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Structural and Functional Analysis of the Human Y-Box Binding Protein (YB-1) Gene Promoter

Structural and Functional Analysis of the Human Y-Box Binding Protein (YB-1) Gene Promoter  1996 Oxford University Press Nucleic Acids Research, 1996, Vol. 24, No. 10 1873–1878 Structural and functional analysis of the human Y-box binding protein (YB-1) gene promoter Yoshinari Makino, Takefumi Ohga, Satoshi Toh, Koji Koike, Katsuzumi Okumura , Morimasa Wada, Michihiko Kuwano and Kimitoshi Kohno* Department of Biochemistry, Kyushu University School of Medicine, Maidashi, Fukuoka 812-82, Japan and Department of Biochemistry, Mie University of Medicine, Edobashi, Tsu-shi 514, Japan Received February 6, 1996; Revised and Accepted April 1, 1996 EMBL accession no. X96666 ABSTRACT stimuli (10,11). Binding activity of MDR NF-1 to this Y-box element is augmented in nuclear extracts prepared from cells treated We have isolated three overlapping genomic clones with either UV irradiation or anticancer agents. It has also been containing the 5′ portion of the human YB-1 gene. These shown that the deletion of the Y-box region leads to markedly clones span ~ 25 kb of contiguous DNA containing reduced expression of the mdr1 promoter/CAT construct (12). We 10 kb of 5′ flanking sequence and 15 kb of the gene. The have previously cloned a cDNA for MDR NF-1 by Southwestern nucleotide sequence of the first exon and of 2000 screening of a human HeLa cell cDNA expression library using an upstream base pairs (bp) was determined. The first exon mdr1 promoter probe. Sequence analysis of the MDR NF-1 cDNA is unusually large and contains a 166 bp coding showed that it was identical to the Y-box binding protein, YB-1 (11). sequence and a 331 bp untranslated region. CpG Using this cDNA, we have demonstrated that YB-1 mRNA sequences cover the 5′-end of the YB-1 gene including accumulation increased when cells were treated with UV irradiation its first exon and intron as well as the upstream regions. or anticancer agents. Isolation of cis-regulatory elements should help The GC content around the first exon is ~ 70% and a clarify the complex regulation of YB-1 expression. CpG-free region was located in the untranslated We report here the isolation and characterization of several sequence. The segment preceding the major tran- overlapping genomic clones containing the 5′-end of the human scription initiation site does not contain a TATA box, YB-1 gene. CCAAT box and the binding sequence for known transcription factors. A transient expression assay using MATERIALS AND METHODS the chloramphenicol acetyltransferase (CAT) gene showed that the sequence from +24 to +281 was critical Cloning and sequencing of a human MDR-NF1 cDNA for CAT expression. Fluorescence in situ hybridization A human colon cDNA library constructed in the λgt 11 expression demonstrated the chromosomal locus of YB-1 gene on vector was obtained from Stratagene. The cDNA library was plated chromosome 1p34. Polymerase chain reaction analysis on LB agar containing ampicillin and overlaid with nitrocellulose on other genomic phage DNAs showed that several membrane saturated with 10 mM IPTG. A total of 2 × 10 plaques clones were derived from pseudogenes. were screened with the end labelled 140 bp XhoI–TaqI mdr1 promoter region. A 384 bp partial cDNA of human origin (originally INTRODUCTION designated as MDR-NF1) encodes a 128 amino acid polypeptide with 99% identity to hYB-1. Plaques (1 × 10 ) from same λgt 11 The human Y-Box binding protein (YB-1) is a member of a cDNA library were screened by DNA hybridization using this DNA-binding protein family with a structurally and functionally clone to isolate full length cDNA. One clone had an insert 1060 bp. conserved cold shock domain. YB-1 has been shown to regulate Sequence analysis showed 99% identity to the hYB-1/dbpB cDNA gene expression through binding to Y-box sequences (1,2). and almost covered the coding region. Several eukaryotic genes contain the consensus Y-box sequence, 5′-CTGATTGG-3′, in their cis-regulatory elements, including Isolation of genomic clones and DNA sequencing major histocompatibility complex class II (3), thymidine kinase, proliferating cell nuclear antigen, DNA polymerase α, epidermal Clones were isolated by screening a human placenta genomic growth factor receptor and multidrug resistance 1 (MDR1) (4). library in EMBL3 with a 1060 bp probe from the YB-1 cDNA The multidrug resistance gene encodes an energy-dependent drug (13,14). Positive phages were mapped with EcoRI and SalI and efflux pump that is overexpressed in cancer cells resistant to multiple hybridized with a 5′ probe, a 350 bp EcoRI–StyI fragment, and a anticancer agents (5). We have previously shown that MDR1 is a 3′ probe, a 300 bp SalI–EcoRI fragment. Several restriction stress responsive gene (6–11). Transfection of an mdr1 promoter/ fragments around the first exon were subcloned in pUC18 and CAT reporter plasmid has shown that an inverted CCAAT box sequenced. All plasmid DNAs were sequenced from both ends (Y-box) is critical for CAT induction by various environmental using a Shimazu DSQ 1000 automated sequencer. The primer for To whom correspondence should be addressed 1874 Nucleic Acids Research, 1996, Vol. 24, No. 10 primer extension analysis, 5′-GCTCATGGTTGCGGTGATGG-3′ transcriptase buffer (BRL). The primer was extended with 20 U was derived from the sequence of the first exon. The primers for MMTV reverse transcriptase (BRL) using 1 mM each of the four PCR analysis C1, 5′-CAACAGGAATGACACCAAGG-3′ and deoxynucleotides. After 1 h at 37C, the reaction was terminated C2, 5′-GATGGTAGAGATGGTAAGCCG-3′ were derived from with 20 mM EDTA. RNA was hydrolyzed with 0.125 M NaOH cDNA sequences. This primer pair was used to analyze genomic for 1 h at 65C. The reaction was neutralized and the DNA was clones containing pseudogenes. Polymerase chain reaction was collected. Sequencing reactions using the same primer were performed in 5 μl reactions containing 100 ng DNA, l μM of each analyzed on a 7 M urea–6% polyacrylamide gel to determine the primer pair, 0.2 mM each of the four deoxynucleotides size of the extended product (13,14). (Pharmacia), and 1 U Taq polymerase (Boehringer Mannheim) in PCR buffer [30 mM Tris–HCl (pH 8.5), 2 mM MgCl , 5 mM Fluorescence in situ hybridization (FISH) β-mercaptoethanol, 0.01% gelatin and 0.1% Thesit] (18–20). Initial Chromosome spreads were obtained from phytohaemaggulutinin- denaturation at 94C for 10 min was followed by 35 amplification stimulated blood lymphocytes of a healthy donor after thymidine cycles of denaturation at 94C for 30 s, annealing at 55C for 30 s, synchronization and bromodeoxyuridine incorporation by the and extension at 72C for 30 s in a Gene Amp PCR System 9600 method of Takahashi et al. (15,16). Genomic DNA fragments were (Perkin Elmer). The product of 773 bp was of the expected size from labelled with biotin-16-dUTP (Boehringer Mannheim) by nick the cDNA sequence. translation. In situ hybridization was performed according to Lichter et al. (17) in the presence of COT-1 DNA (Gibco-BRL, Construction of CAT reporter plasmids Gaithersburg, MD) as a competitor. Hybridized probes were detected with FITC-conjugated avidin (Boehringer Mannheim). The vectors, SVOOCAT and pCH110 (pSV-β-Gal), were purchased Chromosomes were counterstained with 0.2 mg/ml propidium from Nippon gene (Tokyo) and Pharmacia (Sweden), respectively. iodide (PI) for R-banding. Fluorescence signals were imaged using Different truncations and deletions of the 5′ region of the YB-1 gene a Zeiss Axioskop epifluorescence microscope equipped with a were ligated into the HindIII site of pSVOOCA T. The fragment from cooled Charge Coupled Device (CCD) camera (Photometrics, PXL HindIII (–1384) to PvuII (+295) was obtained from a plasmid 1400). Image acquisition was performed on a Macintosh Quadra subclone. From this, fragments from NcoI (–483), ApaI (–119), SalI 840AV computer with the software program IPLabTM (Signal (+24) or BalI (+127) to PvuII (+295) were isolated and filled with Analytics Co.). The images were then pseudocoloured and merged the Klenow fragment of DNA polymerase I. After HindIII linker using Adobe Photoshop TM 2.5J (Adobe Systems Inc.). FITC and ligation, these fragments were ligated into pSVOOCAT and propidium iodide images were shown in white and grey, respective- transformed. The resulting constructs were named pYB-CAT1, ly. The merged images of FITC and PI were directly printed by Fuji pYB-CAT2, pYB-CAT3, pYB-CAT4 and pYB-CAT5, respectively. Pictrography 3000 from a Macintosh computer. pYB-CAT3R and pYB-CAT5R indicate plasmid with the promoter fragment inserted in reverse orientation. To construct pYB-CA T3Δ1, RESULTS pYB-CA T3Δ2 and pYB-CAT3Δ3, pYB-CAT3 was digested with SalI–AvrII, AvrII–SmaI and SalI–SmaI, respectively, blunt ended We have previously isolated and characterized a YB-1 cDNA clone and ligated. Plasmid DNAs were purified by alkaline lysis followed (11) that was then used as a hybridization probe to screen a HeLa by two cycles of equilibrium centrifugation in a CsCl–ethidium cell cDNA library (Clontech). Several positive clones were bromide gradient. isolated, one of which was a nearly full-length cDNA for YB-1, pYB-1-22 (data not shown). To isolate genomic clones encoding Transfection and assays of cell extracts the 5′ region of the YB-1 gene, a human genomic library was screened using the 1060 bp cDNA insert of pYB-1-22. More than T24, a human bladder carcinoma cell line, was grown as previously 50 positive clones were isolated. Twenty-four clones were described (21). The cells were maintained at 37C with 5% CO . characterized by Southern hybridization using two probes derived Exponentially growing cells were trypsinized, seeded at 3 × 10 cells from the cDNA as described in Materials and Methods. Three in 60 mm diameter plates and incubated overnight prior to clones contained non-identical inserts that hybridized with 5′ cDNA transfection. DNA transfection of T24 cells was then carried out by fragments. The restriction maps for these non-identical clones, calcium phosphate precipitation with 5 μg pCH110 (pSV-β-gal) and EMBL-YBG1, YBG2 and YBG3, are shown in Figure 1. 10 μg of a reporter construct. The precipitate was added to the cells Confirmation that these clones encoded YB-1 was obtained by and after an 8 h transfection, the cells were washed twice, sequence analysis as described later. replenished with fresh medium and incubated for another 48 h. At the end of this period, cell extracts were prepared and assayed for both CAT and β-galactosidase activities according to the standard Sequence analysis of the promoter region procedure (22,23). Normalized CA T activities were expressed as the We determined the nucleotide sequence of the promoter region of ratio of that seen with pYB-CAT1. At least four independent the YB-1 gene. A 9 kb EcoRI fragment of EMBL-YBG1 was experiments were performed for each construct and all measure- subcloned into pUC18. To localize the first exon more exactly, ments were determined in duplicate. this plasmid was digested with restriction enzymes and analyzed by Southern blotting using a 350 bp fragment of the cDNA Primer extension by reverse transcriptase containing the 5′-end of the cDNA as a probe. Several fragments The synthetic primer described above was labelled at its 5′-end hybridized with the probe and were subcloned in plasmid pUC18 and hybridized to poly(A) RNA in 80% formamide, 0.4 M NaCl, (data not shown). The nucleotide sequence of the first exon and 40 mM PIPES (pH 6.4) and 1 mM EDTA for 4 h at 50C. After its 5′ flanking region was then determined. This fragment precipitation of nucleic acids, the pellet was dissolved in reverse contained exon(s) with sequences identical to those previously 1875 Nucleic Acids Research, 1996, Vol. 24, No. 10 1875 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 1. Restriction endonuclease cleavage map of three overlapping genomic clones. Restriction enzyme cleavage sites are indicated as follows: E,Eco RI, S, SalI. Overlapping regions within these clones were confirmed by the Southern blotting (data not shown). determined from the cDNA (3). Sequence comparison with the cDNA also indicated that the last codon of this exon is split at the second base. A similar division is also found in the first exon of human dbpA gene (24). The dbpA gene is closely related to the YB-1 gene. The first exon of the dbpA gene is 474 bp long. We therefore assumed that we had identified the first exon of the YB-1 gene. We performed primer extension to define precisely the start site for transcription. The cDNA products extended from the primer were analyzed by electrophoresis beside sequencing reactions using the same primer. Three major and four minor transcription initiation sites were observed. About 60% of the transcripts were initiated at the three major sites indicated in Figure 2. Sequence analysis of the region upstream of exon 1 failed to locate any sequence motifs such as TATA and CCAAT boxes, for the transcription factor binding but identified a highly GC-rich element. There are two GT boxes and three GC boxes in the untranslated region that are binding sites for Sp1 family transcription factors. Multiple E-boxes are found in the promoter region. Characteristics of the promoter sequences are summarized in Table 1. The GC content is ~ 70% around the first exon. Since ‘housekeeping’ genes have a CpG island overlapping the transcription start site, we analyzed the 5′ region of the YB-1 gene for the presence of a CpG island. We plotted the frequency of both CpG and GpC dinucleotides against the position in the sequence. As shown in Figure 3A, CpG dinucleotides are enriched at the 5′-end of the YB-1 gene including its first exon. Table 1. Features of the sequence Figure 2. Primer extension study. Hybridization of primer extension was with Feature From To 5′ labelled oligonucleotide and 2 μg of KB and T24 poly (A) RNA. The same oligonucleotide was used for sequencing reactions. Electrophoresis was Promoter 1 1855 performed on a 7 M urea–8% acrylamide DNA sequencing gel. The Exon 1 1856* 2352 radioactivity of each product was measured and the ratio to total activity is E-box 48 53 expressed as %. E-box 353 358 E-box 458 463 E-box 531 536 E-box 1147 1152 Identification of a YB-1 promoter by analysis of E-box 1201 1206 transcriptional activity GT box 1957 1962 GC box 2001 2006 To determine whether the region upstream of the putative GC box 2024 2029 transcriptional start sites can activate transcription, available GC box 2033 2038 restriction sites were utilized to construct a series of deletion GT box 2100 2105 plasmids (Fig. 3B). These constructs were then tested by transient Start codon 2187 2189 transfection in human bladder carcinoma T24 cells. DNA to only Inverted repeat 1 1904 1914 –119 yielded full promoter activity. Deletion of –119 to +24 Inverted repeat 2 2034 2044 diminished promoter activity by ~ 45%, but this construct was still Other initiation sites 1883, 1886, 1938, able to promote some CAT activity. Further deletion of +24 to 1947, 1959, 1972 +127 or all internal deletions of the untranslated region almost completely abolished promoter activity. Surprisingly, no CAT The most 5′ transcription initiation site is started at 1856 as indicated by an aster- activity was found when cells were transfected with pYBCAT-3Δ2, isk (*) and designated as +1 in the text and Figure 3. 1876 Nucleic Acids Research, 1996, Vol. 24, No. 10 Figure 3. (A) Structure and deletion analysis of the YB-1 promoter. Relevant restriction enzyme cleavage site are as indicated: H,Hin dIII; S, SalI; N, NcoI; A, ApaI; B, BalI; Sm, SmaI; Av, AvrII; P, PvuII. The small black triangles indicate transcriptional start sites determined by primer extension analysis. The diagram shows the distribution of GpC and CpG dinucleotides. Bold line indicates the first exon. (B) Constructs containing various portion or deletions of the 5′ region were subclones in front of a CAT reporter gene, and transiently transfected into T24 cells. The β-galactosidase reporter gene was co-transfected as an internal control. CAT activity was normalized to β-galactosidase activity. At least four independent experiments were done and all observations were performed in duplicate. The results are all normalized to the value of the CAT activity of pYBCAT-1 and are indicated as %. which still contains both the promoter region and all transcription surrounding the 5′-end of the gene and the start site for the initiation sites. transcription. The dbpA gene is an another member of a protein family containing cold shock domains. The human dbpA gene covers 24 Chromosome mapping and analysis of other genomic kb and includes 10 exons (24). Characteristic of its exon–intron clones for YB-1 structure is a large first exon of 474 bp. YB-1 gene may have a FISH was used to determine the chromosome location of YB-1. similar structure. The first exon of YB-1 is 497 bp long resembling Comparison of the fluorescence signals and the banding patterns of that of the dbpA gene (Table 1). The regulatory regions are highly the chromosomes indicated that this region was located on GC rich and the CpG islands are located at 5′ to the first exon and chromosome band 1p34 (Fig. 4). These results were based on intron. Thus, the YB-1 promoter has structural features common observations made on >10 prometaphase chromosomes. Fluor- to those of housekeeping genes. This feature of the YB-1 5′ region escence signals were not consistently observed on the other is consistent with its ubiquitous mRNA expression in all human chromosomes. We mentioned before that >50 clones were isolated tissues (24), and suggests that it encodes a protein assumed with an in the first screening. This might indicate the existence either of a essential cellular function. No typical TATA and CCAAT boxes gene family or of pseudogenes. To test for pseudogenes, all genomic exist in the region preceding the first exon, this may account for the clones were analyzed by a PCR assay using a primer pair derived heterogeneity of transcription initiation sites (Fig. 2). Multiple from the cDNA sequence. The PCR product includes half of the E-boxes are also found in the promoter region between –1855 and mRNA. We used human placenta DNA and cDNAs transcribed –555 similar to in the promoter region of the dbpA gene (Fig. 2). from total RNA of KB and T24 cells as templates (Fig. 5, lanes P, This could be a reflection of factors controlling the tissue specific R1 and R2), and a 773 bp PCR product was consistently observed. expression of these genes. There is no significant homology A similar product was also detected when some phage genomic between the proximal promoter sequences of the YB-1 and dbpA DNA clones were used as the template (lanes 1 and 3). genes. This divergence in the promoter region between YB-1 and dbpA may reflect the presence of different regulatory mechanisms. DISCUSSION Functional analysis of the promoter region in a transient expression system demonstrated significant promoter activity in We have described the isolation and characterization of the 5′ region of the YB-1 gene. We isolated overlapping genomic clones the region upstream of the first exon (Fig. 3B). The promoter encompassing 15 kb of the 5′ part of the YB-1 gene and 10 kb of activity of the YB-1 gene is strong and comparable with that of its 5′ flanking regions. We determined the nucleotide sequence the SV40 promoter. Our initial analysis of this promoter revealed 1877 Nucleic Acids Research, 1996, Vol. 24, No. 10 1877 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 5. Detection of a pseudogene in genomic clones. PCR was performed with a primer pair derived from cDNA on human placenta DNA, total cDNA and genomic phage DNAs. The 1.2% agarose gel shown here displays the following: RT–PCR using total cDNA of KB cells (R1) and T24 cells (R2). P, human placenta DNA; lanes 1–14, genomic phage DNAs (independent positive clones); M, 100 bp DNA size marker. Arrow indicates PCR product. either from intronless pseudogenes or from a single big exon. If the exon–intron structure of YB-1 is similar to that of the dbpA gene, these results indicate the existence of pseudogenes. Kudo et al. (24) have reported that 16 YB-1 genomic clones contained intronless processed genes. Isolation and characterization of these pseudogenes is now in progress in our laboratory. Figure 4. Mapping of the gene region encoding human YB-1 by fluorescence We have previously reported that both steady state level of YB-1 in situ hybridization (FISH). FISH was carried out using phage DNA mRNA and DNA binding activity of YB-1 protein were increased (EMBL-YBG1) as a probe. Fluorescence signals on the R-banded metaphase when cells were treated with either UV irradiation or anticancer chromosomes are indicated by arrow heads. Based on observations made with agents (10,11). YB-1 protein is also known to involve in cell more than 10 metaphase chromosomes, the band is 1p34. proliferation (1,2). YB-1 protein is expected to influence stress response to anticancer agents or UV irradiation as well as cell proliferation through signal transduction cascade. The characteriz- ation of this promoter region allows further exploration of complex regulation. When a region between –119 and +24 was mechanisms governing the YB-1 expression. deleted, CAT activity was diminished to 45%. This result may reflect variation in the amount of transcriptional initiation. Thus, the region between +24 and +127 may act as a promoter of the ACKNOWLEDGEMENTS transcription initiated from minor sites. A unique structure of this We thank Miss Yukiko Mine for editorial assistance. This work was region is that it lacks CpG dinucleotides, as shown in Figure 3A. supported by a grant-in-aid for cancer research from the Ministry of The CAT activity of pYBCAT-3Δ2 was very weak even though Education, Science and Culture, Japan, and the Fukuoka Anti-cancer pYBCAT-3Δ2 contains the promoter region and all sites for Research Fund. transcription, suggesting that the untranslated region is critical for the expression of CAT activity. The untranslated region may play a role in mRNA stability or may confer altered affinities for the translational initiation and elongation machinery. The DNA REFERENCES sequence analysis revealed the presence of inverted repeat in the 1 Wollfe,A.P. (1994) BioEssays, 16, 245–251. 5′ untranslated region. The 11 nucleotides long (+48 to +58) 2 Ladomery,M., Sommerville,J. (1995) BioEssays, 17, 9–11. showed high inverse complementarity to the sequence at position 3 Didier,D.K., Schiffenbauer,J., Woulfe,S.L., Zacheis,M. and Schwartz,B.D. +178 to +188. These sequences could form the stem–loop (1988) Proc. Natl. Acad. Sci. USA, 85, 7322–7326. 4 Kohno,K., Sato,S., Uchiumi,T., Takano,H., Kato,S. and Kuwano,K. (1990) secondary structure, which may affect the translational efficiency. J. Biol. Chem., 265, 19690–19696. More precise analysis is required to define the molecular mechanism 5 Borst,P., Schinkel,A.H., Smit,J.J., Wagenaar,E., Van Deemter,L., of how YB-1 expression is controlled at the translational level. The Smith,A.J., Eijdems,E.W., Baas,F. and Zaman,G.J. (1993) Pharmcol. Ther., EMBL-YBG1 clone was used as a probe for in situ hybridization to 60, 289–99. human metaphase chromosomes. A high specific signal was 6 Kohno,K., Sato,S., Takano,H,. Matsuo,K. and Kuwano,K. (1989) Biochem. Biophys. Res. Commun., 165, 1415–1421. obtained on the short arm of chromosome 1. Our result confirmed 7 Kohno,K., Sato,S., Uchiumi,T., Takano,H., Tanimura,H., Miyazaki,M., mapping data published previously (24). Other genomic clones Matsuo,K., Hidaka,K. and Kuwano,M. (1992) Int. J. Oncol., 1, 73–77 were mapped to various chromosomes (data not shown), and many 8 Tanimura,H., Kohno,K., Sato,S., Uchiumi,T., Miyazaki,M., Kobayashi,M. positive signals were observed in the first screening of a genomic and Kuwano,M. (1992) Biochem. Biophys. Res. Commun., 183, 917–924. library, suggesting the existence of pseudogenes scattered in the 9 Miyazaki,M., Kohno,K,. Uchiumi,T., Tanimura,H., Matsuo,K.,Nasu,M. and Kuwano,M. (1992) Biochem. Biophys. Res. Commun., 187, 677–684. human genome. Primer pairs derived from cDNA sequence 10 Uchiumi,T., Kohno,K,. Tanimura,H., Matsuo,K.,Sto,S.,Uchida,Y. and generated fragments of 773 bp, the expected size of the cDNA, when Kuwano,M. (1993) Cell Growth Differ., 4, 147–157. human placenta DNA or genomic phage DNA were used as 11 Asakuno,K., Kohno,K., Uchiumi,T., Kubo,T., Sato,S., Isono,M. and templates (Fig. 5). This indicated that PCR products are derived Kuwano,M. (1994) Biochem. Biophys. Res. Commun., 199, 1428–1435. 1878 Nucleic Acids Research, 1996, Vol. 24, No. 10 12 Goldsmith,M.E., Madden,M.J., Morrow,C.S. and Cowan,K.H. (1993) J. 19 Kusaba,H., Kohno,K., Asakuno,K., Kuwano,M., Okumura,K., Green,E.D., Biol.Chem., 268, 5856–5860. Schlessinger,D., and Wada,M. (1995) Genome Res., 5, 245–258. 13 Kohno.K., Sullivan,M. and Yamada,Y. (1985) J. Biol.Chem., 260, 20 Torigoe,K., Sato,S., Kusaba,H., Kohno,K., Kuwano,M., Okumura,K., 4441–4447. Green,E.D., Tsui,L.C., Scherer,S.W., Schlessinger,D.and Wada,M. (1995) 14 Kohno.K., Tanimura,H., Sato,S., Nakayama,Y., Makino,Y., Wada,M., Genome. Res., 5, 233–244. Fojo,A.T. and Kuwano,M. (1994) J. Biol.Chem., 269, 20503–20508. 21 Yokomizo,A., Ono,M., Nanri,H., Makino,Y., Ohga,T., Wada,M., 15 Takahashi,E., Hori,T., O’Connell,P., Leppert,M. and White,R. (1990) Hum. Okamoto,T., Yodoi.,J., Kuwano,M. and Kohno,K. (1995) Cancer Res., 55, Genet., 86, 14–16. 4293–4296. 16 Takahashi,E., Yamauchi,M., Tsuji,H., Hitomi,A., Meuth,M. and Hori,T. 22 Gorman,C., Moffat,L.F. and Howard,B.H. (1982) Mol. Cell. Biol., 2, (1991) Hum. Genet., 88, 119–121. 1044–1051. 17 Lichter,P., Cremer,T., Borden,J., Manuelidis,L. and Ward,D.C. (1988) 23 Herbomel,P., Bourachot,B. and Yaniv,M. (1984) Cell, 39, 653–662. Hum. Genet., 80, 224–234. 24 Kudo,S., Mattei,M.J. and Fukuda,M. (1995) Eur. J. Biochem., 231, 72–82. 18 Ponce,M.R., Micol,J.L. (1992) Nucleic Acids Res., 20, 623. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Structural and Functional Analysis of the Human Y-Box Binding Protein (YB-1) Gene Promoter

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Oxford University Press
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© 1996 Oxford University Press
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0305-1048
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10.1093/nar/24.10.1873
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Abstract

 1996 Oxford University Press Nucleic Acids Research, 1996, Vol. 24, No. 10 1873–1878 Structural and functional analysis of the human Y-box binding protein (YB-1) gene promoter Yoshinari Makino, Takefumi Ohga, Satoshi Toh, Koji Koike, Katsuzumi Okumura , Morimasa Wada, Michihiko Kuwano and Kimitoshi Kohno* Department of Biochemistry, Kyushu University School of Medicine, Maidashi, Fukuoka 812-82, Japan and Department of Biochemistry, Mie University of Medicine, Edobashi, Tsu-shi 514, Japan Received February 6, 1996; Revised and Accepted April 1, 1996 EMBL accession no. X96666 ABSTRACT stimuli (10,11). Binding activity of MDR NF-1 to this Y-box element is augmented in nuclear extracts prepared from cells treated We have isolated three overlapping genomic clones with either UV irradiation or anticancer agents. It has also been containing the 5′ portion of the human YB-1 gene. These shown that the deletion of the Y-box region leads to markedly clones span ~ 25 kb of contiguous DNA containing reduced expression of the mdr1 promoter/CAT construct (12). We 10 kb of 5′ flanking sequence and 15 kb of the gene. The have previously cloned a cDNA for MDR NF-1 by Southwestern nucleotide sequence of the first exon and of 2000 screening of a human HeLa cell cDNA expression library using an upstream base pairs (bp) was determined. The first exon mdr1 promoter probe. Sequence analysis of the MDR NF-1 cDNA is unusually large and contains a 166 bp coding showed that it was identical to the Y-box binding protein, YB-1 (11). sequence and a 331 bp untranslated region. CpG Using this cDNA, we have demonstrated that YB-1 mRNA sequences cover the 5′-end of the YB-1 gene including accumulation increased when cells were treated with UV irradiation its first exon and intron as well as the upstream regions. or anticancer agents. Isolation of cis-regulatory elements should help The GC content around the first exon is ~ 70% and a clarify the complex regulation of YB-1 expression. CpG-free region was located in the untranslated We report here the isolation and characterization of several sequence. The segment preceding the major tran- overlapping genomic clones containing the 5′-end of the human scription initiation site does not contain a TATA box, YB-1 gene. CCAAT box and the binding sequence for known transcription factors. A transient expression assay using MATERIALS AND METHODS the chloramphenicol acetyltransferase (CAT) gene showed that the sequence from +24 to +281 was critical Cloning and sequencing of a human MDR-NF1 cDNA for CAT expression. Fluorescence in situ hybridization A human colon cDNA library constructed in the λgt 11 expression demonstrated the chromosomal locus of YB-1 gene on vector was obtained from Stratagene. The cDNA library was plated chromosome 1p34. Polymerase chain reaction analysis on LB agar containing ampicillin and overlaid with nitrocellulose on other genomic phage DNAs showed that several membrane saturated with 10 mM IPTG. A total of 2 × 10 plaques clones were derived from pseudogenes. were screened with the end labelled 140 bp XhoI–TaqI mdr1 promoter region. A 384 bp partial cDNA of human origin (originally INTRODUCTION designated as MDR-NF1) encodes a 128 amino acid polypeptide with 99% identity to hYB-1. Plaques (1 × 10 ) from same λgt 11 The human Y-Box binding protein (YB-1) is a member of a cDNA library were screened by DNA hybridization using this DNA-binding protein family with a structurally and functionally clone to isolate full length cDNA. One clone had an insert 1060 bp. conserved cold shock domain. YB-1 has been shown to regulate Sequence analysis showed 99% identity to the hYB-1/dbpB cDNA gene expression through binding to Y-box sequences (1,2). and almost covered the coding region. Several eukaryotic genes contain the consensus Y-box sequence, 5′-CTGATTGG-3′, in their cis-regulatory elements, including Isolation of genomic clones and DNA sequencing major histocompatibility complex class II (3), thymidine kinase, proliferating cell nuclear antigen, DNA polymerase α, epidermal Clones were isolated by screening a human placenta genomic growth factor receptor and multidrug resistance 1 (MDR1) (4). library in EMBL3 with a 1060 bp probe from the YB-1 cDNA The multidrug resistance gene encodes an energy-dependent drug (13,14). Positive phages were mapped with EcoRI and SalI and efflux pump that is overexpressed in cancer cells resistant to multiple hybridized with a 5′ probe, a 350 bp EcoRI–StyI fragment, and a anticancer agents (5). We have previously shown that MDR1 is a 3′ probe, a 300 bp SalI–EcoRI fragment. Several restriction stress responsive gene (6–11). Transfection of an mdr1 promoter/ fragments around the first exon were subcloned in pUC18 and CAT reporter plasmid has shown that an inverted CCAAT box sequenced. All plasmid DNAs were sequenced from both ends (Y-box) is critical for CAT induction by various environmental using a Shimazu DSQ 1000 automated sequencer. The primer for To whom correspondence should be addressed 1874 Nucleic Acids Research, 1996, Vol. 24, No. 10 primer extension analysis, 5′-GCTCATGGTTGCGGTGATGG-3′ transcriptase buffer (BRL). The primer was extended with 20 U was derived from the sequence of the first exon. The primers for MMTV reverse transcriptase (BRL) using 1 mM each of the four PCR analysis C1, 5′-CAACAGGAATGACACCAAGG-3′ and deoxynucleotides. After 1 h at 37C, the reaction was terminated C2, 5′-GATGGTAGAGATGGTAAGCCG-3′ were derived from with 20 mM EDTA. RNA was hydrolyzed with 0.125 M NaOH cDNA sequences. This primer pair was used to analyze genomic for 1 h at 65C. The reaction was neutralized and the DNA was clones containing pseudogenes. Polymerase chain reaction was collected. Sequencing reactions using the same primer were performed in 5 μl reactions containing 100 ng DNA, l μM of each analyzed on a 7 M urea–6% polyacrylamide gel to determine the primer pair, 0.2 mM each of the four deoxynucleotides size of the extended product (13,14). (Pharmacia), and 1 U Taq polymerase (Boehringer Mannheim) in PCR buffer [30 mM Tris–HCl (pH 8.5), 2 mM MgCl , 5 mM Fluorescence in situ hybridization (FISH) β-mercaptoethanol, 0.01% gelatin and 0.1% Thesit] (18–20). Initial Chromosome spreads were obtained from phytohaemaggulutinin- denaturation at 94C for 10 min was followed by 35 amplification stimulated blood lymphocytes of a healthy donor after thymidine cycles of denaturation at 94C for 30 s, annealing at 55C for 30 s, synchronization and bromodeoxyuridine incorporation by the and extension at 72C for 30 s in a Gene Amp PCR System 9600 method of Takahashi et al. (15,16). Genomic DNA fragments were (Perkin Elmer). The product of 773 bp was of the expected size from labelled with biotin-16-dUTP (Boehringer Mannheim) by nick the cDNA sequence. translation. In situ hybridization was performed according to Lichter et al. (17) in the presence of COT-1 DNA (Gibco-BRL, Construction of CAT reporter plasmids Gaithersburg, MD) as a competitor. Hybridized probes were detected with FITC-conjugated avidin (Boehringer Mannheim). The vectors, SVOOCAT and pCH110 (pSV-β-Gal), were purchased Chromosomes were counterstained with 0.2 mg/ml propidium from Nippon gene (Tokyo) and Pharmacia (Sweden), respectively. iodide (PI) for R-banding. Fluorescence signals were imaged using Different truncations and deletions of the 5′ region of the YB-1 gene a Zeiss Axioskop epifluorescence microscope equipped with a were ligated into the HindIII site of pSVOOCA T. The fragment from cooled Charge Coupled Device (CCD) camera (Photometrics, PXL HindIII (–1384) to PvuII (+295) was obtained from a plasmid 1400). Image acquisition was performed on a Macintosh Quadra subclone. From this, fragments from NcoI (–483), ApaI (–119), SalI 840AV computer with the software program IPLabTM (Signal (+24) or BalI (+127) to PvuII (+295) were isolated and filled with Analytics Co.). The images were then pseudocoloured and merged the Klenow fragment of DNA polymerase I. After HindIII linker using Adobe Photoshop TM 2.5J (Adobe Systems Inc.). FITC and ligation, these fragments were ligated into pSVOOCAT and propidium iodide images were shown in white and grey, respective- transformed. The resulting constructs were named pYB-CAT1, ly. The merged images of FITC and PI were directly printed by Fuji pYB-CAT2, pYB-CAT3, pYB-CAT4 and pYB-CAT5, respectively. Pictrography 3000 from a Macintosh computer. pYB-CAT3R and pYB-CAT5R indicate plasmid with the promoter fragment inserted in reverse orientation. To construct pYB-CA T3Δ1, RESULTS pYB-CA T3Δ2 and pYB-CAT3Δ3, pYB-CAT3 was digested with SalI–AvrII, AvrII–SmaI and SalI–SmaI, respectively, blunt ended We have previously isolated and characterized a YB-1 cDNA clone and ligated. Plasmid DNAs were purified by alkaline lysis followed (11) that was then used as a hybridization probe to screen a HeLa by two cycles of equilibrium centrifugation in a CsCl–ethidium cell cDNA library (Clontech). Several positive clones were bromide gradient. isolated, one of which was a nearly full-length cDNA for YB-1, pYB-1-22 (data not shown). To isolate genomic clones encoding Transfection and assays of cell extracts the 5′ region of the YB-1 gene, a human genomic library was screened using the 1060 bp cDNA insert of pYB-1-22. More than T24, a human bladder carcinoma cell line, was grown as previously 50 positive clones were isolated. Twenty-four clones were described (21). The cells were maintained at 37C with 5% CO . characterized by Southern hybridization using two probes derived Exponentially growing cells were trypsinized, seeded at 3 × 10 cells from the cDNA as described in Materials and Methods. Three in 60 mm diameter plates and incubated overnight prior to clones contained non-identical inserts that hybridized with 5′ cDNA transfection. DNA transfection of T24 cells was then carried out by fragments. The restriction maps for these non-identical clones, calcium phosphate precipitation with 5 μg pCH110 (pSV-β-gal) and EMBL-YBG1, YBG2 and YBG3, are shown in Figure 1. 10 μg of a reporter construct. The precipitate was added to the cells Confirmation that these clones encoded YB-1 was obtained by and after an 8 h transfection, the cells were washed twice, sequence analysis as described later. replenished with fresh medium and incubated for another 48 h. At the end of this period, cell extracts were prepared and assayed for both CAT and β-galactosidase activities according to the standard Sequence analysis of the promoter region procedure (22,23). Normalized CA T activities were expressed as the We determined the nucleotide sequence of the promoter region of ratio of that seen with pYB-CAT1. At least four independent the YB-1 gene. A 9 kb EcoRI fragment of EMBL-YBG1 was experiments were performed for each construct and all measure- subcloned into pUC18. To localize the first exon more exactly, ments were determined in duplicate. this plasmid was digested with restriction enzymes and analyzed by Southern blotting using a 350 bp fragment of the cDNA Primer extension by reverse transcriptase containing the 5′-end of the cDNA as a probe. Several fragments The synthetic primer described above was labelled at its 5′-end hybridized with the probe and were subcloned in plasmid pUC18 and hybridized to poly(A) RNA in 80% formamide, 0.4 M NaCl, (data not shown). The nucleotide sequence of the first exon and 40 mM PIPES (pH 6.4) and 1 mM EDTA for 4 h at 50C. After its 5′ flanking region was then determined. This fragment precipitation of nucleic acids, the pellet was dissolved in reverse contained exon(s) with sequences identical to those previously 1875 Nucleic Acids Research, 1996, Vol. 24, No. 10 1875 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 1. Restriction endonuclease cleavage map of three overlapping genomic clones. Restriction enzyme cleavage sites are indicated as follows: E,Eco RI, S, SalI. Overlapping regions within these clones were confirmed by the Southern blotting (data not shown). determined from the cDNA (3). Sequence comparison with the cDNA also indicated that the last codon of this exon is split at the second base. A similar division is also found in the first exon of human dbpA gene (24). The dbpA gene is closely related to the YB-1 gene. The first exon of the dbpA gene is 474 bp long. We therefore assumed that we had identified the first exon of the YB-1 gene. We performed primer extension to define precisely the start site for transcription. The cDNA products extended from the primer were analyzed by electrophoresis beside sequencing reactions using the same primer. Three major and four minor transcription initiation sites were observed. About 60% of the transcripts were initiated at the three major sites indicated in Figure 2. Sequence analysis of the region upstream of exon 1 failed to locate any sequence motifs such as TATA and CCAAT boxes, for the transcription factor binding but identified a highly GC-rich element. There are two GT boxes and three GC boxes in the untranslated region that are binding sites for Sp1 family transcription factors. Multiple E-boxes are found in the promoter region. Characteristics of the promoter sequences are summarized in Table 1. The GC content is ~ 70% around the first exon. Since ‘housekeeping’ genes have a CpG island overlapping the transcription start site, we analyzed the 5′ region of the YB-1 gene for the presence of a CpG island. We plotted the frequency of both CpG and GpC dinucleotides against the position in the sequence. As shown in Figure 3A, CpG dinucleotides are enriched at the 5′-end of the YB-1 gene including its first exon. Table 1. Features of the sequence Figure 2. Primer extension study. Hybridization of primer extension was with Feature From To 5′ labelled oligonucleotide and 2 μg of KB and T24 poly (A) RNA. The same oligonucleotide was used for sequencing reactions. Electrophoresis was Promoter 1 1855 performed on a 7 M urea–8% acrylamide DNA sequencing gel. The Exon 1 1856* 2352 radioactivity of each product was measured and the ratio to total activity is E-box 48 53 expressed as %. E-box 353 358 E-box 458 463 E-box 531 536 E-box 1147 1152 Identification of a YB-1 promoter by analysis of E-box 1201 1206 transcriptional activity GT box 1957 1962 GC box 2001 2006 To determine whether the region upstream of the putative GC box 2024 2029 transcriptional start sites can activate transcription, available GC box 2033 2038 restriction sites were utilized to construct a series of deletion GT box 2100 2105 plasmids (Fig. 3B). These constructs were then tested by transient Start codon 2187 2189 transfection in human bladder carcinoma T24 cells. DNA to only Inverted repeat 1 1904 1914 –119 yielded full promoter activity. Deletion of –119 to +24 Inverted repeat 2 2034 2044 diminished promoter activity by ~ 45%, but this construct was still Other initiation sites 1883, 1886, 1938, able to promote some CAT activity. Further deletion of +24 to 1947, 1959, 1972 +127 or all internal deletions of the untranslated region almost completely abolished promoter activity. Surprisingly, no CAT The most 5′ transcription initiation site is started at 1856 as indicated by an aster- activity was found when cells were transfected with pYBCAT-3Δ2, isk (*) and designated as +1 in the text and Figure 3. 1876 Nucleic Acids Research, 1996, Vol. 24, No. 10 Figure 3. (A) Structure and deletion analysis of the YB-1 promoter. Relevant restriction enzyme cleavage site are as indicated: H,Hin dIII; S, SalI; N, NcoI; A, ApaI; B, BalI; Sm, SmaI; Av, AvrII; P, PvuII. The small black triangles indicate transcriptional start sites determined by primer extension analysis. The diagram shows the distribution of GpC and CpG dinucleotides. Bold line indicates the first exon. (B) Constructs containing various portion or deletions of the 5′ region were subclones in front of a CAT reporter gene, and transiently transfected into T24 cells. The β-galactosidase reporter gene was co-transfected as an internal control. CAT activity was normalized to β-galactosidase activity. At least four independent experiments were done and all observations were performed in duplicate. The results are all normalized to the value of the CAT activity of pYBCAT-1 and are indicated as %. which still contains both the promoter region and all transcription surrounding the 5′-end of the gene and the start site for the initiation sites. transcription. The dbpA gene is an another member of a protein family containing cold shock domains. The human dbpA gene covers 24 Chromosome mapping and analysis of other genomic kb and includes 10 exons (24). Characteristic of its exon–intron clones for YB-1 structure is a large first exon of 474 bp. YB-1 gene may have a FISH was used to determine the chromosome location of YB-1. similar structure. The first exon of YB-1 is 497 bp long resembling Comparison of the fluorescence signals and the banding patterns of that of the dbpA gene (Table 1). The regulatory regions are highly the chromosomes indicated that this region was located on GC rich and the CpG islands are located at 5′ to the first exon and chromosome band 1p34 (Fig. 4). These results were based on intron. Thus, the YB-1 promoter has structural features common observations made on >10 prometaphase chromosomes. Fluor- to those of housekeeping genes. This feature of the YB-1 5′ region escence signals were not consistently observed on the other is consistent with its ubiquitous mRNA expression in all human chromosomes. We mentioned before that >50 clones were isolated tissues (24), and suggests that it encodes a protein assumed with an in the first screening. This might indicate the existence either of a essential cellular function. No typical TATA and CCAAT boxes gene family or of pseudogenes. To test for pseudogenes, all genomic exist in the region preceding the first exon, this may account for the clones were analyzed by a PCR assay using a primer pair derived heterogeneity of transcription initiation sites (Fig. 2). Multiple from the cDNA sequence. The PCR product includes half of the E-boxes are also found in the promoter region between –1855 and mRNA. We used human placenta DNA and cDNAs transcribed –555 similar to in the promoter region of the dbpA gene (Fig. 2). from total RNA of KB and T24 cells as templates (Fig. 5, lanes P, This could be a reflection of factors controlling the tissue specific R1 and R2), and a 773 bp PCR product was consistently observed. expression of these genes. There is no significant homology A similar product was also detected when some phage genomic between the proximal promoter sequences of the YB-1 and dbpA DNA clones were used as the template (lanes 1 and 3). genes. This divergence in the promoter region between YB-1 and dbpA may reflect the presence of different regulatory mechanisms. DISCUSSION Functional analysis of the promoter region in a transient expression system demonstrated significant promoter activity in We have described the isolation and characterization of the 5′ region of the YB-1 gene. We isolated overlapping genomic clones the region upstream of the first exon (Fig. 3B). The promoter encompassing 15 kb of the 5′ part of the YB-1 gene and 10 kb of activity of the YB-1 gene is strong and comparable with that of its 5′ flanking regions. We determined the nucleotide sequence the SV40 promoter. Our initial analysis of this promoter revealed 1877 Nucleic Acids Research, 1996, Vol. 24, No. 10 1877 Nucleic Acids Research, 1994, Vol. 22, No. 1 Figure 5. Detection of a pseudogene in genomic clones. PCR was performed with a primer pair derived from cDNA on human placenta DNA, total cDNA and genomic phage DNAs. The 1.2% agarose gel shown here displays the following: RT–PCR using total cDNA of KB cells (R1) and T24 cells (R2). P, human placenta DNA; lanes 1–14, genomic phage DNAs (independent positive clones); M, 100 bp DNA size marker. Arrow indicates PCR product. either from intronless pseudogenes or from a single big exon. If the exon–intron structure of YB-1 is similar to that of the dbpA gene, these results indicate the existence of pseudogenes. Kudo et al. (24) have reported that 16 YB-1 genomic clones contained intronless processed genes. Isolation and characterization of these pseudogenes is now in progress in our laboratory. Figure 4. Mapping of the gene region encoding human YB-1 by fluorescence We have previously reported that both steady state level of YB-1 in situ hybridization (FISH). FISH was carried out using phage DNA mRNA and DNA binding activity of YB-1 protein were increased (EMBL-YBG1) as a probe. Fluorescence signals on the R-banded metaphase when cells were treated with either UV irradiation or anticancer chromosomes are indicated by arrow heads. Based on observations made with agents (10,11). YB-1 protein is also known to involve in cell more than 10 metaphase chromosomes, the band is 1p34. proliferation (1,2). YB-1 protein is expected to influence stress response to anticancer agents or UV irradiation as well as cell proliferation through signal transduction cascade. The characteriz- ation of this promoter region allows further exploration of complex regulation. When a region between –119 and +24 was mechanisms governing the YB-1 expression. deleted, CAT activity was diminished to 45%. This result may reflect variation in the amount of transcriptional initiation. Thus, the region between +24 and +127 may act as a promoter of the ACKNOWLEDGEMENTS transcription initiated from minor sites. A unique structure of this We thank Miss Yukiko Mine for editorial assistance. This work was region is that it lacks CpG dinucleotides, as shown in Figure 3A. supported by a grant-in-aid for cancer research from the Ministry of The CAT activity of pYBCAT-3Δ2 was very weak even though Education, Science and Culture, Japan, and the Fukuoka Anti-cancer pYBCAT-3Δ2 contains the promoter region and all sites for Research Fund. transcription, suggesting that the untranslated region is critical for the expression of CAT activity. The untranslated region may play a role in mRNA stability or may confer altered affinities for the translational initiation and elongation machinery. The DNA REFERENCES sequence analysis revealed the presence of inverted repeat in the 1 Wollfe,A.P. 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Journal

Nucleic Acids Research – Oxford University Press

Published: May 1, 1996

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