Human Hypoxic Signal Transduction through a Signature Motif in Hepatocyte Nuclear Factor 4

We identified a human hypoxic signal transduction pathway acting through a signature motif in the carboxyl terminal of hepatocyte nuclear factor 4 (HNF-4), by functional comparison of the transcriptional and protein-protein interaction activities of the wild type and mutants. It was previously shown that HNF-4 functions as a tissue-specific and hypoxia-activated transcription factor for the erythropoietin (Epo) gene. Human HNF-4 (465 amino acid residues) has DNA-binding, ligand-binding, and transactivation domains. The deletion mutant without the carboxyl terminal transactivation domain (amino acids 369-465) has been shown to be a dominant-negative mutant that repressed Epo transcriptional activity in hypoxia. Further characterization of the hypoxia-responsive domain by site-directed mutagenesis indicated that a TKQE motif of the carboxyl terminal (amino acids 460-463) in HNF-4 was essential for hypoxia-inducible Epo gene expression. We also found, by means of immunoprecipitation and a mammalian two-hybrid system, direct interactions between HNF-4 and hypoxia-inducible factor 1 (HIF-1), a heterodimer composed of alpha and beta subunits. HNF-4 was observed to interact with HIF-1alpha and HIF-1beta (arylhydrocarbon receptor nuclear translocator, ARNT) during hypoxia. In addition, the TKQE motif of HNF-4 was essential for protein-protein interactions with HIF-1alpha and ARNT. These results indicate that the human hypoxic signal of HIF-1 is transduced through interactions with the signature TKQE motif of the carboxyl terminal of HNF-4, resulting in Epo gene expression as a response to hypoxia.