A 24-BASE-PAIR SEQUENCE 3' TO THE HUMAN ERYTHROPOIETIN GENE CONTAINS A HYPOXIA-RESPONSIVE TRANSCRIPTIONAL ENHANCER

Erythropoietin (Epo) synthesis increases in response to hypoxia. The h epatoma cell line Hep 3B produces low basal levels of Epo mRNA which i ncrease markedly with hypoxia. To define the sequences necessary for t his response, we linked fragments of the human Epo gene to a luciferas e vector, introduced these plasmids into Hep 3B cells and assayed for luciferase activity after growth in 1 % or 21 % oxygen. A 621-bp Epo p romoter fragment resulted in a 2.4-fold increase in luciferase activit y with hypoxia. We tested several Epo gene fragments upstream of this Epo promoter fragment and found that a 613-bp Bgl II-Pvu II 3' fragmen t had a 10-fold increase in activity with hypoxia regardless of orient ation. This fragment had a similar level of activity when linked to a simian virus 40 promoter. Portions of this fragment retained activity, including a 38-bp Apa I-Taq I fragment that had a 17-fold increase in activity with hypoxia. Deletion of nt 4-13 or 19-28 from this 38-bp f ragment resulted in a loss of activity. The 24-bp upstream portion of the 38-bp fragment showed an 8-fold increase in activity with hypoxia. However, deletion of nt 19-24 or mutagenesis of nt 21 or 22 in this 2 4-bp fragment resulted in loss of activity. Our studies indicate that the transcriptional response of the human Epo gene to hypoxia is media ted in part by promoter sequences and to a greater degree by an enhanc er element located in a 24-bp portion of the 3' flanking sequence of t he gene.