REGULATION OF THE ERYTHROPOIETIN GENE

Erythropoietin (Epo), the hormone that stimulates red blood cell produ ction, is induced by hypoxia. We have utilized the human hepatoma cell line, Hep3B, to investigate the regulation of the Epo gene. We presen t evidence that the oxygen sensor in Hep3B cells is a heme protein. Hy poxic and cobalt induction of Epo protein is paralleled by a 50- to 10 0-fold increase in Epo mRNA which we have accurately quantified by mea ns of an assay based on competitive polymerase chain reaction. This in crease in Epo mRNA is due primarily to increased transcription. Transf ection experiments utilizing the sensitive luciferase reporter gene sh ow that the minimal portions of the Epo gene required for hypoxic indu ction include a 53 bp promoter element and a 43 bp enhancer located do wnstream from the polyadenylation site. Gel shift experiments show tha t these two regions cross-compete for specific DNA binding proteins. T he enhancer contains a hexanucleotide direct repeat with a two bp inse rt which footprints with nuclear extracts from Hep3B cells and, when m utated, results in loss of hypoxic induction. This sequence is likely to bind to a member of the steroid/thyroid hormone receptor family of DNA binding proteins. These enhancer and promoter elements appear to c ooperate in enabling the Epo gene to respond to hypoxia in a physiolog ically appropriate manner.