Abstract
The response of the epidermal growth factor (EGF) receptor gene to phorbol 12-myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run-off experiments showed that PMA increased EGF receptor gene transcription. Cell-free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene.
Highlights
From the Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255
Samples of radiolabeled RNA isolated from the nuclei of KB cells treated with or without 100 nM phorbol 12-myristate 13-acetate (PMA) were hybridized to epidermal growth factor receptor (EGFR) DNA to see if there were any changes in the transcription of this gene (Fig. 1)
I report that the mechanism by which PMA increases EGFR mRNA levels is at least in part through an enhancement of transcription
Summary
From the Laboratory of Molecular Biology, Division of Basic Sciences, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255. A promoter fragment containing nucleotides ؊167 to ؊105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene. One of those additional DNA binding proteins, termed ETF (EGFR transcription factor), has been obtained in a highly purified form Both Sp1 and ETF promote EGFR transcription in cell-free extracts [32, 33]. A GCF cDNA was cloned, and GCF was shown to repress EGFR promoter activity by binding to three sites in the promoter [38]
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