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Characterization of the Promoter Region of the Human C-Erbb-2

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Characterization of the Promoter Region of the Human C-Erbb-2 Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4374-4378, July 1987 Biochemistry Characterization of the promoter region of the human c-erbB-2 protooncogene (growth-factor receptor/transcription regulation/"CAAT box"/transcription factor Spl) SHUNSUKE ISHII*, FuMio IMAMOTO*, YUJI YAMANASHIt, KUMAO TOYOSHIMAt, AND TADASHI YAMAMOTOt *Laboratory of Molecular Genetics, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koya-dai, Yatabe, Tsukuba, Ibaraki 305, Japan; and tInstitute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan Communicated by Charles Yanofsky, March 9, 1987 (received for review December 30, 1986) ABSTRACT Three overlapping genomic clones that con- receptor gene, we identified and characterized the promoter tain the 5'-terminal portion of the human c-erbB-2 gene region of c-erbB-2. (ERBB2) were isolated. The promoter region was identified by nuclease S1 mapping with c-erbB-2 mRNA. Seven transcrip- tional start sites were identified. DNA sequence analysis MATERIALS AND METHODS showed that the promoter region contains a "TATA box" and Cells and Tissues. Human adenocarcinoma MKN-7 cells a "CAAT box" about 30 and 80 base pairs (bp), respectively, were maintained in RPMI 1640 medium with 10% fetal bovine upstream of the most downstream RNA initiation site. Two serum. African green monkey kidney CV-1 cells were culti- putative binding sites for transcription factor Spl were iden- vated in Dulbecco's modified Eagle's medium with 10% fetal tified about 50 and 110 bp upstream of the CAAT box, and six bovine serum. Tissues (brain, lung, liver, and kidney) were GGA repeats were found between the CAAT box and the obtained from a single human fetus at 12 weeks of gestation. TATA box. This region had strong promoter activity when Isolation of Clones. A human genomic library was con- placed upstream of the bacterial chloramphenicol acetyltrans- structed from placental DNA as described (10). The library ferase gene and transfected into monkey CV-1 cells. These data was screened by using DNA fragments from the c-erbB-2 indicate that the promoter of the human c-erbB-2 protoonco- cDNA clone pCER235 (2) as probes in plaque-hybridization gene is different from that of the protooncogene c-erbB-I in 50%o (vol/vol) formamide/6x SSC/5x Denhardt's solu- (epidermal growth factor receptor gene), which does not tion/0.1% NaDodSO4 containing 100 ,ug of denatured salmon contain either a TATA box or a CAAT box. Comparison ofthe sperm DNA per ml. (1x SSC is 0.15 M NaCl/15 mM sodium promoter sequences and activities of the two protooncogenes citrate, pH 7; 1x Denhardt's solution is 0.02% polyvinylpyr- should be helpful in analysis of the regulatory mechanism of rolidone/0.02% Ficoll/0.02% bovine serum albumin.) After expression of their gene products, which are growth-factor hybridization at 42°C for 20 hr, the filters were washed in 2x receptors. SSC/0.1% NaDodSO4 at room temperature and then in 0.2x SSC/0.1% NaDodSO4 at 550C. The human c-erbB-2 gene was identified in the human Nucleotide Sequence Analysis. Nucleotide sequence was genome by its cross-hybridization with v-erbB, an oncogene determined by the dideoxy chain-termination method (11, 12) of avian erythroblastosis virus (1). Nucleotide sequence in conjunction with bacteriophage M13 mpl9 (13). analysis of cDNA clones showed that the c-erbB-2 gene Blot Hybridization Analysis of DNA and RNA. High mo- encodes a 1255 amino acid transmembrane protein similar in lecular weight DNAs were prepared from human placenta overall structure to the epidermal growth factor (EGF) and MKN-7 cells. The DNAs (10 ,g per lane) were digested receptor (2). Moreover, the c-erbB-2 gene product was shown with restriction endonucleases under the conditions recom- to be a 185-kDa glycoprotein that is associated with tyrosine mended by the supplier (Takara Shuzo, Kyoto, Japan), kinase activity (3). Therefore, the c-erbB-2 gene product is fractionated by electrophoresis in a 1% agarose gel, and believed to be a receptor for an as yet unidentified growth transferred to a nitrocellulose filter. Hybridization and wash- factor. The rat c-erbB-2 gene (neu) has been shown to be ing were carried out as described by Southern (14). RNAs activated as a transforming gene by a point mutation (4). were prepared by the guanidinium isothiocyanate/cesium Relatively frequent amplification (10-15%) of the c-erbB-2 chloride method (15). Poly(A)+ RNA was selected by oligo- gene has been observed in human adenocarcinomas, espe- (dT)-cellulose (P-L Biochemical type 7) column chromatog- cially those of the stomach and breast (1, 5, 6). Because raphy, denatured with 50% formamide/2.2 M formaldehyde, human adenocarcinoma MKN-7 cells, in which the c-erbB-2 and then subjected to electrophoresis in a 1% agarose gel gene is amplified, express a large amount ofthe c-erbB-2 gene containing 2.2 M formaldehyde (16). RNAs on the gel were product, we think that amplification of the gene may usually transferred directly to a nitrocellulose filter and subjected to be followed by its overexpression. Therefore, it seems blot hybridization as described (17). plausible that overexpression of the c-erbB-2 gene may Nuclease S1 Mapping. A 5-,g sample of poly(A)+ RNA contribute to the phenotype of cellular transformation. Con- from MKN-7 cells was mixed with single-stranded DNAs sistent with this idea, the EGF receptor, which is very similar labeled at the 5' end, in 20 ,ul of 80% formamide/0.4 M in structure to the c-erbB-2 protein, is also often overpro- NaCl/40 mM Pipes, pH 6.4/1 mM EDTA. The mixture was duced in tumor cells (7, 8). denatured at 75°C for 10 min and allowed to hybridize during The promoter region of the EGF receptor gene does not gradual (over 5 hr) cooling to 40°C. Nuclease S1 digestion and contain either a "TATA box" or a "CAAT box," but has five analysis of Sl-protected fragments were performed as de- "GC boxes" (9). To investigate the regulation of expression scribed (18). of the c-erbB-2 gene and to compare it with that of the EGF Transfection and Assay of Chloramphenicol Acetyltrans- ferase (CAT) Activity. CV-1 cells (4 x 105) were seeded into The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: CAT, chloramphenicol acetyltransferase; EGF, epi- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dermal growth factor; SV40, simian virus 40. Downloaded by guest on September 27, 2021 4374 Biochemistry: Ishii et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4375 1 kb were isolated from a human genomic DNA library by screen- ing with the 672-base-pair (bp) Sma I-Pvu II DNA fragment E S s S E E from the cDNA clone pCER235, which consists of 135 bp of I ll 1 1 1 the 5' untranslated sequence and a 537-bp sequence coding l 3' ACER1 for the NH2-terminal portion ofthe c-erbB-2 gene product (2). Twenty-five positive clones were isolated, and three ipEB1 (XCER1, XCER3, and XCER5) of them were found to hybrid- | pEB2 pEB3 ize with the 370-bp HindIII-Sma I DNA fragment of pEB4 pCER235, which contains a 38-bp sequence of the further ,+ -- upstream 5' untranslated region and part of the Okayama- Berg vector. The restriction map of XCER1 is shown in Fig. 1. The 10-kilobase (kb) EcoRI fragment, the 4.1-kb EcoRI-Sma I fragment, the 4.4-kb Sma I fragment, and the Exon 1 0.5-kb Sma I fragment were subcloned in pUC9 vector 1 00 bp (pEB1, pEB2, PEB3, and pEB4, see Fig. 1) and used for further analyses. FIG. 1. Structure of the human c-erbB-2 genomic clones. Re- On Southern (14) blotting analysis, the 370-bp HindIII- striction endonuclease (E, EcoRI; S, Sma I) cleavage map of the Sma I DNA fragment from the pCER235 cDNA clone, used c-erbB-2 DNA in bacteriophage Xis shown at the top. The subcloned as a probe, was found to hybridize with the 530-bp DNA genomic fragments (pEB1, pEB2, pEB3, and pEB4) are shown below the recombinant X phage. Expanded map of the promoter and exon insert of the pEB4 subclone (data not shown). Hence this 1 region is shown at the bottom. Open box represents the 5' fragment should contain the 5' flanking sequence proximal to untranslated sequence in the first exon. the human c-erbB-2 gene. Nucleotide sequence analysis of both the 530-bp Sma I fragment and the adjacent 3' region revealed that one exon is located in this area and that this 100-mm dishes. After 20 hr, a 1-ml suspension of Ca3(PO4)2/ exon contains the 5' untranslated sequence (Fig. 2). DNA precipitate, containing 18 ,g ofpEB plasmid DNA and The cDNA clone pCER235, from which the hybridization 2 ,pg of pRSV,3-gal DNA, was added to each culture at 37°C. probe used for screening of the genomic clones was isolated, Four hours later the cells were exposed to 15% glycerol for was derived from the poly(A)+ RNA of the gastric adeno- 3 min (19). Plasmid pRSVB-gal, which carries the P3-galacto- carcinoma cell line MKN-7, in which the c-erbB-2 gene is sidase gene linked to the Rous sarcoma virus long terminal amplified (30-fold relative to the placenta) (2). In addition to repeat, was used as an internal standard to normalize the the amplified c-erbB-2 gene, a rearranged c-erbB-2 gene was transfection efficiency (20).
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