Modulation of Epidermal Growth Factor Receptor Gene Transcription by a Polymorphic Dinucleotide Repeat in Intron 1

Frank Gebhardt,Kurt S Zänker,Burkhard Brandt

doi:10.1074/jbc.274.19.13176

Abstract

The influence of a highly polymorphic CA dinucleotide repeat in the epidermal growth factor receptor (EGFR) gene on transcription was examined with a quantitative nuclear run-off method. We could demonstrate that transcription of the EGFR gene is inhibited by approximately 80% in alleles with 21 CA repeats. In experiments with polymerase chain reaction products that spanned a region of more than 4,000 base pairs and contained the promoter, two enhancers, and the polymorphic region in the first intron of the gene, we found that transcription activity declines with increasing numbers of CA dinucleotides. In vivo pre-mRNA expression data from cultured cell lines support these findings, although other regulation mechanisms can outweigh this effect. In addition, we showed that under our experimental conditions RNA elongation terminates at a site closely downstream of the simple sequence repeat and that there are two separate major transcription start sites. Our results provide new insights in individually different EGFR gene expression and the role of the CA repeat in transcription of this proto-oncogene.

Highlights

Epidermal growth factor receptor (EGFR)1 is a membranespanning 170-kDa glycoprotein that stimulates cell growth after binding of specific ligands [1, 2]
To probe a potential regulatory function of this highly polymorphic region, we investigated the influence of the CASSR on transcription activity in vitro and characterized RNA synthesis in the EGFR 5Ј-region in relation to the number of dinucleotide repeats
Sequence of the EGFR Promoter/Enhancer Region Reveals No Mutations in Cell Lines but Contains a Polymorphism in Intron 1—To characterize sequence and function of the EGFR promoter/enhancer region, we amplified a 4,050-bp PCR product from several cell lines. It contained most of the upstream enhancer, the promoter region, exon 1, a polymorphic CA-simple sequence repeat (SSR), and a downstream enhancer site in intron 1 (ϩ1788 to ϩ2318) of the EGFR gene (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and DNA Preparation—A431 (epidermoid carcinoma cell line), MDA-MB-231, MDA-MB-468, BT-474 (cultured in RPMI 1640, 10% FCS), HBL-100 (mammary cell line, cultured in RPMI 1640, 5% FCS), and MCF-7 were purchased from the American Type Culture Collection (ATCC). The 50-␮l PCR reaction mixture contained 200 ng of DNA of cultured cells, 1.5 mM MgCl2, 7.5% dimethyl sulfoxide (Sigma), 100 ␮M dNTP each (Perkin Elmer), 1ϫ PCR amplification buffer, 1.5 units of Taq polymerase (Promega), and light white mineral oil (Sigma). Heterodimer Analysis—Equal amounts of the purified EGFR 4,050-bp PCR products from A431 and each other cell line were combined and denatured for 4 min at 100 °C under a mineral oil layer. DNA was instead digested with 10 units of RNase-free DNase I (Roche Molecular Biochemicals), phenol/chloroform/isoamyl alcohol extracted and precipitated in the presence of 50 ␮g of yeast RNA and a fluoresceinated antisense RNA, transcribed from a 572-bp PCR product plus T7 RNA polymerase promoter at the 3Ј-end. To eliminate the influence of gene amplification or loss of heterozygosity on protein expression, protein concentrations were divided by the EGFR gene dosage

RESULTS

58 Ϯ 19 57 Ϯ 19 34 Ϯ 11 18 Ϯ 6 21 Ϯ 7 attomol

DISCUSSION