ERYTHROPOIETIN GENE-REGULATION DEPENDS ON HEME-DEPENDENT OXYGEN SENSING AND ASSEMBLY OF INTERACTING TRANSCRIPTION FACTORS

Studies on erythropoietin (Epo) gene expression have been useful in in vestigating the mechanism by which cells and tissues sense hypoxia. Bo th in vivo and in Hep3B cells, Epo production is induced not only by h ypoxia but also by certain transition metals (cobalt and nickel) and b y iron chelation. When Hep3B cells were incubated in an iron deficient medium, Epo mRNA expression was enhanced fourfold compared to Hep3B c ells in iron enriched medium. Epo induction by cobalt was inversely re lated to iron concentration in the medium, indicating competition betw een the two metals. Under hyperbaric oxygen, cobalt induction of eryth ropoietin mRNA was modestly suppressed while nickel induction was mark edly enhanced. These recent observations support the proposal that the oxygen sensor is a heme protein in which cobalt and nickel can substi tute for iron in the porphyrin ring. The up-regulation of Epo gene tra nscription by hypoxia depends on al least two known DNA binding transc ription factors, HIF-1 and HNF-4, which bind to cognate response eleme nts in a critical similar to 50 bp 3' enhancer. Hypoxia induces HIF-1 binding. HNF-4, an orphan nuclear receptor constitutively expressed in kidney and liver, binds downstream of HIF-1 and cooperates with HIF-1 , contributing importantly to high level and perhaps tissue specific e xpression. The C-terminal activation domain of HNF-4 binds to the beta subunit of HIF-1. The C-terminal portion of the alpha subunit of HIF- 1 binds specifically to p300, a general transcriptional activator. Hyp oxic induction of the endogenous Epo gene in Hep3B cells as well as an Epo-reporter gene was fully inhibited by E1A, an adenovirus protein t hat binds to and inactivates p300, but only slightly by a mutant E1A t hat fails to bind to p300. Moreover, overexpression of p300 enhanced h ypoxic induction. Thus, it is likely that in hypoxic cells, p300 or a related family member plays a critical role in forming a macromolecula r assembly with HIF-1 and HNF-4, enabling transduction from the Epo 3' enhancer to the apparatus on the promoter responsible for the initiat ion of transcription.