Dimerization, DNA binding, and transactivation properties of hypoxia- inducible factor 1

Bing Hua Jiang, Elizabeth Rue, Guang L. Wang, Rick Roe, Gregg L. Semenza

Research output: Contribution to journalArticle

Abstract

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-1α subunit that are required for heterodimerization, DNA binding, and transactivation. The HIF-1α and HIF-1β (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-1α and HIF-1β 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-1α, amino acids 1-166 mediated heterodimerization with HIF-1β (ARNT), but amino acids 1-390 were required for optimal DNA binding. A deletion involving the basic domain of HIF-1α eliminated DNA binding without affecting heterodimerization. In cotransfection assays, forced expression of recombinant HIF-1α and HIF-1β (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-1α (amino acids 391-826) markedly decreased the ability of recombinant HIF-1 to activate transcription. Overexpression of a HIF-1α construct with deletions of the basic domain and carboxy terminus blocked reporter gene activation by endogenous HIF-1 in hypoxic cells.

Original languageEnglish (US)
Pages (from-to)17771-17778
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number30
DOIs
StatePublished - Aug 10 1996

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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