THE ORPHAN RECEPTOR HEPATIC NUCLEAR FACTOR-4 FUNCTIONS AS A TRANSCRIPTIONAL ACTIVATOR FOR TISSUE-SPECIFIC AND HYPOXIA-SPECIFIC ERYTHROPOIETIN GENE-EXPRESSION AND IS ANTAGONIZED BY EAR3 COUP-TF1/

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-ac ting elements in the promoter and in a 3' enhancer, both of which cont ain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, tr anscribed and translated in vitro, were screened by the electrophoreti c mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR 2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the respons e elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those o bserved in nuclear extracts of the Epo-producing cell line Hep3B. More over, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increa se in the hypoxic induction of a luciferase reporter construct which c ontains the minimal Epo enhancer and Epo promoter, provided that the n uclear hormone receptor consensus DNA elements in both the promoter an d the enhancer were intact. The augmentation by HNF-4 in HeLa cells co uld be abrogated by cotransfection with HNF-4 Delta C, which retains t he DNA binding domain of HNF-4 but lacks the C-terminal activation dom ain. Moreover, the hypoxia induced expression of the endogenous Epo ge ne was significantly inhibited in Hep3B cells stably transfected with HNF-4 Delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter, These ele ctrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and h ypoxia-inducible expression of the Epo gene,whereas the COUP family ha s a negative modulatory role.