A novel hepatocytic transcription factor that binds the alpha-fetoprotein promoter-linked coupling element.
We recently characterized a promoter-linked coupling element (PCE) in the rat alpha-fetoprotein (AFP) gene required for strong transcriptional stimulation by distant enhancers (P. Wen, N. Crawford, and J. Locker, Nucleic Acids Res. 21:1911-1918, 1993). In this study, oligonucleotide gel retardation and competition experiments defined the PCE as a 12-bp binding site, TGTCCTTGAACA, an imperfect inverted repeat from -166 to -155 near the AFP promoter. A factor that bound this site (PCF) was abundant in HepG2 nuclear extracts and detectable in extracts from several other AFP-producing hepatocarcinoma cell lines and fetal liver. Hepatocytic cell lines that did not express AFP, nonhepatocytic cell lines, adult liver, and fetal brain did not show the factor. Experiments excluded the possibility that PCF activity was due to binding of glucocorticoid receptor or an AP1-like factor that bound overlapping sites. Competition experiments with several mutant oligonucleotides determined that the optimum PCF binding site was TGTCCTTGAAC(A/T). Mutations decreased binding or totally abolished binding activity. In expression plasmids, PCE mutations strongly reduced gene expression. UV cross-linking to a PCE probe identified peptide bands near 34 kDa. PCF was purified by heparin-Sepharose chromatography followed by affinity binding to oligomerized PCE DNA. The product resolved as a complex of three peptides (PCF alpha 1, PCF alpha 2, and PCF beta, 32 to 34 kDa) on sodium dodecyl sulfate-acrylamide gels. The peptide sizes and gel patterns are unlike those of any of the well-described hepatic transcription factors, and the binding site has not been previously reported. PCF thus appears to be a novel transcription factor.
Wen
P
P
Department of Pathology, University of Pittsburgh, Pennsylvania 15261.
Locker
J
J
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003432:Cross-Linking Reagents; D004252:DNA Mutational Analysis; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000235:genetics*
D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000509:alpha-Fetoproteins; D015500:Chloramphenicol O-Acetyltransferase