PU.1 Activates Transcription of SHP-1 Gene in Hematopoietic Cells
2007; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês
10.1074/jbc.m607526200
ISSN1083-351X
AutoresPaweł Włodarski, Qian Zhang, Xiaobin Liu, Monika Kasprzycka, Michał Marzec, Mariusz A. Wasik,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoProtein-tyrosine phosphatase SHP-1 is the key negative regulator of numerous signaling pathways. SHP-1 is expressed in the hematopietic and epithelial cells as two structurally similar mRNA transcripts controlled by two different promoters designated P2 and P1, respectively. Whereas the transcriptional regulation of the SHP-1 gene P1 promoter has been partially elucidated, the structure and functional control of the P2 promoter remain unknown despite the critical role played by SHP-1 in the normal and malignant lymphoid and other hematopoetic cells. Using luciferase reporter assays with the set of constructs that contained a gradually truncated intron 1 of the SHP-1 gene, we identified the minimal (<120 bp) fragment that is able to fully activate expression of the reporter gene. Furthermore, we found that PU.1 (a member of the Ets transcription factor family that plays a crucial role in differentiation and function of the lymphoid and myeloid cells) binds to the identified P2 promoter both in vitro and in vivo. PU.1 also activates the promoter in the sequence specific manner and is critical for its expression as evidenced by the profound supression of the SHP-1 gene transcription upon the siRNA-mediated depletion of PU.1. These findings provide an insight into the structure of the hematopoietic cell-specific P2 promoter of the SHP-1 gene and identify PU.1 as the transcriptional activator of the P2 promoter. Protein-tyrosine phosphatase SHP-1 is the key negative regulator of numerous signaling pathways. SHP-1 is expressed in the hematopietic and epithelial cells as two structurally similar mRNA transcripts controlled by two different promoters designated P2 and P1, respectively. Whereas the transcriptional regulation of the SHP-1 gene P1 promoter has been partially elucidated, the structure and functional control of the P2 promoter remain unknown despite the critical role played by SHP-1 in the normal and malignant lymphoid and other hematopoetic cells. Using luciferase reporter assays with the set of constructs that contained a gradually truncated intron 1 of the SHP-1 gene, we identified the minimal (<120 bp) fragment that is able to fully activate expression of the reporter gene. Furthermore, we found that PU.1 (a member of the Ets transcription factor family that plays a crucial role in differentiation and function of the lymphoid and myeloid cells) binds to the identified P2 promoter both in vitro and in vivo. PU.1 also activates the promoter in the sequence specific manner and is critical for its expression as evidenced by the profound supression of the SHP-1 gene transcription upon the siRNA-mediated depletion of PU.1. These findings provide an insight into the structure of the hematopoietic cell-specific P2 promoter of the SHP-1 gene and identify PU.1 as the transcriptional activator of the P2 promoter. SHP-1 tyrosine phosphatase (also known as SHP1-PTP, SH-PTP1, SHP-1C, PTP1C, Hep, HepH, HPTP1C, and HCP) is encoded by the PTPN6 gene and expressed primarily in the hematopoietic and epithelial cells (1Shen S.H. Bastien L. Posner B.I. Chretien P. Nature. 1991; 352: 736-739Crossref PubMed Scopus (336) Google Scholar, 2Yi T.L. Cleveland J.L. Ihle J.N. Mol. Cell. Biol. 1992; 12: 836-846Crossref PubMed Scopus (306) Google Scholar). SHP-1 plays a particularly important role in the maturation and functional differentiation of lymphoid and myeloid cells as evidenced by the aberrant immunoproliferation and impaired hematopoiesis in the “motheaten” mice that display defects in the SHP-1 gene expression (3Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (685) Google Scholar, 4Tsui H.W. Siminovitch K.A. de Souza L. Tsui F.W. Nat. Genet. 1993; 4: 124-129Crossref PubMed Scopus (512) Google Scholar). SHP-1 acts in the immune and other hematopoietic cells by inhibiting signaling through receptors for cytokines, growth factors, and chemokines as well as receptors directly involved in the immune responses and programmed cell death (5Wu C. Sun M. Liu L. Zhou G.W. Gene (Amst.). 2003; 306: 1-12Crossref PubMed Scopus (239) Google Scholar). SHP-1 down-regulates cell activation by binding and de-phosphorylating the receptors, receptor associated tyrosine kinases, and the down-stream signaling molecules such as Vav1 (6Stebbins C.C. Watzl C. Billadeau D.D. Leibson P.J. Burshtyn D.N. Long E.O. Mol. Cell. Biol. 2003; 23: 6291-6299Crossref PubMed Scopus (207) Google Scholar) and src kinase substrates (7Frank C. Burkhardt C. Imhof D. Ringel J. Zchornig O. Wieligmann K. Zacharias M. Bohmer F.D. J. Biol. Chem. 2004; 279: 11375-11383Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). SHP-1 acts as tumor suppressor and loss of its expression has been identified in the whole spectrum of lymphoid and myeloid cell malignancies (8Zhang Q. Raghunath P.N. Vonderheid E. Odum N. Wasik M.A. Am. J. Pathol. 2000; 157: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Oka T. Ouchida M. Koyama M. Ogama Y. Takada S. Nakatani Y. Tanaka T. Yoshino T. Hayashi K. Ohara N. Kondo E. Takahashi K. Tsuchiyama J. Tanimoto M. Shimizu K. Akagi T. Cancer Res. 2002; 62: 6390-6394PubMed Google Scholar, 10Chim C.S. Fung T.K. Cheung W.C. Liang R. Kwong Y.L. Blood. 2004; 103: 4630-4635Crossref PubMed Scopus (201) Google Scholar, 11Mena-Duran A.V. Togo S.H. Bazhenova L. Cervera J. Bethel K. Senent M.L. Nieva J. Sanz M.A. Saven A. Mustelin T. Br. J. Haematol. 2005; 129: 791-794Crossref PubMed Scopus (20) Google Scholar). The SHP-1 gene, located on chromosome 12p13, is comprised of 17 exons and activated from two different promoters (5Wu C. Sun M. Liu L. Zhou G.W. Gene (Amst.). 2003; 306: 1-12Crossref PubMed Scopus (239) Google Scholar). Whereas the distal promoter, P1, located upstream of the very short exon 1 (sometimes designated also exon 1a) is active in epithelial cells, the proximal promoter P2 that initiates gene transcription from exon 2 (alternatively known as exon 1b), is utilized by the hematopoietic cells. Whereas function of the P1 promoter has been partially elucidated (12Tsui H.W. Hasselblatt K. Martin A. Mok S.C. Tsui F.W. Eur. J. Biochem. 2002; 269: 3057-3064Crossref PubMed Scopus (33) Google Scholar), the structure and regulatory mechanisms of the P2 promoter remain essentially unknown. Using a series of reporter assays, we identified the minimal, <120-bp-long, P2 promoter located immediately upstream of the exon 2 that is capable of driving full expression of the reporter luciferase gene. By using the combination of “in silico” analysis, electromobility shift assay (EMSA), 2The abbreviations used are: EMSA, electromobility shift assay; ChIP, chromatin immunoprecipitation; ALK, anaplastic lymphoma kinase; PMSF, phenylmethylsulfonyl fluoride; pcv, volume of pelleted cells; pnv, volume of pelleted nuclei; siRNA, small interfering RNA; RT, reverse transcription.2The abbreviations used are: EMSA, electromobility shift assay; ChIP, chromatin immunoprecipitation; ALK, anaplastic lymphoma kinase; PMSF, phenylmethylsulfonyl fluoride; pcv, volume of pelleted cells; pnv, volume of pelleted nuclei; siRNA, small interfering RNA; RT, reverse transcription. chromatin immunoprecipitation (ChIP), and siRNA-based depletion experiments, we identified PU.1, the member of the Ets factor family important for lymphoid and myeloid cell maturation, as the transcriptional activator of the P2 promoter. This new insight into the structure and regulation of the P2 promoter expression may contribute to a better understanding of the role played by PU.1 and SHP-1 in normal and malignant hematopoietic cells. Cells—Immature T-cell, acute leukemia/lymphoblastic lymphoma-derived Jurkat, mature T-cell, anaplastic lymphoma kinase (ALK)-expressing SUP-M2 and Karpas 299, mature B-cell, Burkitt’s lymphoma-derived BJAB and diffuse large B-cell lymphoma-derived LY18 and VAL, and the breast carcinoma-derived MDA-453 cell lines were cultured in the RPMI medium supplemented with 2 mm l-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (all from Invitrogen) and 10% fetal bovine serum (Cellgro/Mediatech Inc., Herndon, VA). The cells were maintained in 5% CO2 atmosphere at 37 °C. At the time of transfection the cells were cultured in the serum-free Opti-MEM medium (Invitrogen). Reporter Constructs—Fragments of intron 1 and exon 2 were amplified by PCR using specific primers and the Expand High Fidelity PCR System (Roche Applied Bioscience). The forward primers recognized sites located -6266 bp, -5266 bp, -4266 bp, -3266 bp, -2266 bp, -1266 bp, -266 bp, and -66 bp upstream from the transcription initiation site, whereas the reverse primer was common and included the nucleotide 97 (+97) downstream form the site. Later, additional fragments starting at -1066 bp, -876 bp, -766 bp, -266 bp, and -166 bp were generated in the same manner. All forward primers contained KpnI restriction site in their 5′-end. The HindIII restriction site was included in the reverse primer. PCR products were first cloned into the pCR4.0 vector (Invitrogen), then subcloned with KpnI and HindIII into respective sites of pGL3Enh plasmid. The resulting constructs were named according to the nucleotide location of the 5′-end of the forward primer used in the PCR reaction. Site-directed Mutagesis—To analyze the functional role of the identified transcription factor binding site in the SHP-1 gene P2 promoter, the selected PU.1b site was mutated by QuikChange® II site-directed mutagenesis kits (Stratagene, La Jolla, CA) according to the product manual. pGL3Enh -266 +97 construct was used as a template. Plasmids obtained by site directed mutagenesis were purified with a QIAprep Spin Miniprep kit (Qiagen Inc., Valencia, CA) and sequenced to confirm the presence of the mutation. Primers used for the mutagenesis reactions were as follows: 5′-GTG CTC TAA AAC ACA CTA CAA GTG AGT TCC CCC (forward) and 5′-GGG GGA ACT CAC TTG TAG TGT GTT TTA GAG CAC (reverse; with the mutated sequence being underlined). Reporter Assays—Jurkat cells were transiently co-transfected with fragments of SHP-1 gene P2 promoter cloned into pGL3Enh vector and pRL-TK vector (both vectors were from Promega, Madison, WI) using 10:1 molar ratios of these DNA, respectively. All transfections were performed in triplicates. After 24 h, the cells were lysed in Passive Lysis Buffer (Promega). Activity of the firefly and Renilla luciferases was evaluated in 20 μl of the cell lysate using the reagents included in the Dual-luciferase Reporter Assay System (Promega) and the Monolight 2010 luminometer (Analytical Luminiscence Laboratory, San Diego, CA). The results are presented as the activity ratio of firefly luciferase activity induced by the pGL3-SHP-1-promoter to the activity of the Renilla luciferase induced by the constitutively active pRL-TK vector. Nuclear Protein Extraction—Cells were collected by centrifugation and washed with phosphate-buffered saline. Volume of the pelleted cells (pcv) was measured using the graduation on the tube. The cells were next, briefly washed in a hypotonic buffer (10 mm HEPES, 1.5 mm MgCl2, 10 mm KCl, 0.2 mm PMSF, 0.5 mm dithiothreitol) with the volume of the buffer being equal to 5 pcv and centrifuged. The pellets were suspended in a 3-fold pcv volume of hypotonic buffer, incubated for 10 min on ice, transferred into a precooled Dounce homogenizer, and disrupted with a type B pestle. Nuclei were pelleted by centrifugation (15 min, 3300 × g), and their volume (pnv) was determined. Nuclei were then suspended in a 0.5 pnv of the low salt buffer (20 mm HEPES, 25% glycerol, 1.5 mm MgCl2, 0.02 m KCl, 0.2 mm EDTA, 0.2 mm PMSF, 0.5 dithiothreitol). Next, 0.5 pnv of the high salt buffer (20 mm HEPES, 25% glycerol, 1.5 mm MgCl2, 1.2 m KCl, 0.2 mm EDTA, 0.2 mm PMSF, 0.5 dithiothreitol) was added for a 30-min incubation with continuous, gentle mixing. The extracts were cleared by centrifugation (30 min, 25,000 × g). EMSA—The nuclear extracts were prepared as described above. Twenty fmol of biotin-labeled probe was incubated with 6.0 μg of the nuclear extract for 20 min at room temperature. To confirm the specificity of binding, the extract-probe mixtures were either further admixed with a 200-fold excess of the unlabeled, consensus nucleotide or 2 μg of the PU.1-specific antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The formed protein-DNA complexes were resolved from the free probe in the 7% non-denaturing polyacrylamide gel by electrophoresis, transferred onto positively charged Nylon membrane (Amersham Biosciences) and UV-cross-linked (Stratalinker, Stratagene). The membrane was then subjected to a Lightshift chemiluminescent EMSA kit (Pierce) following the manufacturer’s instructions. The nucleotides were custom synthesized by IDT Inc. (Coralville, IA). Probes used in the electrophoretic mobility shift assay were double-stranded biotin-labeled nucleotides. The sequences of the sense strands, 5′-biotinated, were as follows: 5′-GCT CTG CTT CTC TTC CCC (PU.1a/Ets2) and 5′-TAA AAC GAG AAG TAC AAG (PU.1b). The reverse complementary and unlabeled nucleotides were coupled to form double-stranded probes. Competitor unlabeled, double-stranded oligonucleotides (PU.1a and PU.1b cold probes) were generated in the same manner. ChIP Assay—To cross-link DNA and DNA-binding proteins, 1 × 106 of the Jurkat and BJAB cells were fixed with 1% formaldehyde at room temperature for 15 min and then at 4 °C for 45 min. The fixed cells were lysed with the buffer containing protease inhibitors (1% SDS, 10 mm EDTA, 50 mm Tris, pH 8.1, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 mm PMSF). The lysate was sonicated on ice to shear the DNA to fragments of about 500 bp. The resulting DNA-protein preparation was incubated with the antibody against PU.1 (Santa Cruz Biotechnology) or with the control NPM/ALK antibody (BD Biosciences) at 4 °C for 12 h. The DNA-protein-antibody complexes were precipitated with protein A-agarose beads, washed, and treated with RNase A and protease K. The DNA was recovered by purification using phenol/chloroform and ethanol precipitation. Next, the DNA was PCR-amplified in 25 cycles (20 s at 94 °C, 20 s at 55 °C, and 20 s at 72 °C). Products of the reaction were analyzed in 2% agarose gel stained with EtBr. Primers used in the reaction permitted amplification of either the -166 to -59 fragment of the P2 promoter (5′-GCCTTTGATTGCAGACGTG and 5′-ACTCACTTGTACTTCTCGT) or exon 16 (5′-CTGACCCTGTGGAAGCATTT and 5′-AGTGATCCCAGGGCTTTATTT) of the SHP-1 gene. siRNA-mediated RNA Depletion—Jurkat and BJAB cells were treated with siRNA directed against PU.1 or non-targeting control siRNA twice at 24 h intervals. Mixtures of four PU.1-specific siRNAs or non-targeting siRNAs (both mixtures were purchased from Dharmacon Inc, Chicago, IL) were transfected into cells using DMRIE-C liposomal reagent (Invitrogen) according to the manufacturer’s protocol. Briefly, 1 ml of Opti-MEM medium (Invitrogen) containing a 100 nm concentration of the respective siRNA mixture was mixed with 16 μl of DMI-RIE-C and incubated for 30 min at room temperature. One ml of the resulting RNA-lipid complexes was added to 1 ml of the cells seeded at 5 × 105/1 ml of the Opti-MEM medium. The cells were then incubated for 4–5 h at 37 °C in 5% CO2 atmosphere, and the medium was changed to the complete RPMI medium. The procedure was repeated at 24 h, and the cells were harvested 72 h after the initial transfection. Efficiency of the PU.1 depletion was assessed by Western blotting, using expression of β-actin as an internal control. RT-PCR—Total RNA was isolated using RNeasy mini kit (Qiagen) according to the manufacturer’s instruction. Purified RNA was treated with DNase I (Invitrogen), and the reverse transcription was carried out using Thermoscript RT-PCR system (Invitrogen) with random hexamers as the cDNA synthesis primers. PCR was performed with Platinum TaqDNA polymerase (Invitrogen) over 25 (for β-actin) 30 (for SHP-1) or 40 (for SHP-1(I) and SHP-1(II)) cycles: 20 s at 94 °C, 30 s at 58 °C (β-actin SHP-1(I), SHP-1(II)) or 55 °C (SHP-1), and 30 s at 72 °C. The PCR products were visualized by EtBr staining in the 2% agarose gel. The primers used in PCR were: β-actin, 5′-ACCATTGGCAATGAGCGGT (forward) and 5′-GTCTTTGCGGATGTCCACGT (reverse), SHP-1, 5′-GTGACCCATATTCGGATCCAG (forward) and 5′-CTTGAAATGCTCCACCAGGTC (reverse); SHP-1(I), 5′-CACAGCTGTGCCGCTGGCTCA (forward) and 5′-GTGAGTCTGTCCATCGCGA (reverse); and SHP-1(II), 5′-CTGCCTGCCCAGACTAGCTG (forward) and 5′-GTGAGTCTGTCCATCGCGA (reverse). Western Blot—For assessment of the protein expression Jurkat and BJAB cells were lysed with RIPA buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm EDTA, 0.1% SDS) with 50 mm sodium fluoride and 0.5 mm sodium orthovanadate and supplemented with 1× Complete protease inhibitor mixture (Roche Applied Bioscience) and phosphatase inhibitor mixture I (Sigma-Aldrich). Solubilized protein extract was cleared by centrifugation. For normalization of the protein loading, the extracts were assayed by Lowry method (Dc protein assay, Bio-Rad). Typically, 20 μg of protein per lane were subjected to the SDS-PAGE in a 10% polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. After blocking with the 5% nonfat milk solution in TBST (20 mm Tris, pH 7.5, 0.15 m NaCl, 0.1% Tween 20), the blots were probed with the primary and secondary (horseradish peroxidase-conjugated) antibodies. The primary antibodies used were against PU.1, SHP-1, β-actin, and phopshotyrosine 4G10 (all from Santa Cruz Biotechnology). Membranes were washed in TBST buffer, and proteins of interest were detected by ECL Plus chemiluminescence substrate (Amersham Biosciences). In some experiments, intensity of the signal was measured using scanner HP ScanJet3500c and ImageJ 1.32j software developed at the National Institute of Health, Bethesda, MD. Identification of the P2 Promoter of the SHP-1 Gene—As shown in Fig. 1A, exons 1 and 2 of the SHP-1 gene are separated by an intron that contains almost 6300 bp, which is believed to contain the hematopoietic cell-specific promoter P2. We have confirmed the presence of the P2 promoter in the intron 1 by demonstrating that, in contrast to the epithelial MDA-453 cells, the immature Jurkat T lymphocytes and mature BJAB B lymphocytes express SHP-1 mRNA encoded in part by exon 2 but not exon 1 (Fig. 1B). To determine the exact localization and size of P2, we developed a gene expression reporter system comprised of the promoterless vector pGL3Enh containing fragments of the intron 1 cloned in upstream of the luciferase gene. The constructed twelve fragments varied in length from over 6300 bp that cover essentially the entire intron 1 to ∼160 bp (Fig. 2A). They all contained the transcription initiation site (+1) and the adjacent, 97-bp-long fragment of the coding DNA. The pGL3Enh vector that contained only the luciferase gene but no intronic DNA served as a negative control. As shown in Fig. 2A, most of the progressively truncated intron 1 fragments analyzed displayed a very similar, strong transcriptional activity. The only exceptions that generated much weaker signals in a highly reproducible manner were the second longest (-5266 +97) and the shortest (-66 +97) fragments. Whereas the result with the longer fragment suggests the existence of a long range transcriptional repressor, the result with the shortest fragment, in particular when compared with the intact, full transcriptional activity of the second shortest fragment (-166 +97), indicated that the P2 promoter is rather small and located nearby the transcription initiation site. To determine the more exact size of the promoter, we prepared the second set of the intron 1 fragments starting at -141, -116, and -91. The additional fragments starting at -1066 and -166 served as positive controls, and the fragment starting at -66 and the intronless vector served again as negative controls. No fragment that was shorter than the one starting at -66 was prepared based on the premise that the region immediately upstream of the transcription initiation site contains the core promoter that binds the RNA polymerase complex (please see also below). As shown in Fig. 2B, transcriptional activity of the -141 +97 and -116 +97 regions was not significantly diminished when compared with the positive controls. In contrast, the activity of -91 +97 fragment was decreased but still much stronger than that of the -66 +97 core promoter indicating that important regulatory element(s) is (are) contained within the short -116 to -66 region.FIGURE 2Structural delineation of the SHP-1 gene P2 promoter. Progressively truncated fragments of intron 1 of the SHP-1 gene were inserted into the luciferase gene-containing pGL3Enh vector and examined for their ability to activate transcription of the reporter gene in the lymphoid Jurkat cells. As depicted in the figure, the fragments varied in length with the longest covering essentially the entire intron 1 (-6266 +97) and the shortest (-66 +97) containing the RNA polymerase binding complex region located immediately upstream of the transcription initiation site. The construct designated as “promoterless” represents the pGL3Enh vector containing only the luciferase gene but no promoter. The results reflect the calculated ratios of the luciferase activity as compared with activity of the co-transfected renilla gene-containing vector. The depicted data (mean value ± S.E.) are representative of three totally separate experiments performed in triplicates. A, rough characterization of the P2 promoter position using the large, gradually truncated DNA fragments of the intron 1. B, refined analysis of the P2 location and size using the smaller, progressively truncated fragments with the largest one stating at the position -166 bp upstream of the transcription initiation site.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mapping of the Putative Transcription Activator Binding Sites within the SHP-1 Gene P2 Promoter—We next addressed the question of which transcription factor(s) may be involved in activation of the P2 promoter. To accomplish this aim, we performed an “in silico” structural analysis of the identified P2 promoter for potential transcription factor binding sites. Using the transcription element search software, we identified within the key -116 to -66 region three sequences specific for two members of the Ets transcription factor family with two sites potentially binding PU.1 (13O'Prey J. Ramsay S. Chambers I. Harrison P.R. Mol. Cell. Biol. 1993; 13: 6290-6303Crossref PubMed Scopus (44) Google Scholar) and one Ets-2 (14Wen S.C. Ku D.H. De Luca A. Claudio P.P. Giordano A. Calabretta B. Exp. Cell Res. 1995; 217: 8-14Crossref PubMed Scopus (30) Google Scholar). It is noteworthy that in contrast to the well established critical role of PU.1 in normal and malignant hematopoiesis (15Rosmarin A.G. Yang Z. Resendes K.K. Exp. Hematol. 2005; 33: 131-143Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 16Hagman J. Lukin K. Curr. Opin. Immunol. 2006; 18: 127-134Crossref PubMed Scopus (68) Google Scholar), the potential involvement of Ets-2 in the process is less well documented (17Oh I.H. Reddy E.P. J. Biol. Chem. 1997; 272: 21432-21443Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 18Ratajczak M.Z. Perrotti D. Melotti P. Powzaniuk M. Calabretta B. Onodera K. Kregenow D.A. Machalinski B. Gewirtz A.M. Blood. 1998; 91: 1934-1946Crossref PubMed Google Scholar) and seemingly controversial (19Henkel G.W. McKercher S.R. Yamamoto H. Anderson K.L. Oshima R.G. Maki R.A. Blood. 1996; 88: 2917-2926Crossref PubMed Google Scholar). As shown in Fig. 3A, the potential PU.1 binding sites, designated PU.1a and PU.1b, are located at positions -103 -98 and -75 -70, respectively. The hypothetical Ets-2 site is located at -97 -93, directly adjacent to the PU.1a site. The most proximal region of the P2 promoter contained the BRE-TFIIB and INR-TFIID binding sites consistent with its role as the core promoter responsible for docking the RNA polymerase complex involved in the initiation of gene transcription. Expression and Binding of PU.1 to the P2 Promoter—To confirm the expression of PU.1 by the Jurkat cells, we performed Western blot analysis. As shown in Fig. 3B, these immature T-cell line expressed substantial amount of the PU.1 protein comparable with the one seen in the mature B-cell line BJAB and, to a lesser degree, two other mature B-cell lines examined. In contrast, two mature T-cell lines did not express the protein. To determine whether PU.1 binds to the identified PU.1a and PU.1b sites within the P2 promoter, nuclear protein extracts from the lymphoid Jurkat cells were evaluated in EMSA for binding the biotin-labeled probes corresponding to the sites (with the PU.1a probe containing also the putative Ets-2 binding site). Unlabeled PU.1a and PU.1b probes served as controls of the binding specificity. As can be seen in Fig. 3C (the left lanes in both panels), each of the labeled PU.1 probes generated single, distinct band. These unique bands were eliminated when the unlabeled “cold” PU.1 probe was added (the middle lanes). To confirm that the PU.1a and PU.1b sites indeed bind PU.1 and not Ets-2 or another transcription factor possibly present in the nuclear extract, we performed a supershift EMSA using a PU.1-specific antibody. As can be seen (the right lanes), addition of the antibody impeded migration of the entire oligonucleotide probe-protein complexes confirming identity of PU.1 as the nuclear factor binding to both identified sites within the SHP-1 gene P2 promoter. PU.1 Binds and Activates SHP-1 Gene P2 Promoter in Vivo—To demonstrate that PU.1 binds to the P2 promoter also in vivo, we performed a ChIP assay using the PU-1-specific antibody as well as an irrelevant (anti-ALK tyrosine kinase) antibody that served as a negative control. Immunoprecipitation of the protein extracts obtained from Jurkat and another lymphoid cell line BJAB was followed by PCR using a set of primers specific for the -166 -59 P2 promoter region with the centrally located -103 -98 PU.1a and -75 -70 PU.1b binding sites. Another set of primers specific for the distal region of the SHP-1 gene coding for exon 16 (5Wu C. Sun M. Liu L. Zhou G.W. Gene (Amst.). 2003; 306: 1-12Crossref PubMed Scopus (239) Google Scholar) served as a control. Whereas both sets of primers yielded strong PCR bands from the whole cell lysate (input), a strong band could be obtained from the PU.1 antibody precipitate using only the primers reacting with the P2 promoter but not the primers that recognize exon 16 (Fig. 3A). No band was generated from the precipitate obtained with the control anti-ALK antibody confirming specificity of the PU.1 binding. To provide evidence that the identified PU.1 binding sites are transcriptionally active, we performed a luciferase reporter assay with the P2 promoter constructs that contained mutation of the PU.1a or PU.1b binding site. To preserve the overall structure and spatial relations of the site, we performed substitutions rather than deletions of the target binding site nucleotides leaving intact the remaining sequence of the examined -266 + 97 fragment. As shown in Fig. 4B, the targeted six-base substitution of the PU.1a binding site markedly diminished transcriptional activity of the entire fragment. The effect of substitution of the PU1.b site was even more profound and decreased the transcriptional activity almost to the baseline level of the RNA polymerase-binding core promoter. The combination of both PU.1a and PU.1b binding site substitutions had no additional inhibitory effect. These findings indicate that PU.1 is the transcriptional activator of the P2 promoter. PU.1-dependent Transcription of the SHP-1 Gene—To demonstrate that PU.1 binding to the P2 promoter and activation of the promoter as documented in the luciferase gene reporter assay also impacts on the expression of the endogeneous, native SHP-1 gene, we inhibited expression of PU.1 using the siRNA technology. Treatment of both Jurkat and BJAB cells with the PU.1-specific but not the control, non-targetting siRNA markedly diminished expression of PU.1 in these cells. The inhibition of PU.1 expression resulted in the profound suppression of transcriptional activity of the SHP-1 gene as determined by marked decrease in the amount of the SHP-1 mRNA (Fig. 5A) and protein (Fig. 5B). These observations provided the final evidence for the key role of PU.1 in activation of the SHP-1 gene. Of note, the PU.1 depletion and the related down-regulation of SHP-1 expression were associated with the substantial increase in tyrosine phosphorylation of several intracellular proteins (Fig. 5B) in agreement with the well defined role of SHP-1 as the tyrosine phosphatase of a number of key proteins in the in the hematopoietic cells (1Shen S.H. Bastien L. Posner B.I. Chretien P. Nature. 1991; 352: 736-739Crossref PubMed Scopus (336) Google Scholar, 2Yi T.L. Cleveland J.L. Ihle J.N. Mol. Cell. Biol. 1992; 12: 836-846Crossref PubMed Scopus (306) Google Scholar, 3Shultz L.D. Schweitzer P.A. Rajan T.V. Yi T. Ihle J.N. Matthews R.J. Thomas M.L. Beier D.R. Cell. 1993; 73: 1445-1454Abstract Full Text PDF PubMed Scopus (685) Google Scholar, 4Tsui H.W. Siminovitch K.A. de Souza L. Tsui F.W. Nat. Genet. 1993; 4: 124-129Crossref PubMed Scopus (512) Google Scholar, 5Wu C. Sun M. Liu L. Zhou G.W. Gene (Amst.). 2003; 306: 1-12Crossref PubMed Scopus (239) Google Scholar, 6Stebbins C.C. Watzl C. Billadeau D.D. Leibson P.J. Bursht
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