CHARACTERIZATION OF UEF-4, A DNA-BINDING PROTEIN REQUIRED FOR TRANSCRIPTIONAL SYNERGISM BETWEEN 2 AP-1 SITES IN THE HUMAN UROKINASE ENHANCER

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