesis, Protein Conformation, Recombinant Proteins, Sequence Deletion, Subcellular Fractions, Transcription Factors, Transcription, Genetic, Transcriptional Activation
J. Biol. Chem.
Date: Jul. 26, 1996
PubMed ID: 8663540
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