The epidermal growth factor receptor (EGFR) is overexpressed in many human cancers and cancer cell lines due to gene amplification and/or increased gene transcription. We have continued our efforts to identify and characterize transcription factors that regulate EGFR gene expression. In our studies of EGFR gene regulation, we have documented the interaction of transcription factors such as Sp1, AP1, AP2 and p53 with the promoter region. Recent reports that p53-related molecules, p63/p51/p73L/p40/KET, can tranactivate p53 target promoters prompted us to examine whether these molecules are involved in EGFR gene regulation. TAp63/p51A represses basal EGFR promoter activity in contrast to the activation by p53. Additionally, dNp63/p73L, a dominant-negative effector of TAp63/p51A, rescues the repression by dNp63/p73L. The repression by TAp63/p51A is not mediated via the p53 binding site and appears to involve an inhibition of AP-1 activity. We have also shown the nuclear factor kappaB, NF-kappaB, can bind to several sites in the EGFR promoter but does not transactivate. These results add to the complex nature of EGFR gene regulation and its expression in cancer cells. Another factor that we have shown to interact with the EGFR promoter region is the transcriptional repressor GCF2. Genomic DNA from this clone was labeled and used in FISH analysis to determine chromosomal location. The GCF2 gene was localized to chromosome 3 q27 region. This is a region that is frequently overrepresented in malignant lymphomas. This is consistent with our finding of high level GCF2 expression in six Burkitts lymphoma cell lines and in one cell line derived from a T-cell lymphoma. We have recently determined that GCF2 can repress both basal and TAT-mediated activation of the HIV-LTR. GCF2 is able to bind to TAR RNA and other double-stranded RNAs. GCF2 can also bind to TAR DNA. GCF2 repression of the HIV-LTR requires both amino-terminal and carboxy-terminal regions of GCF2. The mechanism of repression appears to involve interactions with TAR RNA and DNA. In collaboration with Levon Khachigan, University of New South Wales, Sidney Australia, we also showed that the platelet-derived growth factor A-chain (PDGF-A) promoter was repressed by GCF2. The binding site was determined by DNase I footprinting analysis and found to be identical in sequence to the site in the EGFR promoter. The GCF2 binding site in the PDGF-A promoter overlaps binding sites for Sp1, Sp3 and Egr-1. GCF2 was shown to compete with these three factors for binding to this site. The EGFR promoter is also regulated by Egr-1 but in a cell dependent manner. In collaboration with Xu-Wen Liu, NICHD, we have isolated and cloned the rat EGFR promoter region. The rat EGFR promoter is also GC-rich and contains 64% homology to the human promoter. The TCCTCCTCC repeats as well as Sp1 binding and multiple transcription initiation sites are features of both promoters. We have shown that the TCCTCCTCC repeats are required for nerve growth factor downregulation of EGFR gene expression. We have identified the Wilms Tumor protein, WT1, as a factor that binds to this region. WT1 binds to this promoter region and enhances promoter activity. Upon nerve growth factor treatment, the binding of WT1 is decreased. Thus, the mechanism for nerve growth factor downregulation of rat EGFR expression may be due to loss of WT1 activity. In collaboration with Howard Cedar, Hebrew University, Jerusalem, Israel, we have shown that GCF2 is able to bind to a repressor element in the upstream region of the gene. GCF2 binding and repressor activity are abrogated by DNA methylation of the site in the maternal IGF2 allele. These results establishes a role of GCF2 in the imprinting of the IGF2 gene.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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Nishi, Hirotaka; Neta, Gila; Nishi, Katsura H et al. (2003) Analysis of the epidermal growth factor receptor promoter: the effect of nuclear factor-kappaB. Int J Mol Med 11:49-55
Rikiyama, Toshiki; Curtis, Joseph; Oikawa, Masaya et al. (2003) GCF2: expression and molecular analysis of repression. Biochim Biophys Acta 1629:15-25
Nishi, H; Senoo, M; Nishi, K H et al. (2001) p53 Homologue p63 represses epidermal growth factor receptor expression. J Biol Chem 276:41717-24
Johnson, A C; Murphy, B A; Matelis, C M et al. (2000) Activator protein-1 mediates induced but not basal epidermal growth factor receptor gene expression. Mol Med 6:17-27
Liu, X W; Katagiri, Y; Jiang, H et al. (2000) Cloning and characterization of the promoter region of the rat epidermal growth factor receptor gene and its transcriptional regulation by nerve growth factor in PC12 cells. J Biol Chem 275:7280-8
Khachigian, L M; Santiago, F S; Rafty, L A et al. (1999) GC factor 2 represses platelet-derived growth factor A-chain gene transcription and is itself induced by arterial injury. Circ Res 84:1258-67