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-1 alpha subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1 alpha and HIF-1 beta (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-1 alpha and HIF-1 beta 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-1 alpha, amino acids 1-166 mediated heterodimerization with HIF-1 beta (ARNT), but amino acids 1-390 were required for optimal DNA binding, A deletion involving the basic domain of HIF-1 alpha eliminated DNA binding without affecting heterodimerization, In cotransfection assays, forced expression of recombinant HIF-1 alpha and HIF-1 beta (ARNT) activated transcription of reporter genes containing EPO enhancer sequences with intact, but not mutant, HIF-1 binding sites, Deletion of the car boxy terminus of HIF-1 alpha (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription, Overexpression of a HIF-1 alpha construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.