OXYGEN-REGULATED ERYTHROPOIETIN GENE-EXPRESSION IS DEPENDENT ON A CPGMETHYLATION-FREE HYPOXIA-INDUCIBLE FACTOR-I 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 (hyp oxia), HIF-1 binds to an hypoxia-response element located 3' to the er ythropoietin 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 ta rget of cytosine methylation. We found that methylation of the HBS abo lishes 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 vari ous human cell lines and mouse organs was assessed by genomic Southern blotting using a methylation-sensitive restriction enzyme. Whereas th is site was essentially methylation-free in the erythropoietin-produci ng cell line Hep3B, a direct correlation between erythropoietin protei n expression and the degree of erythropoietin 3' HBS methylation was f ound in different HepG2 sublines. However, the finding that this site is partially methylation-free in human cell lines and mouse tissues th at do not express erythropoietin suggests that there might be a genera l selective pressure to keep this site methylation-free, independent o f erythropoietin expression.