Erythropoietin 5' – Flanking Sequence–Binding Protein Induced during Hypoxia and Cobalt Exposure1

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The 5'-flanking region of the human erythropoietin (Epo) gene contains a 0.14-kb sequence that is conserved in the Epo gene from mouse and located within a promoter that is activated under hypoxic conditions such as anemia. Using a fragment containing this sequence in DNA mobility shift assays, we found that specific DNA-binding proteins were induced in mouse kidney nuclei under anemic hypoxia. Using synthetic double-stranded oligonucleotides that contain this sequence, the essential binding site was defined to be the -40 to -20 region upstream of the transcription initiation site in the human Epo gene. By DNA affinity chromatography using a column with the immobilized 5'-flanking sequence, two inducible binding proteins with apparent molecular masses of 55 and 45 kDa were identified in the nuclei of mouse kidney and liver under anemic hypoxia. These binding proteins were also induced during cobalt exposure.

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