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• W2014165340 abstract "The glycoprotein gp91 phox is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91 phox gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91 phox promoter in various types of gp91 phox -expressing cells. A 10-base pair (bp) region from bp −105 to −96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp −101 to −96 (−98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the −98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the −105/+12 construct in a −98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91 phox gene in human eosinophils. The glycoprotein gp91 phox is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91 phox gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91 phox promoter in various types of gp91 phox -expressing cells. A 10-base pair (bp) region from bp −105 to −96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp −101 to −96 (−98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the −98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the −105/+12 construct in a −98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91 phox gene in human eosinophils. base pair(s) CCAAT displacement protein major basic protein electrophoretic mobility gel shift assay CCAAT/enhancer-binding protein 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid cAMP responsive element-binding protein-binding protein The large subunit of flavocytochrome b 558(gp91 phox) is an essential component of the phagocyte NADPH oxidase, which produces superoxide anion to kill parasites and microorganisms. Mutations in the gp91 phox gene such as deletions (1.Kumatori A. Faizunnessa N.N. Suzuki S. Moriuchi T. Kurozumi H. Nakamura M. Genomics. 1998; 53: 123-128Crossref PubMed Scopus (27) Google Scholar) and substitutions (2.Ariga T. Sakiyama Y. Furuta H. Matsumoto S. Eur. J. Haematol. 1994; 52: 99-102Crossref PubMed Scopus (18) Google Scholar) result in X-linked chronic granulomatous disease, which is characterized by severe recurrent infections due to the lack of superoxide generation (3.Tauber A.I. Borregaard N. Simons E. Wright J. Medicine (Baltimore). 1983; 62: 286-309Crossref PubMed Scopus (172) Google Scholar). The gp91 phox gene is expressed in terminally differentiated phagocytes and B-lymphocytes (4.Segal A.W. Garcia R. Goldstone H. Cross A.R. Jones O.T. Biochem. J. 1981; 196: 363-367Crossref PubMed Scopus (73) Google Scholar, 5.Kobayashi S. Imajoh O.S. Nakamura M. Kanegasaki S. Blood. 1990; 75: 458-461Crossref PubMed Google Scholar), indicating the expression of the gene to be both lineage- and differentiation stage-specific. Several transcription factors regulate gp91 phox gene expression through cis-elements in a 450-base pair (bp)1 sequence in the 5′-flanking region of the gene. A CCAAT displacement protein (CDP/cut) (6.Blochlinger K. Bodmer R. Jack J. Jan L.Y. Jan Y.N. Nature. 1988; 333: 629-635Crossref PubMed Scopus (224) Google Scholar, 7.Neufeld E.J. Skalnik D.G. Lievens P.M. Orkin S.H. Nat. Genet. 1992; 1: 50-55Crossref PubMed Scopus (186) Google Scholar) suppresses the expression of the gp91 phox gene by binding to four sites at bp −350, −220, −150, and −110 of the gene in immature myeloid cell lines. Overexpression of CDP inhibits the induction of the gene in myeloid differentiation (8.Lievens P.M. Donady J.J. Tufarelli C. Neufeld E.J. J. Biol. Chem. 1995; 270: 12745-12750Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The down-regulation of its DNA binding activity during differentiation allows the expression of the gene (9.Skalnik D.G. Strauss E.C. Orkin S.H. J. Biol. Chem. 1991; 266: 16736-16744Abstract Full Text PDF PubMed Google Scholar, 10.Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). CP1, one of the CCAAT box-binding proteins, and BID proteins (bindingincreased during differentiation) are activators. However, these factors may work after bound CDPs are released because their binding sites overlap with sites for CDP (10.Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 18203-18210Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,11.Eklund E.A. Luo W. Skalnik D.G. J. Immunol. 1996; 157: 2418-2429PubMed Google Scholar). Interferon regulatory factor-2 may have dual functions as a transcriptional activator and repressor depending on binding sites in gp91 phox gene expression (12.Luo W. Skalnik D.G. J. Biol. Chem. 1996; 271: 23445-23451Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 13.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). TF1 phox ,a component of BID-2, displaces CDP and interferon regulatory factor-2, resulting in increased expression of the gp91 phox gene in the myeloid cell lines (13.Eklund E.A. Kakar R. J. Biol. Chem. 1997; 272: 9344-9355Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). We have shown that PU.1 is an essential activator for the expression of the gp91 phox gene in human neutrophils, monocytes, and B-lymphocytes (14.Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). Eklund et al. (15.Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) have proposed a cooperative activation of the gp91 phox gene by PU.1 and interferon regulatory factor-1 in myeloid cell lines. Eosinophils have an important role in the host defense against pathogenic parasites and microorganisms and produce superoxide anion as do other phagocytes (neutrophils, monocytes, and macrophages) and B-lymphocytes by means of NADPH oxidase activity (16.Yazdanbakhsh M. Tai P.C. Spry C.J. Gleich G.J. Roos D. J. Immunol. 1987; 138: 3443-3447PubMed Google Scholar). It has long been thought that the expression mechanism of gp91 phox in eosinophils is the same as that in other phagocytes. However, a restricted expression of gp91 phox in eosinophils from our X-linked chronic granulomatous disease patients (17.Kuribayashi F. Kumatori A. Suzuki S. Nakamura M. Matsumoto T. Tsuji Y. Biochem. Biophys. Res. Commun. 1995; 209: 146-152Crossref PubMed Scopus (28) Google Scholar) 2D. Roos, unpublished data. implied a certain eosinophil-specific expression mechanism of the gene. No positive regulatory mechanisms of the gene have, however, been shown in eosinophils, although an eosinophil-specific GATA-3 suppression mechanism of the gene was recently suggested by us (18.Sadat M.A. Kumatori A. Suzuki S. Yamaguchi Y. Tsuji Y. Nakamura M. FEBS Lett. 1998; 436: 390-394Crossref PubMed Scopus (14) Google Scholar). In this report, we show the eosinophil-specific regulation of the gp91 phox gene by transcription factors GATA-1 and GATA-2 through the GATA-binding site spanning bp −101 to −96 of the gene. To prepare a differentiated eosinophilic cell line (HL-60-C15E), HL-60-C15 cells (a kind gift from Dr. Y. Yamaguchi, Kumamoto University, Kumamoto, Japan), the eosinophil-committed subline of the promyelocytic cell line HL-60, were maintained for >3 months at pH 7.7 in RPMI 1640 medium supplemented with 10% fetal calf serum and 25 mm EPPS (Sigma). They were then treated with 0.5 mm n-butyrate (Sigma) for 5 days for differentiation into eosinophils (19.Fischkoff S.A. Leuk. Res. 1988; 12: 679-686Crossref PubMed Scopus (52) Google Scholar). HL-60 (Riken Cell Bank, Tsukuba, Japan), B-lymphocytic HS-Sultan (Japanese Cancer Center Research Resources Bank, Tokyo), and T-lymphocytic Jurkat (given by Dr. K. Furukawa, Nagoya University) cells were cultured at pH 7.2 in RPMI 1640 medium with 10% fetal calf serum. HL-60 cells were differentiated into neutrophils and monocytes by treatment with 1 μmall-trans-retinoic acid (Sigma) and 1 ng/ml recombinant human transforming growth factor-β1 (Roche Molecular Biochemicals, Tokyo) in combination with 0.1 μm vitamin D3 (1,25-dihydroxyvitamin D; a gift from Chugai Pharmaceutical Co., Ltd., Tokyo), respectively, for 3 days (20.Breitan T.R. Selonick S.E. Collions S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2936-2940Crossref PubMed Scopus (1858) Google Scholar, 41.Tesla U. Masciulli R. Tritarelli E. Pustorino R. Mariani G. Martucci R. Barberi T. Gamagna A. Valtieri M. Peschle C. J. Immunol. 1993; 150: 2418-2430PubMed Google Scholar). COS-7 cells were maintained in Eagle's minimal essential medium supplemented with 10% fetal calf serum. Cells (5 × 105) in 50 μl of phosphate-buffered saline containing 0.5% bovine serum albumin were stained by incubation with an appropriate amount of either one of the following monoclonal antibodies or isotype-matched immunoglobulins on ice for 60 min: R-phycoerythrin-conjugated anti-interleukin-5 receptor α chain (Pharmingen, San Diego, CA), fluorescein isothiocyanate-conjugated anti-CD19 (Pharmingen), and R-phycoerythrin-conjugated anti-CD14 (Dako Japan, Kyoto, Japan). These are lineage-specific to eosinophils, B-lymphocytes, and monocytes, respectively. Surface gp91 phox of the cells was stained as described above with fluorescein isothiocyanate-conjugated 7D5 (22.Yu L. Zhen L. Dinauer M.C. J. Biol. Chem. 1997; 272: 27288-27294Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). 3A. Yamauchi, unpublished data. These cells were washed twice with phosphate-buffered saline containing 0.5% bovine serum albumin, and their fluorescence intensities were analyzed on a FACScan (Becton Dickinson, La Jolla, CA). Total RNA was prepared from cells with Trizol LS reagent (Life Technologies, Inc.) according to the manufacturer's protocol. The RNA (10 μg/lane) was electrophoresed on formaldehyde-containing 0.9% agarose gels, transferred to HybondTM-N+ nylon membranes (Amersham Pharmacia Biotech, Tokyo), and fixed by ultraviolet light. Messenger RNAs were detected by hybridization with probes labeled with [32P]dCTP using a random primer labeling kit (Amersham Pharmacia Biotech). The following DNAs were used as probes for defining cell lineage: a 780-bp full-length cDNA of human major basic protein (MBP; a gift from Dr. I. Nagaoka, Juntendo University, Tokyo), a 725-bp full-length cDNA of human eosinophil cationic protein cloned by polymerase chain reaction, a 500-bp fragment of eosinophil peroxidase (kindly provided by Dr. Y. Yamaguchi), and a 285-bp cDNA for defensin (human neutrophil peptide-3; kindly supplied by Dr. I. Nagaoka). After hybridization patterns by these probes were visualized on a Molecular Imager FX (Bio-Rad), the membrane was washed to remove the first probes and hybridized with a 32P-labeled 800-bp cDNA of rat glyceraldehyde-3-phosphate dehydrogenase as the internal control of RNA loading. Plasmids pGV−986/Luc, pGV−301/Luc, and pGV−267/Luc, with promoter fragments of gp91 phox spanning from the corresponding bp positions to bp +12, were prepared by the exonuclease III deletion method (21.Henikoff S. Methods Enzymol. 1987; 155: 156-165Crossref PubMed Scopus (676) Google Scholar) from pGV−5635/Luc, which has the fragment of the gene extending from its initiation codon to the upstream bp −5635 at the NcoI site of the firefly luciferase gene in the pGV−B2 vector (Toyoink, Tokyo). For making deletion constructs p−267/Luc, p−115/Luc, p−105/Luc, p−95/Luc, and p−84/Luc, we first made fragments spanning from their corresponding positions to bp +12 by the polymerase chain reaction method using pGV−267/Luc as the template and sets of forward primers with KpnI sites and a reverse primer with a BamHI site. These polymerase chain reaction products were digested withKpnI and BamHI and subcloned into theKpnI- and BglII-digested pXP2N of firefly luciferase reporter gene vector, originating from pXP2 (a kind gift from Dr. Y. Yamaguchi). The order of the restriction sites in the multicloning site of pXP2N (5′-BamHI/KpnI/SmaI/SalI/HindII/XhoI/BglII-3′) is different from that of pXP2 (5′-BamHI/HindIII/SmaI/SalI/KpnI/XhoI/BglII-3′). To make p−986/Luc and p−301/Luc, fragments were cut out from pGV−986/Luc and pGV−301/Luc, respectively, at the KpnI sites of their multicloning site and the bp −137 HindIII site of the gp91 phox gene. The correspondingKpnI/HindIII fragment of the p−267/Luc reporter construct was replaced by either one of the above fragments. For making the mutant reporter plasmid p−105M/Luc, a two-point mutation (GA to CT) was introduced into the bp −100 to −99 sequence of p−105/Luc by polymerase chain reaction using a mutated primer. Each cDNA for human GATA-1 and GATA-2 was inserted into the pEF-MCIneo vector with a forward orientation (24.Visvader J.E. Elefanty A.G. Strasser A. Adams J.M. EMBO J. 1992; 11: 4557-4564Crossref PubMed Scopus (162) Google Scholar). For HL-60-C15E and other differentiated HL-60 cells, cells (5 × 106) were incubated with 20 μg of gp91 phox promoter/firefly luciferase plasmid, 10 μg of carrier Bluescript II KS plasmid, and 3 μg of herpes simplex virus thymidine kinase promoter/Renillaluciferase plasmid in 0.25 ml of 20 mm HEPES/RPMI 1640 medium (pH 7.2) at room temperature for 15 min and electroporated at 200 V for 70 ms on an ElectroSquarePorator T820 (BTX, Inc., San Diego, CA). HS-Sultan cells (5 × 106) were incubated with 10 μg of gp91 phox promoter firefly luciferase plasmid and 50 ng of cytomegalo virus promoterRenilla luciferase plasmid as described above, but electroporated with a Gene Pulser II (Bio-Rad) at 950 microfarads and 310 V. Jurkat cells (5 × 106) were electroporated as described for HS-Sultan cells, but in the presence of 5 μg of firefly luciferase plasmid and 20 ng ofRenilla luciferase plasmid at 950 microfarads and 280 V. After HL-60-C15E, HL-60, and HS-Sultan, Jurkat cells were incubated at 37 °C under 5% CO2 and 95% air for 6, 6, 10, and 24 h, respectively, their reporter activities were measured as described previously (14.Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). In the case of cotransfection experiments, various amounts of human GATA expression plasmids were transfected with 5 μg of reporter plasmid into 5 × 106 Jurkat cells. Prior to nuclear extraction, COS-7 cells (107) were transfected with 4 μg of GATA-1 or GATA-2 plasmid in 5 ml of Eagle's minimal essential medium by 20 nmol of L1-Liposome (kindly supplied by Dai-ichi Pharmaceutical Co., Ltd., Tokyo) for 8 h and cultured in 15 ml of 10% fetal calf serum/Eagle's minimal essential medium for 40 h. Preparation of nuclear protein extracts, labeling of the double-stranded oligonucleotides from bp −115 to −90 of the human gp91 phox gene for the probe, and EMSAs were performed as described previously (14.Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). Each reaction mixture (20 μl) containing 0.5–14 μg of nuclear protein extracts, 10,000 cpm of each radiolabeled probe equivalent to 1.16 fmol, and 1 μg of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech) in 20 mm HEPES (pH 7.9), 50 mm KCl, 1 mm MgCl2, 1 mm dithiothreitol, 0.2 mm EDTA, 0.01% Triton X-100, 5% glycerol, and 0.5 mm spermidine was incubated on ice for 15 min. In competition assays, a 300-molar excess of unlabeled competitor oligonucleotides was added prior to the addition of the probe to the mixture, which was then preincubated on ice for 15 min. For the inhibition assay with antibodies, an aliquot of nuclear extracts was incubated on ice with 2–4 μg of goat IgG against human GATA-1, murine IgG against human GATA-2, control goat IgG, or control murine IgG for 1 h before the addition of the probe. Both anti-GATA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Electrophoresis was performed on a native 6% acrylamide gel in 0.4× buffer containing 36 mm Tris, 36 mm boric acid, and 8 mm EDTA (pH 8.3). Upper-strand sequences for the probe and of competitors are as follows: sequence for the probe and of the wild-type competitor (bp −115 to −90 of the normal fragment of the gp91 phox gene), 5′-TATTAGCCAATTTCTGATAAAAGAAA-3′; GATA-binding site competitor of the human T-cell receptor δ gene (25.Redondo J.M. Hata S. Brocklehurst C. Krangel M.S. Science. 1990; 247: 1225-1229Crossref PubMed Scopus (82) Google Scholar), 5′-TTATTTAAGTCTTAGTTGTAG-3′; GATA site mutant of the wild-type competitor (−105M), 5′-AATTTCTCTTAAAAGAAAAGGAA-3′; and a heterologous competitor, 5′-CACAACCACATTCAACCTCTGCCACC-3′. The underlined sequence represents mutated bases. Apparent dissociation constants (K d) were determined according to the methods described by Merika and Orkin (26.Merika M. Orkin S.H. Mol. Cell. Biol. 1993; 13: 3999-4010Crossref PubMed Scopus (564) Google Scholar). The binding and running conditions were same as those described above. Each fixed amount of nuclear protein extract from COS-7 cells expressing GATA-1 (0.5 μg/20 μl) or GATA-2 (14 μg/20 μl) was incubated on ice with serially diluted labeled probe and incubated for 15 min, which was confirmed to be long enough to bring the reaction to equilibrium. Quantitation of free and bound DNAs was performed with a Molecular Imager FX. Scatchard plots for GATA-1 and GATA-2 were accomplished assuming front counts and specifically retarded counts to be proportional to free and bound concentrations, respectively, and also assuming their binding to one DNA duplex to be 1. Each value shown as the mean ± S.D. was obtained from three or more means of independent duplicates or triplicates. Values of p lower than 0.05 in mostly paired Student's t test were taken to be statistically significant. To analyze an eosinophil-specific cis-element of the gp91 phox promoter, we differentiated HL-60-C15 cells, an eosinophil-committed subline of the promyelocytic HL-60 leukemia cell line, by treatment with butyrate under alkaline pH. As shown in Fig.1, these treated cells, but not parental HL-60 cells, expressed mRNAs for eosinophil lineage-specific MBP, eosinophil cationic protein, and eosinophil peroxidase. These results support that the cells are phenotypically eosinophilic. We named the cells HL-60-C15E and used them in further experiments as differentiated human eosinophilic cells. Next, we transiently transfected the p−986/Luc construct, which contains the gp91 phox gene fragment from bp −986 to bp +12 in front of a luciferase reporter gene, into HL-60-C15E cells. As shown in Fig. 2, the p−986/Luc construct had a significantly higher reporter activity than the promoterless pXP2N construct. This result indicates that the bp −986 fragment contains positive regulatory elements for gp91 phox gene expression in eosinophilic cells. To clarify the elements, a series of progressive deletion mutants was generated, and the promoter activity of each was examined (Fig. 2). Deletions of sequences −986/−302 (between bp −986 and −301), −301/−116, and −115/−106 produced no significant changes in promoter activity (p−301/Luc, p−115/Luc, and p−105/Luc). However, a further 10-bp deletion from bp −105 to −96 significantly decreased promoter activity by 40% (p < 0.01; compare p−105/Luc and p−95/Luc). An additional 11-bp deletion resulted in a further decrease in activity (p < 0.01; compare p−95/Luc and p−84/Luc). These results suggest that positive regulatory elements exist in the regions from bp −105 to −96 and bp −95 to −85 of the gp91 phox promoter. To determine the lineage specificity of the first region from bp −105 to −96, we examined the relative promoter activity of p−105/Lucversus p−95/Luc in non-eosinophilic gp91 phox -expressing cells as well as eosinophilic HL-60-C15E cells (Fig. 3 A). The ratio of the promoter activity of p−105/Luc to that of p−95/Luc was significantly higher than 1.0 in HL-60-C15E cells (1.7 ± 0.19;p < 0.001), but not in neutrophilic HL-60, monocytic HL-60, or B-lymphocytic HS-Sultan cells. Therefore, at least one eosinophil-specific positive cis-element should exist in the region from bp −105 to −96 of the gp91 phox promoter. On the other hand, the −95/−84 region had no specificity for eosinophilic cells (data not shown). The lineage specificities of these gp91 phox -expressing cells were confirmed by particular expressions of CD14, CD19, and the interleukin-5 receptor on surfaces of transforming growth factor-β1/vitamin D3-treated HL-60, HS-Sultan, and HL-60-C15E cells, respectively, and the only significant expression of defensin mRNA in all-trans-retinoic acid-treated HL-60 cells (Fig.3 B). To determine cis-elements in the −105/−96 region of the gp91 phox promoter, we searched transcription factor-binding sites in the region. A sequence between bp −101 and −96 (5′-TGATAA-3′) perfectly matched with the GATA consensus sequence ((A/T)GATA(A/G)) (27.Evans T. Reitman M. Felsenfeld G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5976-5980Crossref PubMed Scopus (360) Google Scholar). To determine whether this putative GATA-binding site (−98GATA site) actually contributed to activation of the gp91 phox promoter by the −105/−96 region, we examined the effect of a GATA site mutation (GA to CT) that abolishes the ability to bind GATA proteins from a GATA site (26.Merika M. Orkin S.H. Mol. Cell. Biol. 1993; 13: 3999-4010Crossref PubMed Scopus (564) Google Scholar) on activation in HL-60-C15E cells. As shown in Fig. 4, the mutation completely abolished all the activity dependent on the region, clearly demonstrating that the −98GATA site is the essentialcis-element for the eosinophil-specific activation of the gp91 phox promoter by the −105/−96 region. To identify nuclear protein complexes that specifically bind to the −98GATA site in HL-60-C15E cells, we performed EMSAs with the probe from bp −115 to −90 encompassing the −98GATA site (Fig. 5). Two nuclear protein complexes (C1 and C2) specifically bound to the probe because binding was abolished by a 300-fold molar excess of wild-type competitor with sequence identical to that of the probe, but not by an excess of the heterologous sequence oligonucleotide (lanes 1–3). Binding was abolished to a similar extent even by a 120-fold molar excess of wild-type competitor (data not shown). An excess of GATA site-mutated oligonucleotide spanning bp −105 to −85 of the promoter (Mt), which has the same mutation as p−105M/Luc in Fig. 4, failed to abolish the binding of the C1 and C2 complexes (lane 4), indicating that these protein complexes bind to the −98GATA site. An excess of a GATA-binding sequence derived from the T-cell receptor δ gene completely abolished the binding of these complexes to the probe (lane 5), as did the wild-type competitor, suggesting that the C1 and C2 complexes include members of the GATA family. To further characterize these complexes, we performed gel shift immunoassays with anti-GATA-1 and anti-GATA-2 antibodies because mRNAs of GATA-1 and GATA-2 are abundantly expressed in HL-60-C15E cells as well as in human peripheral eosinophils (28.Zon L.I. Yamaguchi Y. Yee K. Albee E.A. Kimura A. Bennett J.C. Orkin S.H. Ackerman S.J. Blood. 1993; 81: 3234-3241Crossref PubMed Google Scholar).2 C1 and the lower part of C2 (L) disappeared when anti-GATA-1 antibody was added (lane 6). The upper part of C2 (U), but not others, was abolished by the addition of anti-GATA-2 antibody (lane 7). The formation of both C1 and C2 bands was mostly inhibited by a combination of both antibodies (lane 8). Neither control goat IgG (lane 9) nor control murine IgG (lane 10) disrupted the complexes. These results suggest that C1 and C2 include GATA-1 alone and GATA-1 and GATA-2, respectively. Although a small amount of mRNA for GATA-3 was expressed in HL-60-C15E cells as well as in peripheral eosinophils, an antibody against GATA-3 exhibited no effects on C1 or C2 (data not shown). These results demonstrate that GATA-1 and GATA-2 specifically bind to the −98GATA site of the gp91 phox promoter in HL-60-C15E cells. The different mobilities of GATA-1 and GATA-2 shown above are consistent with those reported by Briegelet al. (29.Briegel K. Lim K.C. Plank C. Beug H. Engel J.D. Zenke M. Genes Dev. 1993; 7: 1097-1109Crossref PubMed Scopus (174) Google Scholar) and Yamaguchi et al. (30.Yamaguchi Y. Ackerman S.J. Minegishi N. Takiguchi M. Yamamoto M. Suda T. Blood. 1998; 91: 3447-3458Crossref PubMed Google Scholar). To characterize the individual binding of GATA-1 and GATA-2 to the −98GATA site in the gp91 phox promoter, EMSA was performed with nuclear extracts from COS-7 cells that transiently expressed either human GATA-1 or GATA-2 cDNA and the probe spanning bp −115 to −90 of the gp91 phox promoter (Fig.6). The binding of GATA-1 (G1) and GATA-2 (G2) was abolished by the unlabeled wild-type competitor (Wt; lanes 3 and 10) and the GATA-binding site of the T-cell receptor δ gene (GA;lanes 5 and 12), but not by a heterologous sequence (He; lanes 4 and 11) or the GATA site-mutated competitor (Mt; lanes 6 and13). Furthermore, the GATA-1 complexes were supershifted by anti-GATA-1 antibody (SS; lane 8), and the GATA-2 complexes were abolished by anti-GATA-2 antibody (lane 15), contrasting with no effects by corresponding control antibodies (lanes 7 and 14). Nuclear extracts from untransfected COS-7 cells made no such complexes (lane 1). These results confirm that GATA-1 and GATA-2 specifically bind to the −98GATA site and support the use of these nuclear extracts in the kinetic analysis of the interaction between the site and these GATA proteins. To determine the relative binding affinities of GATA-1 and GATA-2 for the −98GATA site, we determined the apparent K dvalues from Scatchard plots obtained from EMSA data (Fig.7). The amount of bound probe increased with increases in the concentration of the probe (panels A1and B1). Scatchard plots for GATA-1 (panel A2) and GATA-2 (panel B2) revealed their apparentK d values to be 0.13 and 0.10 nm, respectively, suggesting that both GATA proteins have similar affinities for the −98GATA site. Accordingly, GATA-1 and GATA-2 may efficiently compete with each other for binding to the site. The above results indicate that both GATA-1 and GATA-2 similarly bind to the −98GATA site of the gp91 phox promoter. To determine whether either one or both of the GATA proteins transactivate the gp91 phox promoter, we individually cotransfected various amounts of their expression vectors with wild-type p−105/Luc into Jurkat cells, which express neither of the endogenous GATA proteins (Fig. 8). Up to 4 μg cotransfected GATA-1 and GATA-2 cDNAs dose-dependently and significantly (p < 0.01 and 0.05, respectively) increased gp91 phox promoter activity. At doses higher than 6 μg, the activity decreased similarly in both plasmids (panel A). The GA-to-CT mutation at the −98GATA site of p−105/Luc significantly decreased the GATA-1- and GATA-2-mediated promoter activities, to 30.5 ± 13.6 and 16.4 ± 16.2%, respectively (panels B and C). These results indicate that individually expressed GATA-1 and GATA-2 transactivate the gp91 phox promoter mostly through the −98GATA site. To clarify each role of GATA-1 and GATA-2 in cells expressing both GATA proteins as eosinophils, GATA-1 and GATA-2 plasmids were cotransfected individually or in combination with the wild-type p−105/Luc reporter construct into Jurkat cells (Fig. 9). The amount of GATA-1 plasmid and that of GATA-2 were fixed to 2 μg, which gave 85 and 80%, respectively, of their maximal activities (Fig.8 A). Transiently expressed GATA-1 (Fig. 9 A,second bar) produced a (15.5 ± 1.5)-fold increase above vehicle activity (p < 0.001 versusthe first bar), and transiently expressed GATA-2 (third bar) produced a (1.0 ± 0.2)-fold increase (p < 0.001 versus the first bar). The combined expression of the two GATA proteins (fourth bar) exhibited an (8.3 ± 0.6)-fold increase, which is significantly lower than the increase obtained by GATA-1 alone (p < 0.005), suggesting that GATA-2 inhibits the transactivation of the gp91 phox promoter by GATA-1. This was confirmed in Fig. 9 B. The amount of GATA-1 plasmid was also fixed here to 2 μg, but the amount of GATA-2 plasmid was increased from 0 to 4 μg to reach its maximal activity (Fig. 8 A). Total amounts of GATA plasmids were 6 μg or less for avoiding their inhibitory effects. The combined expression of increased amounts of GATA-2 along with a fixed amount of GATA-1 impaired the ability of GATA-1 to transactivate the gp91 phox promoter in a dose-dependent fashion. Therefore, GATA-1 is the principal activator for the gp91 phox promoter, and GATA-2 is a relative competitive inhibitor in the presence of both GATA proteins, as in eosinophils. In a previous report, we suggested the existence of an eosinophil-specific mechanism of gp91 phox gene expression (17.Kuribayashi F. Kumatori A. Suzuki S. Nakamura M. Matsumoto T. Tsuji Y. Biochem. Biophys. Res. Commun. 1995; 209: 146-152Crossref PubMed Scopus (28) Google Scholar). In this study, we have shown that the −98GATA site works as a positivecis-element for the gp91 phox promoter in an eosinophil lineage, but not in other lineages expressing gp91 phox. EMSAs have shown that GATA-1 and GATA-2 are the only specific DNA-binding proteins for the GATA site in eosinophilic cells. These results suggest that expression of the gp91 phox gene is regulated by the combination of GATA-1 and GATA-2 through the −98GATA site, particularly in eosinophils. We also found that GATA-1 and GATA-2 individually bind to the −98GATA site with similar apparent dissociation constants and can individually work as positive regulators for the gp91 phox gene. However, GATA-2 inhibits the transactivation of the gene by GATA-1 in coexisting conditions, as in eosinophils. Taken together, our findings suggest that GATA-1 works as a principal activator and that GATA-2 works as a competitive inhibitor of GATA-1 for the expression of the gp91 phox gene in eosinophils. The negative regulation of the gene by GATA-2 can be done simply by competitive binding to the −98GATA site with GATA-1 with an assumption that GATA-2 is one order less effective than GATA-1 for the transactivation of the gene. However, inhibition of the interaction between GATA-1 and some protein factors by GATA-2 may also contribute to decreasing the GATA-1-dependent promoter activity of the gene. At least six members have been identified in the GATA transcription factor family, which recognizes the consensus motif ((A/T)GATA(A/G)) through a highly conserved zinc finger DNA-binding domain (27.Evans T. Reitman M. Felsenfeld G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5976-5980Crossref PubMed Scopus (360) Google Scholar). In hematopoietic tissues, GATA-1, GATA-2, and GATA-3 are expressed in distinct but overlapping patterns (31.Weiss M.J. Orkin S.H. Exp. Hematol. 1995; 23: 99-107PubMed Google Scholar): GATA-1 in erythrocytes, megakaryocytes, mast cells, basophils, and eosinophils; GATA-2 in all these cells and neutrophils; and GATA-3 in T-lymphocytes, mast cells, and eosinophils. Only eosinophils among gp91 phox -expressing lineages express GATA-1. We can therefore conclude that the positive regulation of the gp91 phox gene by GATA-1 is an eosinophil-specific mechanism. We can also conclude that the negative regulation by GATA-2 is unique in eosinophils, which are the only cells that express both GATA-1 and GATA-2 in gp91 phox -expressing cells. Recently, mechanisms for positive regulation by GATA-1 have been detected in two eosinophil-specific genes, the chicken EOS47gene (32.McNagny K.M. Sieweke M.H. Doderlein G. Graf T. Nerlov C. EMBO J. 1998; 17: 3669-3680Crossref PubMed Scopus (107) Google Scholar) and the human MBP gene (30.Yamaguchi Y. Ackerman S.J. Minegishi N. Takiguchi M. Yamamoto M. Suda T. Blood. 1998; 91: 3447-3458Crossref PubMed Google Scholar). The MBP gene is, in particular, regulated positively by GATA-1 and negatively by GATA-2, as is the mechanism presented here for the gp91 phox gene (30.Yamaguchi Y. Ackerman S.J. Minegishi N. Takiguchi M. Yamamoto M. Suda T. Blood. 1998; 91: 3447-3458Crossref PubMed Google Scholar), although GATA-2 by itself cannot activate the MBP gene. Different from theEOS47 and MBP genes, the gp91 phox gene is not specifically expressed in eosinophil lineages, but its expression is definitely supported by GATA-1, which is particular to eosinophils in gp91 phox -expressing cells. The gp91 phox gene is highly expressed in terminally differentiated peripheral eosinophils,2 which still express GATA-1 at a high level (28.Zon L.I. Yamaguchi Y. Yee K. Albee E.A. Kimura A. Bennett J.C. Orkin S.H. Ackerman S.J. Blood. 1993; 81: 3234-3241Crossref PubMed Google Scholar), but the MBP gene at a residual level (33.Gruart V. Truong M.J. Plumas J. Zandecki M. Kusnierz J.P. Prin L. Vinatier D. Capron A. Capron M. Blood. 1992; 79: 2592-2597Crossref PubMed Google Scholar, 34.Li M.S. Sun L. Satoh T. Fisher L.M. Spry C.J. Biochem. J. 1995; 305: 921-927Crossref PubMed Scopus (25) Google Scholar). Some critical factor(s) for the expression of the MBP gene, but not that of the gp91 phox gene, might have disappeared in the late stage of terminal differentiation to peripheral eosinophils. The expression of the gp91 phox gene is regulated by various endogenous and exogenous mediators, including interferon-γ, tumor necrosis factor-α, and lipopolysaccharide (35.Newburger P.E. Dai Q. Whitney C. J. Biol. Chem. 1991; 266: 16171-16177Abstract Full Text PDF PubMed Google Scholar). Some factors related to allergy and parasitic infections may specifically regulate the gp91 phox gene in eosinophils through the GATA-1 system. How does GATA-1 activate the gp91 phox gene? GATA-1 directly interacts with transcription factors such as Sp1, EKLF (36.Gregory R.C. Taxman D.J. Seshasayee D. Kensinger M.H. Bieker J.J. Wojchowski D.M. Blood. 1996; 87: 1793-1801Crossref PubMed Google Scholar, 42.Merika M. Orkin S.H. Mol. Cell. Biol. 1995; 15: 2437-2447Crossref PubMed Scopus (437) Google Scholar), and FOG-1 (37.Tsang A.P. Visvader J.E. Turner C.A. Fujiwara Y., Yu, C. Weiss M.J. Crossley M. Orkin S.H. Cell. 1997; 90: 109-119Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). An acetylase of p300/CBP (38.Blobel G.A. Nakajima T. Eckner R. Montminy M. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2061-2066Crossref PubMed Scopus (317) Google Scholar) also directly associates with GATA-1 as a coactivator. The p300/CBP acetylates GATA-1 and increases its binding to DNA, resulting in stimulation of GATA-1-dependent transcription (39.Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (634) Google Scholar, 40.Hung H.L. Lau J. Kim A.Y. Weiss M.J. Blobel G.A. Mol. Cell. Biol. 1999; 19: 3496-3505Crossref PubMed Scopus (221) Google Scholar). This mechanism of the ubiquitous cofactor may participate in the activation of the gp91 phox promoter by GATA-1. Expression of myeloid-specific genes is regulated by a combination of transcription factors generally expressed in hematopoietic cells (c-Myb, AML-1/CBFβ, and Ets-1) and lineage-specific or -restricted factors (GATA-1, PU.1, and C/EBP) (23.Kulessa H. Frampton J. Graf T. Genes Dev. 1995; 9: 1250-1262Crossref PubMed Scopus (359) Google Scholar). Recently, Yamaguchi et al. (30.Yamaguchi Y. Ackerman S.J. Minegishi N. Takiguchi M. Yamamoto M. Suda T. Blood. 1998; 91: 3447-3458Crossref PubMed Google Scholar) demonstrated the transactivation of the MBP P2 promoter by C/EBPs, especially by C/EBPβ. C/EBPβ may also play a role in the activation of the gp91 phox promoter in eosinophils because possible binding sites of C/EBP are found in the promoter. Previously, we reported the deficient expression of gp91 phox in the neutrophils, monocytes, and B-lymphocytes, but not in the eosinophils, of an X-linked chronic granulomatous disease patient (17.Kuribayashi F. Kumatori A. Suzuki S. Nakamura M. Matsumoto T. Tsuji Y. Biochem. Biophys. Res. Commun. 1995; 209: 146-152Crossref PubMed Scopus (28) Google Scholar). Further analyses suggested that the deficient binding of PU.1 to the gp91 phox promoter due to the point mutation (−53C to T) at its PU.1 motif results in the deficient expression of the gp91 phox gene in the patient (14.Suzuki S. Kumatori A. Haagen I.A. Fujii Y. Sadat M.A. Jun H.L. Tsuji Y. Roos D. Nakamura M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6085-6090Crossref PubMed Scopus (93) Google Scholar). These findings suggest that eosinophils have their own gp91 phox gene expression mechanism independent of PU.1. The GATA-mediated mechanism presented here is a strong candidate for it because the transactivation of the gp91 phox gene by a combination of GATA-1 and GATA-2 has occurred in Jurkat cells, which have no PU.1. We thank Tosiyuki Moriuchi for assistance in DNA sequencing." @default.
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• W2014165340 title "Eosinophil-specific Regulation of gp91 Gene Expression by Transcription Factors GATA-1 and GATA-2" @default.
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• W2014165340 cites W1602266368 @default.
• W2014165340 cites W1626483287 @default.
• W2014165340 cites W1820378590 @default.
• W2014165340 cites W1959970548 @default.
• W2014165340 cites W1971688493 @default.
• W2014165340 cites W1981676369 @default.
• W2014165340 cites W1982809272 @default.
• W2014165340 cites W199379996 @default.
• W2014165340 cites W1995134513 @default.
• W2014165340 cites W2020847300 @default.
• W2014165340 cites W2022942610 @default.
• W2014165340 cites W2023986228 @default.
• W2014165340 cites W2024590318 @default.
• W2014165340 cites W2034694756 @default.
• W2014165340 cites W2036998973 @default.
• W2014165340 cites W2037330840 @default.
• W2014165340 cites W2047791752 @default.
• W2014165340 cites W2048437870 @default.
• W2014165340 cites W2054338068 @default.
• W2014165340 cites W2064443141 @default.
• W2014165340 cites W2065855627 @default.
• W2014165340 cites W2073434161 @default.
• W2014165340 cites W2089019735 @default.
• W2014165340 cites W2089269522 @default.
• W2014165340 cites W2090524722 @default.
• W2014165340 cites W2104378983 @default.
• W2014165340 cites W2122616238 @default.
• W2014165340 cites W2141083396 @default.
• W2014165340 cites W2168508124 @default.
• W2014165340 cites W2293440579 @default.
• W2014165340 cites W335876340 @default.
• W2014165340 cites W4253353778 @default.
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