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 transcription al stimulation by distant enhancers (P. Wen, N. Crawford, and J. Locke r, Nucleic Acids Res. 21:1911-1918, 1993). In this study, oligonucleot ide gel retardation and competition experiments defined the PCE as a 1 2-bp binding site, TGTCCTTGAACA, an imperfect inverted repeat from -16 6 to -155 near the AFP promoter. A factor that bound this site (PCF) w as 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. Experime nts excluded the possibility that PCF activity was due to binding of g lucocorticoid receptor or an AP1-like factor that bound overlapping si tes. Competition experiments with several mutant oligonucleotides dete rmined that the optimum PCF binding site was TGTCCTTGAAC(A/T). Mutatio ns decreased binding or totally abolished binding activity. In express ion 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 bind ing to oligomerized PCE DNA. The product resolved as a complex of thre e 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 pat terns are unlike those of any of the well-described hepatic transcript ion factors, and the binding site has not been previously reported. PC F thus appears to be a novel transcription factor.