RAPID COMMUNICATION Characterization of Hypoxia-Responsive Enhancer in the Human Erythropoietin Gene Shows Presence of Hypoxia-Inducible 120-Kd Nuclear DNA-Binding Protein in Erythropoietin-Producing and Nonproducing Cells

Erythropoietin (Epo) production in response to hypoxia or cobalt is primarily mediated by activation of transcription of the Epo gene. Recently an hypoxia responsive enhancer was identified in the 3 flanking region of the mouse and human Epo genes. Using functional analysis in Hep 38 cells we define here the minimal enhancer element as a 29-bp segment starting at the Apal site in the 3 flanking region of the human Epo gene. Mutagenesis studies of the minimal element identified three different areas that are necessary for full enhancer activity. Electrophoretic mobility shift assays show the presence of hypoxiaand/or cobalt-inducible nuclear DNA-binding proteins that bind to one of the active sites of the enhancer. Induction of hyp-