Proc. Natl. Acad. Sci. USA Vol. 84, pp. 4374-4378, July 1987 Biochemistry Characterization of the region of the human c-erbB-2 protooncogene (growth-factor receptor/ regulation/"CAAT box"/ 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 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 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). 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). After incubation at 37°C for 40 hr also found in the MKN-7 cells (5). Therefore, the 5' region of in Dulbecco's modified Eagle's medium supplemented with the c-erbB-2 gene cloned in XCER1 might not have been the 10% fetal bovine serum, the cells were harvested. Cell DNAs of MKN-7 extracts were prepared, and the total protein concentration authentic one. To exclude this possibility, was measured with protein assay reagents (Bio-Rad). CAT cells and human placenta were analyzed by Southern blot assays were performed as described (21, 22). Reaction hybridization using the 530-bp Sma I DNA fragment, the mixtures (170,l) containing 200,g ofcell-extract protein, 0.8 insert of pEB4, as a probe. If the 5' region of the c-erbB-2 ,Ci of [14C]chloramphenicol (Amersham; 1 uCi = 37 kBq), gene in the pEB4 genomic clone were not the authentic one, and 4 mM acetyl coenzyme A were incubated at 37°C for 3 hr. this probe should hybridize with different DNA fragments of the MKN-7 and placenta. However, as shown in Fig. 3, the MKN-7 DNA and the human placenta DNA contained the RESULTS same hybridizable DNA fragment generated by various Isolation of 5'-Specific Human c-erbB-2 Genomic Clones. restriction enzymes, although MKN-7 DNA also contained Genomic clones containing the 5' end of the c-erbB-2 gene small amounts of another rearranged fragment. From these CCCGGGGGTCCTGGAAGCCACAAGGTAAACACAACACATCCCCCTCCTTGACTATCAATTTTACTAGAGG -400 ATGTGGTGGGAAAACCATTATTTGATATTAAAACAAATAGGCTTGGGATGGAGTAGGATGCAAGCTCCCA -300 GGAAAGTTTAAGATAAAACCTGAGACTTAAAAGGGTGTTAAGAGTGGCAGCCTAGGGAATTTATCCCGGA

CTCCGGGGGAGGGGGCAGAGTCACCAGCCTCTGCATTTAGGGATTCTCCGAGGAAAAGTGTGAGAACGGC -200 TGCAGGCAACCCAGCTTCCCGGCGCTAGGAGGGACGCACCCAGGCCTGCGCGAAGAGAGGGAGAAAGTGA -100 4 AGCTGGGAGTTGCCACTCCCAGACTTGTTGGAATGCAGTTGGAGGGGGCGAGCTGGGAGCGCGCTTGCTC - - - . . -. *4 C ~CAGGAGAAGGAGGAGGTGGAGGAGGAGGGCTGCTTGAGGAAGA~GAATGAAGTTGTGAAG 444+2 CTGAGATTCCCCTCCATTGGGACCGGAGAAACCAGGGAGCCCCCCCGGG

FIG. 2. Nucleotide sequence of the 5' portion of the human c-erbB-2 gene. The DNA sequence of the 530-bp Sma I-Sma I fragment, the insert of pEB4, is shown, where + 1 corresponds to the most downstream RNA start site and residues preceding this position are represented by negative numbers. Sequence that is consistent with that of the cDNA clone is indicated by broken underlining (positions -2 to +42) (2). The sequence of the 5' portion of the cDNA reported previously (2) is corrected here. The multiple initiation sites demonstrated by nuclease S1 mapping are indicated by vertical arrows. The TATA and CAAT sequences are boxed, and the Spl binding sequences are underlined. Horizontal arrows indicate inverted repeats (dyad symmetry elements). Downloaded by guest on September 27, 2021 4376 Biochemistry: Ishii et al. Proc. Natl. Acad. Sci. USA 84 (1987)

A B 1 2 1 2 3 4 5 1 2 3 4 5 Probe- Iwna - kb _0 "MM --23 - 9.4 .' - 6.6 0, . 4. -4.4 6 .9 0 WV 4ow *.Ills - 2.3 - 2.0 S .. 3~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

125 -- e

I .. S ' 4 11 0 . 110 0 0

FIG. 3. Southern blot analysis of the promoter region of the human c-erbB-2 gene. Genomic DNAs from MKN-7 cells (1 ,ug per lane) (A) and human placenta (10 ,ug per lane) (B) were digested with various restriction enzymes before being electrophoresed in 1% agarose. The fractionated DNAs were transferred to a nitrocellulose filter and probed with 32P-labeled pEB4 insert (specific activity, 2 x 108 cpm/Ag). Restriction enzymes used were BamHI (lanes 1), Pvu II (lanes 2), EcoRI (lanes 3), HindIII (lanes 4), and Pst I (lanes 5). Positions offragments ofHindIII-digested X DNA used as standards are shown at right.

results we concluded that the genomic clone pEB4 contains the authentic 5' region of the c-erbB-2 gene. 4039 Nuclease S1 Mapping. For identification of the 5' boundary of the c-erbB-2 RNA, a 32P-labeled Sma I-Sma I (Fig. 2, nucleotides +39 to -493) single-stranded DNA probe was prepared from plasmid pEB4 and used for nuclease S1 protection studies. The probe was hybridized with MKN-7 Sma Sma poly(A)+ RNA and digested with nuclease S1, and the 3' i 51 protected products were fractionated in a denaturing poly- acrylamide gel. Seven protected products-39, 40, 41, 108, 5' .- 3' 110, 111, and 125 nucleotides long-were detected (Fig. 4). These results show that the transcription ofthe c-erbB-2 gene FIG. 4. Nuclease S1 mapping of the 5' portion of c-erbB-2 RNA. starts at nucleotides +1, -1, -2, -69, -71, -72, and -86. The 550-nucleotide (Sma I-Sma I; nucleotides +39 to -493) single- The three most downstream RNA start sites (nucleotides + 1, stranded DNA was labeled with 32P at the 5' end and was used as a -1, and -2) and the four most upstream RNA start sites probe. Five micrograms of MKN-7 poly(A)+ RNA (lane 1) or 5 ,ug (nucleotides -69, -71, -72, and -86) were clustered. of calf liver tRNA (lane 2) was hybridized with the probe. The DNA Sequence of the c-erbB-2 Promoter Region. Preceding nuclease S1-resistant products were analyzed by electrophoresis in the most start sites for is the 8% polyacrylamide/7 M urea gels. Their sizes (in nucleotides) were downstream transcription determined by using products ofthe sequencing reaction ofthe DNA sequence 5' TATA 3' at nucleotides -28 to -25. This fragments by the method ofMaxam and Gilbert (23) and are indicated "TATA box" is typically found about 30 bp upstream of at left. The probe is illustrated below the autoradiogram. Broken RNA start site (24), and in vitro transcriptional studies have lines represent mRNA; the asterisk shows the position of shown that it serves to fix the transcription initiation site (25). 32P-labeling. A typical "CAAT box" is found at -76 to -63, about 75 bp upstream of the most downstream RNA start site. Hence to -127 (5' GCCACTCCCA 3') and at -192 to -183 (5' transcription from the three downstream initiation sites TAGGAGGGAC 3'). (nucleotides +1, -1, and -2) seems to be controlled by a Further analysis of the 5' flanking region of the c-erbB-2 typical TATA box and CAAT box. The promoter region of gene showed that the GGA sequence is repeated seven times the c-erbB-2 gene was suggested to contain the four other in the region between the CAAT box and the TATA box. In RNA start sites further upstream of the three RNA start sites the region upstream of the CAAT box, three dyad symmetry described above. Therefore, there should be another regu- structures were found, at -75 to -94 (dyad symmetry I), -84 latory element for these RNA start sites. Recently the to -100 (dyad symmetry II), and -126 to -145 (dyad symmetry III). The dyad symmetries I and II overlap, and (26) 3' for 5' GGG C Sp only one of them can form a stem-and-loop structure in a recognition was deduced by comparison of different promot- single molecule. Dyad symmetries I and III both contain the ers ( 27). Originally, the transcription factor Spl was pre- sequence 5' TGGGAG ... CTCCCA 3'. These dyad sym- pared from HeLa cells and shown to bind to the G+C-rich metry structures may regulate the expression of the c-erbB-2 regions (28) ("GC boxes") ofthe simian virus 40 (SV40) early gene by binding to specific factors (see Discussion). promoter. Five Spl molecules could bind to the tandemly Expression of Chloramphenicol Acetyltransferase Directed repeated GC boxes ofthe SV40 early promoter and stimulate by the c-erbB-2 Promoter. The results described above transcription in vitro (28, 29). DNA sequences that are showed that authentic c-erbB-2 mRNA is initiated at seven consistent with the consensus Spl binding sequence were sites between positions -86 and +1. The region upstream found in two different parts ofthe c-erbB-2 promoter: at -136 from these start sites contains the typical TATA box and Downloaded by guest on September 27, 2021 Biochemistry: Ishii et al. Proc. Natl. Acad. Sci. USA 84 (1987) 4377 CAAT box and two putative Spi binding sites. The promoter 1 2 3 4 5 activity of this region was examined by inserting the putative A promoter sequences upstream of the bacterial chloramphen- icol acetyltransferase (CAT) gene (19). The 530-bp Sma I-Sma I DNA fragment (nucleotides +39 to -493) was placed kb on the 5' side of the CAT gene derived from the plasmid - 4.6 pSVOcat (19), in either orientation (Fig. 5), and the abilities ofthese constructs (pEBCAT1-1 and pEBCAT1-2) to express CAT activity was tested by transfection of calcium phos- phate-precipitated DNA into African green monkey CV-1 cells. After 48 hr, cells transfected with pEBCAT1-1, in which transcription from the putative promoter proceeds into the CAT gene, expressed about 5 times more CAT activity than that obtained with the SV40 promoter [pSV2cat (19)]. B On the other hand, the plasmid pEBCAT1-2, in which the direction of transcription from the putative promoter is the opposite to that in pEBCAT1-1, did not induce CAT activity. *- 10.0 For confirmation that the region further upstream does not - 5.6 I DNA show promoter activity, the 1.5-kb HindIII-Sma - 2.9 fragment was placed on the 5' side of the CAT gene. Cells 1 - 2.7 transfected with this CAT construct (pEBCAT2) showed no CAT activity, suggesting that this region does not contain a promoter sequence. For more exact localization of the promoter region, the 280-bp Sma I-Pst I (nucleotides -493 to -214) or 250-bp Pst I-Sma I (nucleotides -213 to +39) DNA fragment was inserted into the 5' side of the CAT gene and FIG. 6. Analysis of c-erbB-2 RNA in various human tissues. the abilities ofthese constructs (pEBCAT3 and pEBCAT4) to Poly(A)+ RNAs (2 ,.tg per lane) were subjected to blot hybridization express CAT activity were tested. CV-1 cells transfected with 32P-labeled c-erbB-2 probe (pCER204, ref. 2) (A) and EGF- with pEBCAT4 expressed almost the same CAT activity as receptor probe (pE7, ref. 17) (B). RNAs were isolated from fetal lung cells transfected with pEBCAT1-1, whereas cells transfected (lanes 1), brain (lanes 2), liver (lanes 3), and kidney (lanes 4) and from not express this activity. Therefore, we placenta at term (lanes 5). 32P-labeled fragments from a HindIII with pEBCAT3 did digest ofphage X DNA were used as size markers. The same blot was concluded that the 250-bp sequence from nucleotide +39 to washed and reprobed. -213, where a TATA box, a CAAT box, two putative Spl F4, binding sites, and some dyad symmetry structure are present, Hind I11 Smal Pstl Smal contains a functional promoter of the c-erbB-2 gene. Genomic clone Levels of c-erbB-2 and c-erbB-1 (EGF Receptor) RNAs in Various Tissues. The tissue-specific expressions of the c- CAT gene erbB-2 and c-erbB-J (EGF receptor) genes, the products of ..4 pEBCAT2 which have very similar structures, were compared by pEBCATI-I measuring the RNA transcripts of these protooncogenes in various human fetal tissues (Fig. 6). The transcription ofboth {==| ~~PEBCATI-2 c-erbB-2 and c-erbB-J was relatively high in the placenta and | | pEBCAT3 low in the lung and liver. In the brain, c-erbB-2 mRNA was expressed at a very low level. Similarly, the expression of I_4IIZI |pEBCAT4 5.6- and 10-kb c-erbB-J mRNAs in the brain was detected only after 10 times longer exposure of the film. The nature of the 2.7- and 2.9-kb c-erbB-J mRNAs in the brain is unclear. Although c-erbB-2 mRNA is intermediately expressed in the -AcCM kidney relative to other tissues, the level of c-erbB-J mRNA 0 in the kidney was as low as that in the brain. Thus, the - AcCM expressions ofthese protooncogenes have similar but distinct tissue specificities. Moreover, data on the expression of c-erbB-2 (Fig. 6A) were obtained after 5 times longer expo- sure of the film than for expression of c-erbB-J (Fig. 6B), indicating that the tissues examined contained less c-erbB-2 1P 1P Ir -CM mRNA than c-erbB-J mRNA. These data suggested that the mechanisms controlling the transcriptions of these two 1 2 3 4 5 protooncogenes are different. FIG. 5. Transient expression of the CAT gene directed by the c-erbB-2 promoter. Plasmid DNA was transfected into CV-1 cells DISCUSSION and CAT activity was measured as described in Materials and Methods. The autoradiogram shown was exposed for 10 hr. CM and In this work we identified and characterized the c-erbB-2 AcCM represent chloramphenicol and acetylated chloramphenicol. gene promoter. This promoter region possesses a typical Recombinant plasmids used were pEBCAT2 (lane 1), pEBCAT1-1 TATA box, a typical CAAT box, and two putative Spl- (lane 2), pEBCAT1-2 (lane 3), pEBCAT3 (lane 4), and pEBCAT4 binding sequences. Transcriptional initiation of the c-erbB-2 (lane 5). The structures of these plasmids are shown above the A TATA box autoradiogram. Open box represents the CAT gene. Lines show the gene was shown to occur at multiple sites. DNA fragments prepared from the genomic subclones. Arrows to left serves to fix the transcription initiation site (25), and most of DNA fragments show the direction of transcription from the genes whose promoter regions contain a TATA box have a c-erbB-2 promoter. single RNA start site. Therefore, it is unusual that the Downloaded by guest on September 27, 2021 4378 Biochemistry: Ishii et al. Proc. Natl. Acad. Sci. USA 84 (1987) promoter region of the c-erbB-2 gene has a TATA box and 3. Akiyama, T., Sudo, C., Ogawara, H., Toyoshima, K. & multiple RNA start sites. In the case of c-erbB-2 transcrip- Yamamoto, T. (1986) Science 232, 1622-1646. tion, the function of TATA box-binding protein could be 4. Bargmann, C. I., Hung, M.-C. & Weinberg, R. A. (1986) Cell 45, 649-657. affected by the DNA sequence around the TATA box or by 5. Fukushige, S., Matsubara, K., Yoshida, M. C., Sasaki, M., other factor(s) bound to the promoter region. Suzuki, T., Semba, K., Toyoshima, K. & Yamamoto, T. An interesting feature of the human c-erbB-2 gene promot- (1986) Mol. Cell. Biol. 6, 955-958. er is the presence of two different types of regulatory 6. Yokota, J., Yamamoto, T., Toyoshima, K., Terada, M., elements, a CAAT box and Spl binding sites, Transcription Sugimura, T., Battifora, H. & Cline, M. J. (1986) Lancet i, from the three most downstream RNA start sites appeared to 765-767. be controlled by the CAAT box and the TATA box, because 7. Xu, Y.-H., Richert, N., Ito, S., Merlino, G. T. & Pastan, I. these are, respectively, about 30 bp and 80 bp upstream of (1984) Proc. Nail. Acad. Sci. USA 81, 7308-7312. these RNA start sites, and these distance. s are consistent with 8. Yamamoto, T., Kamata, N., Kawano, H., Shimizu, S., Kuroki, T., Toyoshima, K., Rikimaru, K., Nomura, N., those in many other eukaryotic promoters (24). On the other Ishizaki, R., Pastan, I., Gamou, S. & Shimizu, N. (1986) hand, transcription from the four other RNA start sites Cancer Res. 46, 414-416. located further upstream seemed to be controlled at least 9. Ishii, S., Xu, Y.-H., Stratton, R. H., Roe, B. A., Merlino, partly by Spl. In contrast with the c-erbB-2 gene promoter, G. T. & Pastan, I. (1985) Proc. Natl. Acad. Sci. USA 82, the promoter region of the human EGF receptor gene does 4920-4924. not contain either a TATA box or a CAAT box but has five 10. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connel, Spl binding sites (9). Therefore, the expression of the C., Quon, D., Sim, G. K. & Etstratiadis, A. (1978) Cell 15, c-erbB-2 gene may be regulated by the transcription factor 687-701. Spl, a CAAT box-binding protein (27, 30) and a TATA 11. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. box-binding protein (31), whereas the expression of the EGF 12. Messing, J., Crea, R. & Seeburg, P. H. (1981) Nucleic Acids receptor gene seems to be regulated by Spl but not by the Res. 9, 309-321. latter two proteins. 13. Messing, J. & Vieira, J. (1982) Gene 19, 269-276. Seven repeats of the GGA sequence were found between 14. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. the CAAT box and the TATA box in the c-erbB-2 gene 15. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, promoter. A purine-rich sequence between the CAAT box W. J. (1979) Biochemistry 18, 5294-5299. and TATA box of the human HSP70 gene promoter was 16. Lehrach, H., Diamond, K., Wozney, J. M. & Boedtker, H. reported to be required for serum stimulation (32). Four (1977) Biochemistry 18, 5294-5299. repeats ofTCC, an inverted sequence ofGGA, were found in 17. Xu, Y.-H., Ishii, S., Clark, A. J. L., Sullivon, M., Wilson, R. K., Ma, D. P., Roe, B. A., Merlino, G. T. & Pastan, I. the promoter region of the EGF receptor gene (9). Two of (1984) Nature (London) 309, 806-810. these pyrimidine-rich regions were found to bind nuclear 18. Berk, A. & Sharp, P. (1977) Cell 12, 721-732. proteins (I. Pastan, personal communication). These sites are 19. Gorman, C. M., MQffat, L. M. & Howard, B. H. (1982) Mol. in sufficiently close proximity to form a "slipped" structure Cell. Biol. 2, 1044-1051. under certain conditions (33, 34). The potential for a switch 20. Edlund, T., Walker, M. D., Barr, P. J. & Rutter, W. J. (1985) in DNA conformation raises the possibility that these sites Science 230, 912-916. are involved in regulation of the EGF receptor gene (I. 21. Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I. Pastan, personal communication). If the binding of nuclear & Howard, B. H. (1982) Proc. Natl. Acad. Sci. USA 79, proteins to these sequences is independent of the DNA 6777-6781. 22. Gorman, C. M., Padmanabhan, R. & Howard, B. H. (1983) sequence orientation, the same factor(s) could bind to GGA Science 221, 551-553. repeats of the c-erbB-2 gene and regulate the expression of 23. Maxam, A. & Gilbert, W. (1980) Methods Enzymol. 65, this gene, too. 499-560. Two dyad symmetry structures containing the, same se- 24. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. quence (5' CTGGGAGT . .. ACTCCCAG 3'; positions -145 50, 349-383. to -126 and -94 to -75) are found in the c-erbB-2 promoter. 25. Fromm, M. & Berg, P. (1982) J. Mol. Appl. Genet. 1, 457-481. Dyad symmetry structures of the promoter region are be- 26. Briggs, M. R., Kadonaga, J. T., Bell, S. P. & Tjian, R. (1986) lieved to regulate the expression of the corresponding genes. Science 234, 47-52. One is the heat consensus element 27. Jones, K. A., Yamamoto, K. R. & Tjian, R. (1985) Cell 42, example shock sequence 559-572. (5' CTNGAANNTTCNAG 3') found in the promoter of the 28. Dynan, W. S. & Tjian, R. (1983) Cell 32, 669-680. heat shock genes (35, 36). 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