Molecular cloning and characterization of a transcription regulator with homology to GC-binding factor.
GC-binding factor (GCF) represses transcription of certain genes and is encoded by a 3.0-kilobase mRNA (Kageyama, R., and Pastan, I. (1989) Cell 59, 815-825). The GCF cDNA hybridizes to two additional mRNA species, 4.2 and 1.2 kilobases. We have used differential hybridization to identify a cDNA clone (termed GCF2) for the 4. 2-kilobase mRNA and find that it is highly expressed in HUT-102 cells. The open reading frame consists of 2256 nucleotides and encodes a protein of 752 amino acids with a calculated molecular mass of 83 kilodaltons. GCF2 expressed in vitro using reticulocyte lysates and Escherichia coli migrates as a 160-kilodalton protein in SDS-polyacrylamide gel electrophoresis but has a molecular mass of 83 kilodaltons as determined by mass spectrum analysis. GCF2 binds to epidermal growth factor receptor promoter fragments, and the major binding site is located between nucleotides -249 and -233. Cotransfection assays show that GCF2 acts to repress transcription from the epidermal growth factor receptor promoter in constructs containing the major GCF2 binding site and not when the site had been mutated. Thus, GCF2 is a newly identified transcriptional repressor with aberrant electrophoretic mobility.
Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
A L
AL
Reed
H
H
Yamazaki
J D
JD
Kaufman
Y
Y
Rubinstein
B
B
Murphy
A C
AC
Johnson
D000595:Amino Acid Sequence; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014407:Tumor Cells, Cultured
C072278:GCFC2 protein, human; D012333:RNA, Messenger; D012097:Repressor Proteins; D004247:DNA; D066246:ErbB Receptors