Characterization of UEF-4, a DNA-binding protein required for transcriptional synergism between two AP-1 sites in the human urokinase enhancer

The enhancer of the inducible urokinase gene depends on three essential but not sufficient transactivating elements, an upstream PEA3/AP-1A and a downstream AP-1B site. Enhancer activity also requires the interposed 74-base pair-long cooperation mediator (COM) region that allows transcriptional synergism between the transactivating sites. The 5'-half of COM (uCOM) forms four retarded complexes with HeLa or Hep-G2 nuclear proteins (UEF-1-4). We have identified the binding sequence for UEF-4 and generated uCOM elements uniquely mutated in the UEF-4-binding site or uniquely binding UEF-4. Introduction of these and other mutations in the context of the urokinase enhancer showed that all uCOM sites are important for enhancer activity but that UEF-4 and UEF-1 plus UEF-2/3 can substitute for each other, suggesting functional redundancy of urokinase enhancer factors. UEF-4 was purified from HeLa nuclear extract by affinity chromatography and shown to contain two polypeptides of 105 and 65 kDa, respectively, of which at least the former was endowed with DNA binding activity.