Transitional Change in Interaction Between HIF-1 and HNF-4 in Response to Hypoxia

The roles of the erythropoietin (Epo) 3' enhancer in the activation of gene expression in response to hypoxia were investigated. The enhancer contains hypoxia-inducible enhancer binding site 1 (HIF-1 element) and two direct repeats of hexanucleotide consensus nuclear receptor half site (HNF-4 element). HIF-1, which is a heterodimeric complex of HIF-1 alpha and aryl hydrocarbon receptor nuclear translecator (ARNT), binds to HIF-1 element. HNF-4 binds to HNF-4 element as a homodimeric complex. Studies on mutant reporter plasmids demonstrated that both HIF-1 alpha and HNF-4 elements were necessary for augmentation of the enhancer activity, since mutation of either the HIF-1 or the HNF-4 element caused loss of inducibility under hypoxic conditions. Mammalian two-hybrid experiments in vivo revealed that transitional change took place from the interaction of HNF-4 with ARNT to that with HIF-1 alpha in response to hypoxia. Such interactive domains were identified in amino acids 369-465 containing the C-terminal of HNF-4 and amino acids 1-458 containing basic helix-loop-helix (bHLH) and Per-ARNT-AHR-Sim (PAS) domains of ARNT in normoxia. Also, an extended sequence containing ligand and dimerization domains, and the C-terminal of HNF-4 (amino acids 135-465), and the PAS domain (amino acids 106-526) of HIF-1 alpha were used for the interaction between the two transcription factors in hypoxia. From these data, the functional significance of the transitional change in the augmentation of gene expression by the Epo enhancer in hypoxia is discussed.