Purification and characterization of UEF3, a novel factor involved in the regulation of the urokinase and other AP-1 controlled promoters.
Basal as well as induced transcription from the human urokinase-type plasminogen activator gene requires an enhancer containing two elements, a combined PEA3/AP-1 and a consensus AP-1 site. The integrity of each of these binding sites as well as their cooperation is required for activating transcription. The two elements are separated by a 74-base pair cooperation mediating (COM) region required for the cooperation between the transactivating sites. The COM region contains binding sites for four different unidentified urokinase-type plasminogen activator enhancer factors (UEF 1 to 4), all four required for correct COM activity. We have purified UEF3 from HeLa nuclear extracts by several chromatographic steps including DNA affinity purification. Purification and UV cross-linking data showed that UEF3 is a complex of three polypeptides (p40, p50, and p64). Amino acid sequence from one peptide of p64 was obtained, which showed no homology to other known proteins. Both crude and purified UEF3 specifically bound to the sequence TGACAG as shown by electrophoretic mobility shifts and methylation interference studies. DNA-binding specificity of purified UEF3 was identical to that of NIP, a non-characterized factor binding and regulating multiple AP-1-regulated promoters like stromelysin and interleukin-3. Thus UEF3 appears to be a general DNA-binding factor involved in modulating the transcriptional response of AP-1 containing promoters.
Berthelsen
J
J
Department of Genetics and Biology of Microorganisms, University of Milano, Via Celoria 26, 20135 Milano, Italy.
Vandekerkhove
J
J
Blasi
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D007377:Interleukin-3 / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
D004268:DNA-Binding Proteins; D007377:Interleukin-3; D009838:Oligodeoxyribonucleotides; D018808:Transcription Factor AP-1; D014568:Urokinase-Type Plasminogen Activator; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3