Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U69609
Differential Hybridization of GC-binding factor (GCF) cDNA Fragments—We have previously reported that GCF cDNA hybridizes to three mRNA species of 4.5, 3.0, and 1.2 kb in several cell lines
We found that various fragments of the cDNA hybridized differently to the three mRNA species [28]
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U69609. Sp1, wild type p53, EGFR transcription factor, and AP2 have been shown to activate EGFR gene transcription (20 –23). EGFR transcriptional repressor, GC-binding factor (GCF), and the Wilms’ tumor supressor, bind to sites within the EGFR promoter (24 –26). Cotransfection experiments have shown that GCF can repress transcription of the EGFR promoter and several other growth-related gene promoters such as transforming growth factor-␣ and insulin-like growth factor II [27]. We present results on isolation of a cDNA that hybridizes to the larger mRNA and has homology to the 5Ј-end of the GCF cDNA. We show that the protein encoded by this mRNA binds to the EGFR promoter and represses the activity of the EGFR, SV40, and RSV promoters
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