Oxygen-regulated erythropoietin gene expression is dependent on a CpG methylation-free hypoxia-inducible factor-1 DNA-binding site.
The hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in the expression of oxygen-regulated genes such as that for erythropoietin. Following exposure to low oxygen partial pressure (hypoxia), HIF-1 binds to an hypoxia-response element located 3' to the erythropoietin gene and confers activation of erythropoietin expression. The conserved core HIF-1 binding site (HBS) of the erythropoietin 3' enhancer (CGTG) contains a CpG dinucleotide known to be a potential target of cytosine methylation. We found that methylation of the HBS abolishes HIF-1 DNA binding as well as hypoxic reporter gene activation, suggesting that a methylation-free HBS is mandatory for HIF-1 function. The in vivo methylation pattern of the erythropoietin 3' HBS in various human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas this site was essentially methylation-free in the erythropoietin-producing cell line Hep3B, a direct correlation between erythropoietin protein expression and the degree of erythropoietin 3' HBS methylation was found in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues that do not express erythropoietin suggests that there might be a general selective pressure to keep this site methylation-free, independent of erythropoietin expression.
Wenger
R H
RH
Institute of Physiology, University of Zürich-Irchel, Zürich, Switzerland. labbauer@physiol.unizh.ch
Kvietikova
I
I
Rolfs
A
A
Camenisch
G
G
Gassmann
M
M
D000818:Animals; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D015687:Cell Hypoxia; D002467:Cell Nucleus / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004921:Erythropoietin / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007668:Kidney / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D007739:L Cells; D007938:Leukemia; D008099:Liver / Q000196:embryology / Q000254:growth & development / Q000378:metabolism; D008113:Liver Neoplasms; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D009447:Neuroblastoma; D009687:Nuclear Proteins / Q000378:metabolism*; D009928:Organ Specificity; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D004921:Erythropoietin; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; C015772:CpG dinucleotide