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Identification of PU.1 and Sp1 as Essential Transcriptional Factors for the Promoter Activity of MousetecGene
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
234, 376–381 (1997)
RC976650
Identification of PU.1 and Sp1 as Essential Transcriptional Factors for the Promoter Activity of Mouse tec Gene Hiroaki Honda,* Keiya Ozawa,† Yoshio Yazaki,* and Hisamaru Hirai*,1 *Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; and †Department of Molecular Biology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan
Received April 9, 1997
Tec is a cytoplasmic protein-tyrosine kinase abundantly expressed in hematopoietic precursor cells. To investigate the mechanism regulating the expression of Tec molecule, we cloned and analysed 5* flanking region of mouse tec gene up to 02kb from the transcriptional initiation site. Luciferase assays using successive deletion mutants demonstrated that regions from 0364 to 0323 and from 0122 to 063, which contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively, are important for the transcriptional activity. Gel-shift and supershift assays revealed that PU.1 and Sp1 bind to the these regions through their consensus binding motifs. In addition, introduction of mutations into these motifs resulted in marked decrease in the promoter activity. These results indicate that PU.1 and Sp1 are essential for the transcriptional activity of the tec promoter and suggest that the cooperation of PU.1 and Sp1 plays a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineages. q 1997 Academic Press
Tec is a cytoplasmic-type protein-tyrosine kinase (PTK) originally cloned from a mouse liver cDNA library using v-fps as a probe (1). The predicted amino acid sequence of the cDNA revealed that the Tec contains a src-homology 2 (SH2) domain followed by an SH3 and a kinase domains. The structure of Tec resembles to those of Src family PTKs. However, Tec lacks the myristylation site at the N-terminus and the negative1 Corresponding author: Hisamaru Hirai, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-5689-7286. Abbreviations: RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; IL, interleukin; DMEM, Dulbecco’s modified Eagle’s medium.
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regulatory tyrosine residues at the C-terminus (2,3) which are the characteristics of Src family PTKs. Tec is thus considered to be a new member of non-Src family PTKs. Recently, a number of Tec-related genes have been cloned. The Itk (also termed as Tsk) was cloned from murine T-cells (4,5). Subsequently, Btk was identified as a responsible gene for the Bruton-type X-chromosome-linked agammaglobulinemia (XLA) (6,7). Furthermore, Bmx and Txk were cloned from human bone marrow cells and from CD19-positive peripheral blood mononuclear cells, respectively (8,9). Since these Tecrelated PTKs share a similar structural characteristics of having a unique N-terminal region containing the recently described pleckstrin homology domain (PH) (10) and a conserved amino acid stretch located between the PH and SH3 domains, they are considered to consist of the Tec family of cytoplasmic PTKs. Considering that Tec family PTKs are abundantly expressed in hematopoietic cells and that BTK was cloned as a responsible gene for the X-linked agammaglobulinemia (6,7), all five Tec-related kinases seem to be deeply implicated in a signal transduction and play an important role in the regulation of the hematopoietic system. Indeed, Tec was demonstrated to be involved in IL-3-mediated signal transduction in an IL3-dependent mouse cell line, FDC-P1 (11). Subsequent studies revealed that Tec is physiologically associated with Lyn through its N-terminal region (12). To investigate the transcriptional mechanism regulating the expression of the Tec molecule, it is necessary to isolate and analyse the promoter region. We previously cloned 5* flanking region of the mouse tec gene approximately 400bp, determined the transcriptional initiation site, and confirmed its promoter activity in tec-expressing leukemic cells (13). In this report, we cloned and sequenced mouse tec promoter up to approximately 02kb from the transcription initiation
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site, determined the regions essential for the promoter activity, identified the binding motifs and transcription factors, and demonstrated that the binding motifs are critical for the promoter activity. MATERIALS AND METHODS Cells. BA/F3 (14) cells and 32D (15) cells which express tec mRNA at a high level were maintained in RPMI1640 medium with 10% fetal calf serum (FCS) supplemented with 25U/ml of recombinant murine IL-3 (supplied by Kirin Brewery Co. Ltd.) at 377C in a humidified atmosphere. NIH 3T3 (16) cells were cultured in DMEM medium with 10% FCS. Screening of the mouse tec promoter. A total of 51105 independent phage plaques of BA/F3 genomic library were screened with 32Plabeled AccIII-Eco473 fragment which corresponds to the nucleotide sequences from 0241 to 023 of the tec promoter (13). The hybridization and washing conditions were as previously described (17). DNAs of the inserts of the positive phage clones were subjected to restriction enzyme mapping. The approximately 2kb PstI-PstI fragment which hybridized to the AccIII-Eco473 fragment was blunt ended with T4 DNA polymerase (Takara, Kyoto, Japan) and subcloned into the filled HindIII site in pBluescript (Stratagene). Construction of deletion mutants and sequencing of the promoter. The pBluescript containing the above approximately 2kb tec gene was double digested with PstI and EcoRV (Takara) and the 3 * deletion mutants of the cloned tec gene were generated using DoubleStranded Nested Deletion Kit (Pharmacia) according to the manufacturer’s instructions. An insert of a deletion mutant whose 3 * end was deleted to /22 within the first exon of the tec gene (designated the major transcription initiation site defined by the RACE-PCR (13) as /1) was excised and subcloned into a promoterless expression vector, pUC00Luc (18) to give a plasmid, mtecLuc. The mtecLuc was digested with KpnI and SalI (Takara) and serial 5* deletion mutants of the tec gene were generated as described above. The deletion mutants were subjected to the DNA sequencing. DNA sequencing was performed by the dideoxy chain method (19) using an oligonucleotide 5*-ACGACGTTGTAAAACGACGGCCAGT-3* as a primer, which was derived from the nucleotide sequence just upstream of the multiple cloning site of pUC00Luc (18). The promoter regions of D4-D9 deletion constructs were generated by PCR as follows. The 5* primer sequences were 5*-CCGGTACCACCCACAAGTGCTATTGCTA-3* (0323 to 0304) for D4, 5*-CCGGTACCTTTTCTGGACGGTTCTCTTA-3* (0272 to 0253) for D5, 5*-CCGGTACCACGCCGCGTCTGTCTGGATA-3* (0222 to 0203) for D6, 5*-CCGGTACCACCGCGAGCTCCGATTCTTA-3* (0172 to 0153) for D7, 5*-CCGGTACCGGGACACTGGCGCTGTGGGCA-3* (0122 to 092) for D8, 5*-CCGGTACCGAGGAGCCGGGCGGTGGGCGT-3* (061 to 040) for D9 and the 3* primer sequence was 5*-CCGTCGACCAGAGCGACGTCCAAACTGC-3* (/2 to /20). The PCR amplification was carried out as previously described (20) using 10ng of mtecLuc as a template. The PCR products of the expected size were digested with KpnI and SalI (Takara) and subcloned to pUC00Luc to generate D4-D9. The nucleotide sequences of the inserts were verified by sequencing. DNA transection and luciferase assays. DNA transection and the luciferase assays were performed essentially as previously described (18). In brief, 51106 to 11107 cells in the growing phase were washed with PBS and incubated with 500mg of DEAE-dextran (Pharmacia) and 10mg of the reporter plasmid DNA for 25min at room temperature. Subsequently, the cells were incubated in the culture medium containing 100mM chloroquine at 377C in 5% CO2 for 1hr. The cells were washed with PBS and harvested after 48hrs’ incubation in the culture medium. The cell lysates were subjected to the luciferase assays using Luciferase Assay System (Promega) according to the manufacturer’s instructions.
Gel-shift and supershift assays. Nuclear extracts were prepared from the cells as described (21). Four different double-stranded oligonucleotide probes (termed PU.1-1, PU.1-2, Sp1-1, and Sp1-2, overlined in Fig. 1B) were generated for the gel-shift assays. The sequences of the probes are as follows: PU.1-1, 5*-GGGCTAAGCGGAAGTGGAGGTC-3* (0356 to 0325); PU.1-2, 5*-GTTCTCTTAGGATGGGAAGTCCGGAC-3* (0261 to 0236); Sp1-1, 5*-GCGAGGGGGCGGGGCCAGGGAGGAGC-3* (080 to 055); Sp1-2, 5*-GGAGGAGCCGGGCGGTGGGCGTGGC-3* (062 to 038). For the competition experiment, mutated probes for PU.1-1 and Sp1-1 (termed PU.1-1M and Sp1-1M) were also synthesized, in which the PU.1 binding site GGAA and the Sp1 binding site GGGCGG were substituted to TAGC and AATAAT, respectively. The sequences of the mutated probes are as follows (the substituted nucleotides are underlined): PU.1-1M, 5*-GGGCTAAGCTAGCGTGGAGGTC-3*; Sp1-1M, 5*-GCGAGGAATAATGGCCAGGGAGGAGC-3*. Twenty ng of each oligonucleotide was radiolabeled at the 5* end with g32P-ATP (Amersham) using T4 polynucleotide kinase (Takara). The oligonucleotides were annealed with the complementary strands and the radiolabeled probes were purified through a Sephadex G-50 column (Pharmacia). For the gel-shift assays, approximately 31104 cpm of the probe was incubated with 3mg of nuclear extract, 5ml of D-buffer (20mM Hepes (pH 7.9), 20% Glycerol, 100mM KCl, 0.2mM EDTA, 0.2mM PMSF, and 0.5mM DTT), 1ml of 0.5% bovine serum albumin, and 2ml of 0.8mg/ml calf thymus DNA at 307C for 15min. For the competition experiments, 20ng of wild or mutated unlabeled oligonucleotides (approximately 100-fold excess of the radiolabeled probe) were added to the above reaction solution. For the supershift assays, 3mg of nuclear extract was pre-incubated with 1 or 2ml of anti-PU.1 (PU.1 (Spi-1)(T-21), Santa Cruz Biochemistry, Inc., Santa Cruz, CA) or anti-Sp1 (Sp1(PEP2), Santa Cruz Biochemistry, Inc.) in the presence of 5ml of D-buffer at 47C for one hour. After pre-incubation, 1ml of 0.5% BSA, 2ml of calf thymus DNA, and 31104 cpm of the probe were added to the above solution and the samples were incubated at 307C for 15min. All samples were electrophoresed on 4.2% polyacrylamide gel using a high-ionic-strength buffer (50mM Tris-Cl, 380mM Glycine, and 2mM EDTA) at 40mA for approximately 1hr. The gels were dried and autoradiographed. In vitro mutagenesis. To introduce mutations in PU.1-1 and Sp11 sites in the promoter, the promoter region of 0362 to /22 was subcloned into M13 vector (Stratagene) and single strand DNA was rescued as described (18). The rescued DNA was annealed with mutated oligonucleotides (PU.1-1M, or Sp1-1M, or both) and the in vitro mutagenesis was performed essentially as described (22). The introduced mutations were verified by sequencing.
RESULTS Cloning and sequencing of mouse tec promoter. To obtain the promoter region of mouse tec gene that contains several kilobase upstream of the transcription initiation site (13), we screened BA/F3 genomic library with the AccIII-Eco473 fragment of the tec gene as a probe (13). As a result, we isolated several phage clones and identified an approximately 2kb PstI-PstI fragment that was overlapped among the positive clones, was hybridized to the AccIII-Eco473 fragment, and contained the same restriction enzyme mapping (Fig 1A). Sequencing of the fragment revealed that it contained the promoter region to 01948 and harbored the first exon and a part of the first intron. The restriction enzyme mapping and the nucleotide sequence of the promoter is shown in Fig. 1A and 1B, respectively.
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FIG. 1. (A) Restriction enzyme mapping of mouse tec promoter. Restriction enzyme sites are P; PstI, A; ApaI, S; SalI, X; XhoI, Sm; SmaI, Nc; NcoI, Nh; NheI, Ac; AccIII, and Sa; SacI. The first exon is shown as a shaded box and the promoter and the first intron are shown with thick and thin bars, respectively. (B) Nucleotide sequence of the promoter region. The 5* ends of the cloned promoter (D0) and the nested deletion mutants (D1-D9) used for the luciferase assays are indicated. The transcription initiation site determined by RACEPCR (13) is indicated by an asterisk. Nucleotide sequences used as probes for gel shift assays are overlined and the putative binding motifs in the probes are underlined. The virtual binding sites for nuclear proteins including PU.1 and Sp1 are shown in bold face.
Characterization of mouse tec promoter by successive deletion mutants. To examine the promoter activity of the region, 5* deletion mutants were fused to the luciferase gene and the constructs were transfected to tec-expressing cells, BA/F3 and 32D, or tec-non-expressing cells, NIH 3T3. The original fragment initiating at 01948 (named D0, see Fig. 1B) and three successive deletion constructs initiating at 01006, 0624, and 0364bp (named D1, D2, and D3, respectively, see Fig. 1B) were subjected to the luciferase assays. The result showed that the sequence(s) essential for the promoter activity existed within the proximal 364bp of the region (Fig. 2A). To determine the sequence(s) critical for the promoter activity more precisely, the luciferase assays were performed using several promoter constructs generated by PCR, in which the 5* ends were successively
deleted with approximately 50bp (named D4-D9, see Fig. 1B). The result showed that marked decrease in luciferase activity occurred between D3 and D4 and between D8 and D9, indicating that the sequences of 0364 to 0323 and 0122 to 063 are essential for the promoter activity (Fig. 2B). In comparison to BA/F3 cells, 32D cells showed essentially the same pattern as BA/F3 cells but the luciferase activities were approximately half of those observed in BA/F3 cells, probably reflecting the lower expression level of tec gene (data not shown). In any experiment, NIH 3T3 cells showed no significant luciferase activity (data not shown). Nuclear proteins specifically bind to the regions essential for the promoter activity. The above results demonstrated that the regions of 0364 to 0323 and 0122 to 063 are functionally important for the promoter ac-
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FIG. 2. Construction of the reporter plasmids and results of the luciferase assays in BA/F3 cells. (A) Luciferase activities of the original promoter (01948, shown in Fig. 1B as D0) and three successive constructs with large deletions (01006, 0624, and 0364, shown in Fig. 1B as D1, D2, and D3, respectively). (B) Luciferase activites of serial deletion constructs (D3-D9). The putative PU.1 binding sites (PU.1-1 and PU.1-2) and Sp1 binding sites (Sp1-1 and Sp1-2) used for the gel shift assays are indicated by circles and squares, respectively. The regions that serve as the binding sites for PU.1 and Sp1 are indicated by bold and shaded faces. In (A) and (B), the luciferase activity of each deletion mutant is shown as folds over the activity obtained with the promoterless plasmid, pUC00Luc (equal to 1.00) and is the mean of three independent experiments.
tivity. Inspection of the regions revealed that the regions contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively (shown in bold face and underlined in Fig. 1B). To address the possibility that those transcription factors interact with the tec promoter through the regions, gel-shift assays were performed using oligonucleotide probes PU.1-1 and Sp1-1 (overlined in Fig. 1B), in which the binding sites were located in the center of the sequences. Oligonucleotide probes PU.1-2 and Sp1-2 (also overlined in Fig. 1B) derived from regions that also contained the binding motifs (underlined in Fig. 1B) but had no positive effect on the luciferase assays (Fig. 2B) were also used as negative controls. As shown in Fig. 3A, a set of proteins were observed to bind to PU.11 and Sp1-1 probes, whereas no proteins were shown to interact with PU.1-2 or Sp1-2 (Fig. 3A). This result demonstrated that nuclear proteins selectively bind to the regions essential for the promoter activity in reference to the adjacent sequences.
FIG. 3. The results of the gel shift and supershift assays. (A) Gel shift assays of PU.1-1, PU.1-2, Sp1-1, and Sp1-2 probes with (/) or without (0) nuclear extracts (N.E.). The differently migrating bands observed in PU.1-1 and Sp1-1 are labeled as P1-P4 and S1-S3, respectively. (B) Competition experiments of PU.1-1 and Sp1-1. The radiolabeled probes were incubated with approximately 100-fold excess of wild (W.) or mutated (M.) cold competitors. (C) Supershift assays of PU.1-1 and Sp1-1. Nuclear extracts were preincubated with antibody (Ab.) for PU.1 (aPU.1) or Sp1 (aSp1) and the protein-antibody complexes were subjected to the gel shift assays. The bands identified as complexes for PU.1 and Sp1 are indicated by arrows and the supershifted band observed in Sp1-1 is indicated by an arrowhead (SS).
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moter activity. The results indicate that the above binding motifs are essential for the transcriptional activity of the tec promoter and suggest that they play a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineage. DISCUSSION FIG. 4. Introduction of mutations in the PU.1 and Sp1 binding sites in the promoter region and the results of luciferase assays. The locations of PU1.1 and Sp1-1 that serve as PU.1 and Sp1 binding sites are shown as a shaded circle and a shaded square, respectively. The wild promoter and three mutants that have a mutation in PU.11 (PU.1-1M), in Sp1-1 (Sp1-1M), or in both regions (PU1.1M/Sp11M) were subjected to luciferase assays. The luciferase activity of each mutant is shown as folds over the activity obtained with the promoterless plasmid, pUC00Luc (equal to 1.00) and is the mean of three independent experiments.
Identification of PU.1 and Sp1 as important transcriptional factors for tec gene. To examine the binding specificity of the proteins for PU.1-1 and Sp1-1 probes, competition experiments were performed. Approximately 100-fold unlabeled wild or mutated probes were added to the reaction. As shown in Fig. 3B, the wild competitor completely blocked the binding of the nuclear proteins to the radiolabeled probes. In contrast, the mutated probes PU.1-1M and Sp1-1M, in which the PU.1 binding sequence, GGAA, and Sp1 binding sequence, GGGCGG, were substituted to TAGC and AATAAT, respectively, did not affect the protein-DNA binding (Fig. 3B). These results indicated that the binding of proteins to the probes was specific and that DNAbinding proteins interacted with the probes through the consensus binding sequences of PU.1 and Sp1. To further examine whether PU.1 and Sp1 were virtually involved in the protein-DNA complexes, supershift assays were carried out using specific antibodies for PU.1 and Sp1. As shown in Fig. 3C, the band of P4 (Fig. 3C) was ablated in the presence of anti-PU.1 although the supershifted band was not clearly detected. Furthermore, by the addition of anti-Sp1, the band S1 was observed to be clearly supershifted. These results demonstrated that PU.1 and Sp1 bind to the PU.1-1 and Sp1-1 regions through their consensus sequences. PU.1 and Sp1 binding motifs are essential for the promoter activity of Tec molecule. Finally, to confirm that PU.1- and Sp1-binding sequences in PU1.1 and Sp1-1 regions are essential for the promoter activity, mutations were introduced in the motifs and the mutated promoters were subjected to the luciferase assays. As shown in Fig.4, introduction of mutation in the PU.1 binding motifs decreased the transcriptional activity to approximately half of the original promoter. In addition, introduction of mutation in the Sp1 binding motif resulted in significant decrease in the pro-
In this article, we identified PU.1 and Sp1 as essential transcriptional factors for the expression of the Tec molecule in the hematopoietic cells. PU.1 belongs to the Ets family transcription factors and was originally isolated from the integration site in murine erythroleukemia induced by the spleen focus forming virus (SSFV) (23). Given that PU.1 is restrictedly expressed in hematopoietic tissues (24-26), PU.1 is regarded as an important transcription factor for genes preferentially expressed in hematopoietic lineages. Indeed, subsequent studies demonstrated that PU.1 plays an essential role for genes including immunoglobulin heavy and light chains, macrophage colonystimulating factor promoter, and CD11b promoter (2731). Furthermore, a direct evidence of the biological significance of PU.1 was provided by targeting the PU.1 gene in mice (32). One of the phenotypic characteristics of PU.1-deficient mice is the absence of myeloid/ lymphoid progenitor cells. This finding strongly suggests that PU.1 functions at a level of precursor cells committed to myeloid and lymphoid lineages. Considering that tec is abundantly expressed in hematopoietic progenitor cells of the same developmental stage, it is reasonable that PU.1 plays a critical role for Tec expression. Sp1 was initially identified through its ability initiating transcription from SV40 promoter in Hela cells (33). Sp1 binds to G/C-rich box that is frequently observed in TATA-less promoters. Since tec promoter does not have TATA motif (13), it is likely that Sp1 plays as a transcriptional factor for tec gene. In contrast to PU.1, Sp1 is rather ubiquitously expressed and is considered to contribute to a basal transcriptional activity. However, in some cases, Sp1 is reported to play a role in tissue-specific expression of the target genes, such as CD11b (34). Therefore, Sp1 as well as PU.1 might contribute to the preferential expression of the Tec molecule in the hematopoietic cells. Recently, the analysis of the Btk promoter was reported (35). Interestingly, as observed in the case of tec promoter, PU.1 and Sp1 were demonstrated to be critical for the transcriptional activity of the btk promoter. Therefore, the cooperation of PU.1 and Sp1 might play a role in the preferential expression of Tec family PTKs in the hematopoietic tissues. However, Btk is mainly expressed in B-lymphoid cells, while Tec is predominantly expressed in myeloid cells. Thus, other transcription factors that bind to more upstream
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region(s) of the promoter or to introns might account for the lineage-preferential expression of Tec-family PTKs. To further investigate whether the promoter is functionally active in vivo, we fused the longest form of the promoter (01948 to /22) to p210bcr/abl, a chimeric oncogene responsible for causing human chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) (36, 37), and generated transgenic mice expressing p210bcr/abl driven by the tec promoter. The transgenic mice developed CML- and ALL-like diseases (manuscript in preparation), providing direct evidence that the cloned tec promoter is functional in hematopoietic progenitor cells, presumably in stem cell level. In summary, we described the identification, characterization, and functional analysis of mouse tec promoter. The findings will help us to understand the precise transcriptional mechanism of the Tec molecule, which is highly expressed in hematopoietic lineages. In addition, the promoter shown in this report would be useful and applicable for expressing a gene of interest in vivo, especially in hematopoietic precursor cells. ACKNOWLEDGMENTS We thank Dr. Toshio Inaba for the technical assistance of the gelshift and supershift assays. This work was supported in part by Grants-in-Aids from the Ministry of Education, Science and Culture of Japan.
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Identification of PU.1 and Sp1 as Essential Transcriptional Factors for the Promoter Activity of Mouse tec Gene Hiroaki Honda,* Keiya Ozawa,† Yoshio Yazaki,* and Hisamaru Hirai*,1 *Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; and †Department of Molecular Biology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-04, Japan
Received April 9, 1997
Tec is a cytoplasmic protein-tyrosine kinase abundantly expressed in hematopoietic precursor cells. To investigate the mechanism regulating the expression of Tec molecule, we cloned and analysed 5* flanking region of mouse tec gene up to 02kb from the transcriptional initiation site. Luciferase assays using successive deletion mutants demonstrated that regions from 0364 to 0323 and from 0122 to 063, which contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively, are important for the transcriptional activity. Gel-shift and supershift assays revealed that PU.1 and Sp1 bind to the these regions through their consensus binding motifs. In addition, introduction of mutations into these motifs resulted in marked decrease in the promoter activity. These results indicate that PU.1 and Sp1 are essential for the transcriptional activity of the tec promoter and suggest that the cooperation of PU.1 and Sp1 plays a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineages. q 1997 Academic Press
Tec is a cytoplasmic-type protein-tyrosine kinase (PTK) originally cloned from a mouse liver cDNA library using v-fps as a probe (1). The predicted amino acid sequence of the cDNA revealed that the Tec contains a src-homology 2 (SH2) domain followed by an SH3 and a kinase domains. The structure of Tec resembles to those of Src family PTKs. However, Tec lacks the myristylation site at the N-terminus and the negative1 Corresponding author: Hisamaru Hirai, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Fax: 81-3-5689-7286. Abbreviations: RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; IL, interleukin; DMEM, Dulbecco’s modified Eagle’s medium.
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regulatory tyrosine residues at the C-terminus (2,3) which are the characteristics of Src family PTKs. Tec is thus considered to be a new member of non-Src family PTKs. Recently, a number of Tec-related genes have been cloned. The Itk (also termed as Tsk) was cloned from murine T-cells (4,5). Subsequently, Btk was identified as a responsible gene for the Bruton-type X-chromosome-linked agammaglobulinemia (XLA) (6,7). Furthermore, Bmx and Txk were cloned from human bone marrow cells and from CD19-positive peripheral blood mononuclear cells, respectively (8,9). Since these Tecrelated PTKs share a similar structural characteristics of having a unique N-terminal region containing the recently described pleckstrin homology domain (PH) (10) and a conserved amino acid stretch located between the PH and SH3 domains, they are considered to consist of the Tec family of cytoplasmic PTKs. Considering that Tec family PTKs are abundantly expressed in hematopoietic cells and that BTK was cloned as a responsible gene for the X-linked agammaglobulinemia (6,7), all five Tec-related kinases seem to be deeply implicated in a signal transduction and play an important role in the regulation of the hematopoietic system. Indeed, Tec was demonstrated to be involved in IL-3-mediated signal transduction in an IL3-dependent mouse cell line, FDC-P1 (11). Subsequent studies revealed that Tec is physiologically associated with Lyn through its N-terminal region (12). To investigate the transcriptional mechanism regulating the expression of the Tec molecule, it is necessary to isolate and analyse the promoter region. We previously cloned 5* flanking region of the mouse tec gene approximately 400bp, determined the transcriptional initiation site, and confirmed its promoter activity in tec-expressing leukemic cells (13). In this report, we cloned and sequenced mouse tec promoter up to approximately 02kb from the transcription initiation
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site, determined the regions essential for the promoter activity, identified the binding motifs and transcription factors, and demonstrated that the binding motifs are critical for the promoter activity. MATERIALS AND METHODS Cells. BA/F3 (14) cells and 32D (15) cells which express tec mRNA at a high level were maintained in RPMI1640 medium with 10% fetal calf serum (FCS) supplemented with 25U/ml of recombinant murine IL-3 (supplied by Kirin Brewery Co. Ltd.) at 377C in a humidified atmosphere. NIH 3T3 (16) cells were cultured in DMEM medium with 10% FCS. Screening of the mouse tec promoter. A total of 51105 independent phage plaques of BA/F3 genomic library were screened with 32Plabeled AccIII-Eco473 fragment which corresponds to the nucleotide sequences from 0241 to 023 of the tec promoter (13). The hybridization and washing conditions were as previously described (17). DNAs of the inserts of the positive phage clones were subjected to restriction enzyme mapping. The approximately 2kb PstI-PstI fragment which hybridized to the AccIII-Eco473 fragment was blunt ended with T4 DNA polymerase (Takara, Kyoto, Japan) and subcloned into the filled HindIII site in pBluescript (Stratagene). Construction of deletion mutants and sequencing of the promoter. The pBluescript containing the above approximately 2kb tec gene was double digested with PstI and EcoRV (Takara) and the 3 * deletion mutants of the cloned tec gene were generated using DoubleStranded Nested Deletion Kit (Pharmacia) according to the manufacturer’s instructions. An insert of a deletion mutant whose 3 * end was deleted to /22 within the first exon of the tec gene (designated the major transcription initiation site defined by the RACE-PCR (13) as /1) was excised and subcloned into a promoterless expression vector, pUC00Luc (18) to give a plasmid, mtecLuc. The mtecLuc was digested with KpnI and SalI (Takara) and serial 5* deletion mutants of the tec gene were generated as described above. The deletion mutants were subjected to the DNA sequencing. DNA sequencing was performed by the dideoxy chain method (19) using an oligonucleotide 5*-ACGACGTTGTAAAACGACGGCCAGT-3* as a primer, which was derived from the nucleotide sequence just upstream of the multiple cloning site of pUC00Luc (18). The promoter regions of D4-D9 deletion constructs were generated by PCR as follows. The 5* primer sequences were 5*-CCGGTACCACCCACAAGTGCTATTGCTA-3* (0323 to 0304) for D4, 5*-CCGGTACCTTTTCTGGACGGTTCTCTTA-3* (0272 to 0253) for D5, 5*-CCGGTACCACGCCGCGTCTGTCTGGATA-3* (0222 to 0203) for D6, 5*-CCGGTACCACCGCGAGCTCCGATTCTTA-3* (0172 to 0153) for D7, 5*-CCGGTACCGGGACACTGGCGCTGTGGGCA-3* (0122 to 092) for D8, 5*-CCGGTACCGAGGAGCCGGGCGGTGGGCGT-3* (061 to 040) for D9 and the 3* primer sequence was 5*-CCGTCGACCAGAGCGACGTCCAAACTGC-3* (/2 to /20). The PCR amplification was carried out as previously described (20) using 10ng of mtecLuc as a template. The PCR products of the expected size were digested with KpnI and SalI (Takara) and subcloned to pUC00Luc to generate D4-D9. The nucleotide sequences of the inserts were verified by sequencing. DNA transection and luciferase assays. DNA transection and the luciferase assays were performed essentially as previously described (18). In brief, 51106 to 11107 cells in the growing phase were washed with PBS and incubated with 500mg of DEAE-dextran (Pharmacia) and 10mg of the reporter plasmid DNA for 25min at room temperature. Subsequently, the cells were incubated in the culture medium containing 100mM chloroquine at 377C in 5% CO2 for 1hr. The cells were washed with PBS and harvested after 48hrs’ incubation in the culture medium. The cell lysates were subjected to the luciferase assays using Luciferase Assay System (Promega) according to the manufacturer’s instructions.
Gel-shift and supershift assays. Nuclear extracts were prepared from the cells as described (21). Four different double-stranded oligonucleotide probes (termed PU.1-1, PU.1-2, Sp1-1, and Sp1-2, overlined in Fig. 1B) were generated for the gel-shift assays. The sequences of the probes are as follows: PU.1-1, 5*-GGGCTAAGCGGAAGTGGAGGTC-3* (0356 to 0325); PU.1-2, 5*-GTTCTCTTAGGATGGGAAGTCCGGAC-3* (0261 to 0236); Sp1-1, 5*-GCGAGGGGGCGGGGCCAGGGAGGAGC-3* (080 to 055); Sp1-2, 5*-GGAGGAGCCGGGCGGTGGGCGTGGC-3* (062 to 038). For the competition experiment, mutated probes for PU.1-1 and Sp1-1 (termed PU.1-1M and Sp1-1M) were also synthesized, in which the PU.1 binding site GGAA and the Sp1 binding site GGGCGG were substituted to TAGC and AATAAT, respectively. The sequences of the mutated probes are as follows (the substituted nucleotides are underlined): PU.1-1M, 5*-GGGCTAAGCTAGCGTGGAGGTC-3*; Sp1-1M, 5*-GCGAGGAATAATGGCCAGGGAGGAGC-3*. Twenty ng of each oligonucleotide was radiolabeled at the 5* end with g32P-ATP (Amersham) using T4 polynucleotide kinase (Takara). The oligonucleotides were annealed with the complementary strands and the radiolabeled probes were purified through a Sephadex G-50 column (Pharmacia). For the gel-shift assays, approximately 31104 cpm of the probe was incubated with 3mg of nuclear extract, 5ml of D-buffer (20mM Hepes (pH 7.9), 20% Glycerol, 100mM KCl, 0.2mM EDTA, 0.2mM PMSF, and 0.5mM DTT), 1ml of 0.5% bovine serum albumin, and 2ml of 0.8mg/ml calf thymus DNA at 307C for 15min. For the competition experiments, 20ng of wild or mutated unlabeled oligonucleotides (approximately 100-fold excess of the radiolabeled probe) were added to the above reaction solution. For the supershift assays, 3mg of nuclear extract was pre-incubated with 1 or 2ml of anti-PU.1 (PU.1 (Spi-1)(T-21), Santa Cruz Biochemistry, Inc., Santa Cruz, CA) or anti-Sp1 (Sp1(PEP2), Santa Cruz Biochemistry, Inc.) in the presence of 5ml of D-buffer at 47C for one hour. After pre-incubation, 1ml of 0.5% BSA, 2ml of calf thymus DNA, and 31104 cpm of the probe were added to the above solution and the samples were incubated at 307C for 15min. All samples were electrophoresed on 4.2% polyacrylamide gel using a high-ionic-strength buffer (50mM Tris-Cl, 380mM Glycine, and 2mM EDTA) at 40mA for approximately 1hr. The gels were dried and autoradiographed. In vitro mutagenesis. To introduce mutations in PU.1-1 and Sp11 sites in the promoter, the promoter region of 0362 to /22 was subcloned into M13 vector (Stratagene) and single strand DNA was rescued as described (18). The rescued DNA was annealed with mutated oligonucleotides (PU.1-1M, or Sp1-1M, or both) and the in vitro mutagenesis was performed essentially as described (22). The introduced mutations were verified by sequencing.
RESULTS Cloning and sequencing of mouse tec promoter. To obtain the promoter region of mouse tec gene that contains several kilobase upstream of the transcription initiation site (13), we screened BA/F3 genomic library with the AccIII-Eco473 fragment of the tec gene as a probe (13). As a result, we isolated several phage clones and identified an approximately 2kb PstI-PstI fragment that was overlapped among the positive clones, was hybridized to the AccIII-Eco473 fragment, and contained the same restriction enzyme mapping (Fig 1A). Sequencing of the fragment revealed that it contained the promoter region to 01948 and harbored the first exon and a part of the first intron. The restriction enzyme mapping and the nucleotide sequence of the promoter is shown in Fig. 1A and 1B, respectively.
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FIG. 1. (A) Restriction enzyme mapping of mouse tec promoter. Restriction enzyme sites are P; PstI, A; ApaI, S; SalI, X; XhoI, Sm; SmaI, Nc; NcoI, Nh; NheI, Ac; AccIII, and Sa; SacI. The first exon is shown as a shaded box and the promoter and the first intron are shown with thick and thin bars, respectively. (B) Nucleotide sequence of the promoter region. The 5* ends of the cloned promoter (D0) and the nested deletion mutants (D1-D9) used for the luciferase assays are indicated. The transcription initiation site determined by RACEPCR (13) is indicated by an asterisk. Nucleotide sequences used as probes for gel shift assays are overlined and the putative binding motifs in the probes are underlined. The virtual binding sites for nuclear proteins including PU.1 and Sp1 are shown in bold face.
Characterization of mouse tec promoter by successive deletion mutants. To examine the promoter activity of the region, 5* deletion mutants were fused to the luciferase gene and the constructs were transfected to tec-expressing cells, BA/F3 and 32D, or tec-non-expressing cells, NIH 3T3. The original fragment initiating at 01948 (named D0, see Fig. 1B) and three successive deletion constructs initiating at 01006, 0624, and 0364bp (named D1, D2, and D3, respectively, see Fig. 1B) were subjected to the luciferase assays. The result showed that the sequence(s) essential for the promoter activity existed within the proximal 364bp of the region (Fig. 2A). To determine the sequence(s) critical for the promoter activity more precisely, the luciferase assays were performed using several promoter constructs generated by PCR, in which the 5* ends were successively
deleted with approximately 50bp (named D4-D9, see Fig. 1B). The result showed that marked decrease in luciferase activity occurred between D3 and D4 and between D8 and D9, indicating that the sequences of 0364 to 0323 and 0122 to 063 are essential for the promoter activity (Fig. 2B). In comparison to BA/F3 cells, 32D cells showed essentially the same pattern as BA/F3 cells but the luciferase activities were approximately half of those observed in BA/F3 cells, probably reflecting the lower expression level of tec gene (data not shown). In any experiment, NIH 3T3 cells showed no significant luciferase activity (data not shown). Nuclear proteins specifically bind to the regions essential for the promoter activity. The above results demonstrated that the regions of 0364 to 0323 and 0122 to 063 are functionally important for the promoter ac-
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FIG. 2. Construction of the reporter plasmids and results of the luciferase assays in BA/F3 cells. (A) Luciferase activities of the original promoter (01948, shown in Fig. 1B as D0) and three successive constructs with large deletions (01006, 0624, and 0364, shown in Fig. 1B as D1, D2, and D3, respectively). (B) Luciferase activites of serial deletion constructs (D3-D9). The putative PU.1 binding sites (PU.1-1 and PU.1-2) and Sp1 binding sites (Sp1-1 and Sp1-2) used for the gel shift assays are indicated by circles and squares, respectively. The regions that serve as the binding sites for PU.1 and Sp1 are indicated by bold and shaded faces. In (A) and (B), the luciferase activity of each deletion mutant is shown as folds over the activity obtained with the promoterless plasmid, pUC00Luc (equal to 1.00) and is the mean of three independent experiments.
tivity. Inspection of the regions revealed that the regions contain the consensus binding sequences for PU.1 (GGAA) and Sp1 (GGGCGG), respectively (shown in bold face and underlined in Fig. 1B). To address the possibility that those transcription factors interact with the tec promoter through the regions, gel-shift assays were performed using oligonucleotide probes PU.1-1 and Sp1-1 (overlined in Fig. 1B), in which the binding sites were located in the center of the sequences. Oligonucleotide probes PU.1-2 and Sp1-2 (also overlined in Fig. 1B) derived from regions that also contained the binding motifs (underlined in Fig. 1B) but had no positive effect on the luciferase assays (Fig. 2B) were also used as negative controls. As shown in Fig. 3A, a set of proteins were observed to bind to PU.11 and Sp1-1 probes, whereas no proteins were shown to interact with PU.1-2 or Sp1-2 (Fig. 3A). This result demonstrated that nuclear proteins selectively bind to the regions essential for the promoter activity in reference to the adjacent sequences.
FIG. 3. The results of the gel shift and supershift assays. (A) Gel shift assays of PU.1-1, PU.1-2, Sp1-1, and Sp1-2 probes with (/) or without (0) nuclear extracts (N.E.). The differently migrating bands observed in PU.1-1 and Sp1-1 are labeled as P1-P4 and S1-S3, respectively. (B) Competition experiments of PU.1-1 and Sp1-1. The radiolabeled probes were incubated with approximately 100-fold excess of wild (W.) or mutated (M.) cold competitors. (C) Supershift assays of PU.1-1 and Sp1-1. Nuclear extracts were preincubated with antibody (Ab.) for PU.1 (aPU.1) or Sp1 (aSp1) and the protein-antibody complexes were subjected to the gel shift assays. The bands identified as complexes for PU.1 and Sp1 are indicated by arrows and the supershifted band observed in Sp1-1 is indicated by an arrowhead (SS).
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moter activity. The results indicate that the above binding motifs are essential for the transcriptional activity of the tec promoter and suggest that they play a substantial role in the preferential expression of the Tec molecule in the hematopoietic lineage. DISCUSSION FIG. 4. Introduction of mutations in the PU.1 and Sp1 binding sites in the promoter region and the results of luciferase assays. The locations of PU1.1 and Sp1-1 that serve as PU.1 and Sp1 binding sites are shown as a shaded circle and a shaded square, respectively. The wild promoter and three mutants that have a mutation in PU.11 (PU.1-1M), in Sp1-1 (Sp1-1M), or in both regions (PU1.1M/Sp11M) were subjected to luciferase assays. The luciferase activity of each mutant is shown as folds over the activity obtained with the promoterless plasmid, pUC00Luc (equal to 1.00) and is the mean of three independent experiments.
Identification of PU.1 and Sp1 as important transcriptional factors for tec gene. To examine the binding specificity of the proteins for PU.1-1 and Sp1-1 probes, competition experiments were performed. Approximately 100-fold unlabeled wild or mutated probes were added to the reaction. As shown in Fig. 3B, the wild competitor completely blocked the binding of the nuclear proteins to the radiolabeled probes. In contrast, the mutated probes PU.1-1M and Sp1-1M, in which the PU.1 binding sequence, GGAA, and Sp1 binding sequence, GGGCGG, were substituted to TAGC and AATAAT, respectively, did not affect the protein-DNA binding (Fig. 3B). These results indicated that the binding of proteins to the probes was specific and that DNAbinding proteins interacted with the probes through the consensus binding sequences of PU.1 and Sp1. To further examine whether PU.1 and Sp1 were virtually involved in the protein-DNA complexes, supershift assays were carried out using specific antibodies for PU.1 and Sp1. As shown in Fig. 3C, the band of P4 (Fig. 3C) was ablated in the presence of anti-PU.1 although the supershifted band was not clearly detected. Furthermore, by the addition of anti-Sp1, the band S1 was observed to be clearly supershifted. These results demonstrated that PU.1 and Sp1 bind to the PU.1-1 and Sp1-1 regions through their consensus sequences. PU.1 and Sp1 binding motifs are essential for the promoter activity of Tec molecule. Finally, to confirm that PU.1- and Sp1-binding sequences in PU1.1 and Sp1-1 regions are essential for the promoter activity, mutations were introduced in the motifs and the mutated promoters were subjected to the luciferase assays. As shown in Fig.4, introduction of mutation in the PU.1 binding motifs decreased the transcriptional activity to approximately half of the original promoter. In addition, introduction of mutation in the Sp1 binding motif resulted in significant decrease in the pro-
In this article, we identified PU.1 and Sp1 as essential transcriptional factors for the expression of the Tec molecule in the hematopoietic cells. PU.1 belongs to the Ets family transcription factors and was originally isolated from the integration site in murine erythroleukemia induced by the spleen focus forming virus (SSFV) (23). Given that PU.1 is restrictedly expressed in hematopoietic tissues (24-26), PU.1 is regarded as an important transcription factor for genes preferentially expressed in hematopoietic lineages. Indeed, subsequent studies demonstrated that PU.1 plays an essential role for genes including immunoglobulin heavy and light chains, macrophage colonystimulating factor promoter, and CD11b promoter (2731). Furthermore, a direct evidence of the biological significance of PU.1 was provided by targeting the PU.1 gene in mice (32). One of the phenotypic characteristics of PU.1-deficient mice is the absence of myeloid/ lymphoid progenitor cells. This finding strongly suggests that PU.1 functions at a level of precursor cells committed to myeloid and lymphoid lineages. Considering that tec is abundantly expressed in hematopoietic progenitor cells of the same developmental stage, it is reasonable that PU.1 plays a critical role for Tec expression. Sp1 was initially identified through its ability initiating transcription from SV40 promoter in Hela cells (33). Sp1 binds to G/C-rich box that is frequently observed in TATA-less promoters. Since tec promoter does not have TATA motif (13), it is likely that Sp1 plays as a transcriptional factor for tec gene. In contrast to PU.1, Sp1 is rather ubiquitously expressed and is considered to contribute to a basal transcriptional activity. However, in some cases, Sp1 is reported to play a role in tissue-specific expression of the target genes, such as CD11b (34). Therefore, Sp1 as well as PU.1 might contribute to the preferential expression of the Tec molecule in the hematopoietic cells. Recently, the analysis of the Btk promoter was reported (35). Interestingly, as observed in the case of tec promoter, PU.1 and Sp1 were demonstrated to be critical for the transcriptional activity of the btk promoter. Therefore, the cooperation of PU.1 and Sp1 might play a role in the preferential expression of Tec family PTKs in the hematopoietic tissues. However, Btk is mainly expressed in B-lymphoid cells, while Tec is predominantly expressed in myeloid cells. Thus, other transcription factors that bind to more upstream
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region(s) of the promoter or to introns might account for the lineage-preferential expression of Tec-family PTKs. To further investigate whether the promoter is functionally active in vivo, we fused the longest form of the promoter (01948 to /22) to p210bcr/abl, a chimeric oncogene responsible for causing human chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) (36, 37), and generated transgenic mice expressing p210bcr/abl driven by the tec promoter. The transgenic mice developed CML- and ALL-like diseases (manuscript in preparation), providing direct evidence that the cloned tec promoter is functional in hematopoietic progenitor cells, presumably in stem cell level. In summary, we described the identification, characterization, and functional analysis of mouse tec promoter. The findings will help us to understand the precise transcriptional mechanism of the Tec molecule, which is highly expressed in hematopoietic lineages. In addition, the promoter shown in this report would be useful and applicable for expressing a gene of interest in vivo, especially in hematopoietic precursor cells. ACKNOWLEDGMENTS We thank Dr. Toshio Inaba for the technical assistance of the gelshift and supershift assays. This work was supported in part by Grants-in-Aids from the Ministry of Education, Science and Culture of Japan.
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