Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements.
Transcription of the human erythropoietin (Epo) gene is stimulated by exposure to hypoxia and/or cobalt in whole animals and in Hep3B cells. We have systematically investigated the promoter and 3' enhancer elements necessary for this induction by transient transfection of Hep3B cells. We define a promoter region of 53 bp and an enhancer region of 43 bp that confer hypoxia and cobalt inducibility. Each element gives rise to a 6- to 10-fold induction alone. In combination they produce a 50-fold induction after stimulation, similar to the 50- to 100-fold induction of the endogenous Epo gene. Two areas of DNA sequence homology are present in these regions. We demonstrate specific DNA-protein interactions in the enhancer and the ability of the promoter element to compete with these interactions in electrophoretic mobility shift assays. DNase I footprinting and methylation interference data further refine the cis-acting element in the 43-bp enhancer to a short region containing a direct repeat of a steroid/thyroid hormone receptor response element half-site separated by a 2-bp gap. Two half-site consensus sequences are also present in the 53-bp promoter. Site-specific mutation of the half-site sequences in the enhancer destroys the functional activity of the enhancer.
Blanchard
K L
KL
Division of Hematology/Oncology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Acquaviva
A M
AM
Galson
D L
DL
Bunn
H F
HF
D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D004921:Erythropoietin / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010100:Oxygen / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011987:Receptors, Steroid / Q000378:metabolism*; D017385:Sequence Homology
D011987:Receptors, Steroid; D004921:Erythropoietin; D004247:DNA; D003850:Deoxyribonuclease I; D010100:Oxygen