THE TRANSCRIPTION FACTORS ATF-1 AND CREB-1 BIND CONSTITUTIVELY TO THEHYPOXIA-INDUCIBLE FACTOR-I (HIF-1) DNA RECOGNITION SITE

The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA bi nding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription. Sub sequently HIF-1 activity has also been found in cell lines which do no t express erythropoietin, suggesting that HIF-1 is part of a widesprea d oxygen sensing mechanism. In electrophoretic mobility shift assays H IF-1 DNA binding activity is only detectable in nuclear extracts of ce lls cultivated in a low oxygen atmosphere. In addition to HIF-1, a con stitutive DNA binding activity also specifically binds the HIF1 probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HI F-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the cons titutive factor, while Jun and Fos family members, which constitute th e AP-1 factor, were immunologically undetectable. Recombinant ATF-1 an d CREB-1 proteins bound HIF1 probes either as homodimers or as heterod imers, indicating a new binding specificity for ATF-1/CREB-1. Finally reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling p athway is involved in oxygen sensing.