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Cdna Cloning and Transcriptional Properties of a Novel GO Box-Binding Protein, BTEB2

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Cdna Cloning and Transcriptional Properties of a Novel GO Box-Binding Protein, BTEB2 Nucleic Acids Research, 1993, Vol. 21, No. 7 1527-1532 cDNA cloning and transcriptional properties of a novel GO box-binding protein, BTEB2 Kazuhiro Sogawa, Hiroaki Imataka1, Yuichi Yamasaki, Hiroshi Kusume, Hisaku Abe and Yoshiaki Fujii-Kuriyama Department of Chemistry, Faculty of Science, Tohoku University, Sendai 980 and 'institute for Virus Research, Kyoto University, Kyoto 606, Japan Received February 5, 1993; Revised and Accepted March 8, 1993 DDBJ accession no. D14520 ABSTRACT We have cloned a cDNA for a novel GC box-binding transcription of eukaryotic genes through a limited number of protein designated BTEB2 from a human placenta cis-acting elements located in their promoter region. cDNA library using rat BTEB cDNA (Imataka et al. GC box sequence is one of the most widely distributed (1992). EMBO J. 11,3663-3671. as a hybridization promoter elements in cellular and viral genes (1, 2). Tjian and probe. BTEB2 consists of 219 amino acids and contains his colleagues found Spl as the GC box-binding factor, and three contiguous zinc finger motifs at its C-terminus. established its role on transcription by elucidating its molecular The zinc finger domains showed 59% and 64% properties (3, 4). SpI contains three contiguous zinc finger motifs sequence similarity to those of Spl and BTEB, and glutamine-rich domains as the DNA-binding and respectively. Adjacent to the N-terminal of the zinc transcriptional activation domains, respectively. Although Spl finger motifs, a short sequence rich in basic amino has long been thought to be a unique GC box-binding protein, acids is conserved between BTEB2 and Spl. we have recently cloned a cDNA for a GC box-binding protein Furthermore, This basic sequence concurs with the N- designated BTEB (BTE-binding protein) (5). BTEB has three- terminal half of the consensus sequence for basic time repeated zinc finger motifs like Spl. Investigation of domains of the proteins containing both helix-loop-helix transcriptional activity of BTEB indicated that BTEB stimulates and leucine zipper motifs. The other region of BTEB2 promoters with repeated GC box sequences, while promoters with is notably rich in proline, serine, threonine, and alanine a single GC box sequence are not activated by BTEB (5). Finding residues. BTEB2 expressed in Escherichia coil showed of BTEB encouraged us to explore further the presence of GC- DNA-binding activity whose specificity was closely box binding factors other than Spl and BTEB. similar to that of Spl. Cotransfection experiments using Here we report the isolation ofhuman cDNA encoding a novel Hepa-1 cells (a mouse hepatoma cell line) with a BTEB2 GC box binding protein designated BTEB2. BTEB2 consists of expression plasmid and GC box-containing reporter 219 amino acids and contains three contiguous zinc finger motifs plasmids revealed that BTEB2 apparently activated the at its C-terminus. Transient expression of BTEB2 in eukaryotic expression of the CAT activity. Moreover, when BTEB2 cells activates the expression of the CAT (chloramphenicol was fused to GAL4 DNA-binding domain, the chimeric acetyltransferase) activity from a reporter gene consisting of a protein could enhance the transcription through GC-box promoter and CAT structural gene. Transcriptional promoters containing GAL4-binding sites. Analysis of activation by BTEB2 was further confirmed by a fusion protein the BTEB2 mRNA by RNA blot analysis demonstrated of BTEB2 and GALA DNA binding segment which enhances the that the mRNA was expressed specifically in testis and transcription in a GAL4 binding site-dependent manner. placenta with different sizes, 20S and 28S, respectively, among various organs examined. MATERIALS AND METHODS Cloning of cDNAs INTRODUCTION cDNA clones for BTEB2 were isolated from a human placenta The regulation of eukaryotic transcription is governed by the cDNA library in Xgtl 1 by the method of Benton and Davis (6) combined action of various sequence-specific DNA-binding using BTEB cDNA, XBP26 (5), as a probe. Plaque hybridization factors. Extensive studies on the DNA-binding properties of these was carried out with the nick-translated cDNA (specific activity, factors revealed that most of target sequences are recognized by 8x107cpm/,4g) as the probe at 650C in 20mM Tris buffer, multiple DNA-binding proteins acting positively or negatively pH7.5, containing 1M NaCl, lOmM EDTA, 0.1% sodium N- on transcription. Presence of a set of factors which interact with lauroylsarcosinate, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, common binding sites seemed favorable for the fine control of 0.2% bovine serum albumin, and 100/Ag/ml salmon sperm DNA. 1528 Nucleic Acids Research, 1993, Vol. 21, No. 7 The filters were washed twice at 65°C for 30min in 2 x SSC A (lxSSC, 0.15M NaCl and 0.015M sodium citrate) and 0.1% GGGCACGCGCACCACCGC 18 sodium N-lauroylsarcosinate. Positive clones were plaque-purified CAGAGGACCTGGTCCAGAC 108 'ATAGACGAGACAGTGCCTC 198 four times and inserted DNAs were subcloned into pUC18 ACATCACTCACCTGAGAAC 288 'AGAGTGA"ACGACTGCCCC 378 plasmid vector at the EcoRI site. iGGTGAACAATATTTTCATC 468 'GAATACACCGGATCTAGAT 558 ATGCCCAGTTCTACAAATCAGACAGCAGCAAIGGACACTCTTAATGTTTCTATGTCAGCTGCCATGGCAGG ;CCTTAACACACACACCTCT 648 DNA sequence analysis etProS-rS rThrAsnGInThrAl AleAspThrLouA*nV-lS-rStSerAl AlalXtAlGlIyLeuAsnThrHisThrSer 30 GCTGTTCCGCAGACTGCAGTGAAACAATTCCAGGGCATGCCCCCTTGCACATACACAATGCCAAGTCAGTTI'TCTTCCACAACAGGCCACT 738 AlaValProGlnThrA1aValLysG1nPheG1nGlyMetProProCysThrTyrThrtbtProSerGInPh *LeuProG1nG1nAlaThr 60 The cDNA sequence was determined by the dideoxynucleotide TACTTTCCCCCGTCACCACCAAGCTCAGAGCCTGGAAGTCCAGATAGACAAGCAGAGATGCTCCAGAATTTJ!AACCCCACCTCCATCCTAT 828 as vectors TyrPh-ProProS-rProProS-rS-rGluProGlySerProAspArgGlnAlaGlulbtL uGlnAonL tuThrProProProSerTyr 90 method using Ml3mpl8 and Ml3mpl9 cloning (7). GCTGCTACAATTGCTTCTAAACTGGCAATTCACAATCCAAATTTACCCACCACCCTGCCAGTTAACTCACA AAACATCCAACCTGTCAGA 918 of were determined the chemical AlaAlaThrIleAlaSerLysLeuAlaIleHisAsnProAsnLeuProThrThrLeuProValAsnS-rGl nAsnIleGlnProValArg 120 Some parts the sequence by TACAATAGAAGGAGTAACCCCGATTTGGAGAAACGACGCATCCACTACTGCGATTACCCTGGTTGCACAAAAGTTTATACCAAGTCTTCT '1008 Gilbert TyrAsnArgArgS-rAsnProAspLouGluLy AraArgI l-HiTYrc aksDTyrProGlyCvsThrLysV&lTyrThrLysS-rS-r 150 method of Maxam and (8). CATTTAAAAGCTCACCTGAGGACTCACACTGGTGAAAAGCCATACAAGTGTACCTGGGAAGGCTGCGACTGGAGGTTCGCGCGATCGGAT '1098 Hi*sLuLyvA1aHisLeuArqThrHisThrGlyGluLysProTyv¶yaCysThrTrpGluGlyCysAspTrpArgPheAl ArgSerAso o 180 GAGCTGACCCGCCACTACCGGAAGCACACAGGCGCCAAGCCCTTCCAGTGCGGGGTGTGCAACCGCAGCTTCTCGCGCTCTGACCACCTG i1188 Construction of plasmids GluLeuThrArgaHisTyrArqLYsHisThrGlyAlaLysProPheGlnCYaG1YVa1CYsAsnArqSerPheSerArqS*rAsDHisLeu ! 210 GCCCTGCATATGAAGAGGCACCAGAACTGAGCACTGCCCGTGTGACCCGTTCCAGGTCCCCTGGGCTCCCTCAAATGACAGACCTAACTA C1278 For the expression of BTEB2 in E.coli, 832bp BglII/BamHI AlaLeuHisNetLysArgHisGlnAsnStop 219 fragment (The BamHI site is located at the polylinker site of the TTCCTGTGTAAAAACAACAACCC 1301 plasmid vector) was cloned into the BamHI site of pAR2106 (9) which has the T7 promoter to generate pT7BTEB2. For the expression ofBTEB2 in cultured cells, 775bp Xba fragment (The downstream XbaI site is located at the polylinker site of the plasmid vector) was cloned into the Xba I site of pCMSV (10) B which has the cytomegalovirus enhancer and promoter. An Xho fragment which covers the entire protein-cding sequence of Spl Prokv rich so c Zk,C *VS was isolated from pRSVSpl (5) and cloned into the Xho I site Mi2- I=- -GOOH ofpCMSV. A GAL4-BTEB2 chimeric plasmid was constructed Tram-advalon DNA- Bing by inserting 462bp XbaI/DraI fragmant ofBTEB2 at the BamHI site in pSG424(l 1) after filled in with E. coli DNA polymerase 1 50 100 150 200 219 I, Klenow fragment. Gel mobility shift assay Figue 1. (A) Nucleotide and deduced amino acid sequences of human BTEB2 cDNA. The amino acid sequence is numbered from the presumed initiator E.coli strain BL21 was transformed with pT7BTEB2, and the methionine. The zinc finger motifs and a short basic sequence were underlined. transformant was grown in L-broth. Induction with 0.4mM IPTG (B) Schematic repesentation ofstucu ofhuman BTEB2, The proline-rich region (isopropyl-,3-D(-)-thiogalactopyranoside) was performed when and the basic region are indicated by shaded and solid boxes, respectively. Three the culture reached an A6w of 0.1. Three hours after induction, zinc finger motifs are shown by striped boxes. extracts were prepared by treatment with lysozyme (lmg/ml) in 20mM Tris buffer, pH7.5, containing 20mM NaCl at 4°C for 30 min followed by sonication. After centrifugation, the A supernatant was used for gel mobility shift assay. Hepa-1 cells BTEB2 E TG K BTEB GKVYGKSSHL TGERPF from a mouse hepatoma (12) were maintained in Dulbecco's Spl IQ GKVYGe H L TGERPF modified Eagle's medium supplemented with 10% fetal calf serum and 0.35% glucose. Nuclear extracts from Hepa-1 cells TE P.~ 5DELTYRTHTGET were prepared by the method ofDignam et al. (13). Gel mobility shift assays were performed as described previously (14). R5 DHL DNA transfection and CAT assay _ kFRDHL tr DNA transfection was performed by the calcium phosphate-DNA coprecipitation method as described (15). CAT assays were carried out by the method of Gorman et al. (16). B RNA blot analysis BTEB2 SNPDLEKRRIHYC---Zinc Finger Spi GSGDPGKKKQHIC---Zinc Finger Total RNA was isolated using the guanidine thiocyanate-CsCl c-Myc NTEENVKRRTHNVLERQRRNE procedure (17). RNA (15,gg) was electrophoresed on 0.8% TFEB LAKERQKKDNHNLIERRRRFN E KK
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