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• W1991298889 abstract "Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and maturation of myeloid progenitor cells both in vitro and in vivo. We showed that G-CSF rapidly and transiently induces expression of egr-1 in the NFS60 myeloid cell line. Transient transfections of NFS60 cells with recombinant constructs containing various deletions of the humanegr-1 promoter identified the serum response element (SRE) between nucleotides (nt) –418 and –391 as a critical G-CSF-responsive sequence. The SRE (SRE-1) contains a CArG box, the binding site for the serum response factor (SRF), which is flanked at either side by an ETS protein binding site. We demonstrated that a single copy of the wild-type SRE-1 in the minimal promoter plasmid, pTE2, is sufficient to induce transcriptional activation in response to G-CSF and that both the ETS protein binding site and the CArG box are required for maximal transcriptional activation of the pTE2-SRE-1 construct. In electromobility shift assays using NFS60 nuclear extracts, we identified SRF and the ETS protein Fli-1 as proteins that bind the SRE-1. We also demonstrated through electrophoretic mobility shift assays, using an SRE-1 probe containing a CArG mutation, that Fli-1 binds the SRE-1 independently of SRF. Our data suggest that SRE-binding proteins potentially play a role in G-CSF-induced egr-1expression in myeloid cells. Granulocyte colony-stimulating factor (G-CSF) stimulates the proliferation and maturation of myeloid progenitor cells both in vitro and in vivo. We showed that G-CSF rapidly and transiently induces expression of egr-1 in the NFS60 myeloid cell line. Transient transfections of NFS60 cells with recombinant constructs containing various deletions of the humanegr-1 promoter identified the serum response element (SRE) between nucleotides (nt) –418 and –391 as a critical G-CSF-responsive sequence. The SRE (SRE-1) contains a CArG box, the binding site for the serum response factor (SRF), which is flanked at either side by an ETS protein binding site. We demonstrated that a single copy of the wild-type SRE-1 in the minimal promoter plasmid, pTE2, is sufficient to induce transcriptional activation in response to G-CSF and that both the ETS protein binding site and the CArG box are required for maximal transcriptional activation of the pTE2-SRE-1 construct. In electromobility shift assays using NFS60 nuclear extracts, we identified SRF and the ETS protein Fli-1 as proteins that bind the SRE-1. We also demonstrated through electrophoretic mobility shift assays, using an SRE-1 probe containing a CArG mutation, that Fli-1 binds the SRE-1 independently of SRF. Our data suggest that SRE-binding proteins potentially play a role in G-CSF-induced egr-1expression in myeloid cells. colony-stimulating factor β-galactosidase bovine serum albumin chloramphenicol acetyltransferase cytomegalovirus ETS protein binding site electrophoretic mobility shift assay granulocyte CSF granulocyte-macrophage CSF interleukin nucleotide(s) serum response factor serum response element signal transducers and activators of transcription Myeloid blood cell production is controlled by cytokines such as colony-stimulating factors (CSFs)1 and interleukins (ILs). Granulocyte colony-stimulating factor (G-CSF) stimulates survival, proliferation, and differentiation of granulocytic precursors and activation of neutrophils (1.Demetri G.D. Griffin J. Blood. 1991; 78: 2791-2808Crossref PubMed Google Scholar, 2.Avalos B.R. Blood. 1996; 88: 761-777Crossref PubMed Google Scholar). G-CSF mediates its cellular effects through binding the G-CSF receptor, a member of the cytokine receptor superfamily (2.Avalos B.R. Blood. 1996; 88: 761-777Crossref PubMed Google Scholar, 3.Nicola N.A. J. Cell. Physiol. Suppl. 1987; 5: 9-14Crossref PubMed Scopus (24) Google Scholar). Growth factor-mediated signals promoting cell proliferation or differentiation incite rapid induction of a family of genes termed immediate early genes (4.McMahon S.B. Monroe J.G. FASEB. 1992; 6: 2707-2715Crossref PubMed Scopus (121) Google Scholar, 5.Herschman H. Trends Biochem. Sci. 1989; 14: 455-458Abstract Full Text PDF PubMed Scopus (76) Google Scholar), which include c-fos (6.Greenberg M. Ziff E. Nature. 1984; 311: 433-438Crossref PubMed Scopus (2009) Google Scholar) c-jun (7.Ryder K. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8454-8467Crossref PubMed Scopus (22) Google Scholar), and the early growth response geneegr-1 (8.Varnum B.C. Lim R.W. Kujubu D.A. Luner S.J. Kaufman S.E. Greenberger J.S. Gasson J.C. Herschman H. Mol. Cell. Biol. 1989; 9: 3580-3583Crossref PubMed Scopus (64) Google Scholar) (also known as Tis 8, Krox 24, NFGIA, and zif/268). Egr-1 is a ubiquitously expressed zinc finger transcription factor (59 kDa) (9.Cao X. Koski R.A. Gashler A. McKiernan M. Morris C.F. Gaffney R. Hay R.V. Sukhatme V.P. Mol. Cell. Biol. 1990; 10: 1931-1939Crossref PubMed Scopus (298) Google Scholar) that can act to either positively or negatively regulate gene transcription (10.Gashler A. Swaminathan S. Sukhatme V. Mol. Cell. Biol. 1993; 13: 4556-4571Crossref PubMed Scopus (213) Google Scholar, 11.Lin J.-X. Leonard W.J. Mol. Cell. Biol. 1997; 17: 3714-3722Crossref PubMed Google Scholar). Egr-1 has been demonstrated to be a critical upstream mediator of proliferation (12.Perez-Castillo A. Pipaon C. Garcia I. Alemany S. J. Biol. Chem. 1993; 268: 19445-19450Abstract Full Text PDF PubMed Google Scholar, 13.Hu R. Levin E. J. Clin. Inv. 1994; 93: 1820-1827Crossref PubMed Scopus (50) Google Scholar), differentiation (14.Karabanda S. Nakamura T. Stone R. Hass R. Bernstein S. Datta R. Sukhatme V. Kufe D. J. Clin. Inv. 1991; 88: 571-577Crossref PubMed Scopus (93) Google Scholar, 15.Nguyen H. Hoffman-Liebermann B. Liebermann D. Cell. 1993; 72: 197-209Abstract Full Text PDF PubMed Scopus (353) Google Scholar, 16.Krishnaraju K. Hoffman B. Liebermann D.A. Blood. 1998; 92: 1957-1966Crossref PubMed Google Scholar, 17.Dinkel A. Warnatz K. Ledermann B. Rolink A. Zipfel P.F. Burki K. Eibel H. J. Exp. Med. 1998; 188: 2215-2224Crossref PubMed Scopus (47) Google Scholar), and apoptosis (18.Muthukkumar S. Han S.-S. Muthukkumar S. Rangnekar V.M. Bondada S. J. Biol. Chem. 1997; 272: 27987-27993Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19.Nair P. Muthukkumars S. Sells S. Hans S. Sukhatme V. Rangnekar V. J. Biol. Chem. 1997; 272: 20131-20138Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Treatment of myeloid cells with G-CSF results in rapid and transient expression of egr-1 independently of protein synthesis. G-CSF induces egr-1 expression and granulocyte differentiation in 32Dcl3 cells (20.Kreider B.L. Rovera G. Oncogene. 1992; 7: 135-140PubMed Google Scholar) and also stimulates proliferation and egr-1 expression in human UT-7 epo cells overexpressing the wild-type G-CSF receptor (21.Tian S.-S. Tapley P. Sincich C. Stein R.B. Rosen J. Lamb P. Blood. 1996; 88: 4435-4444Crossref PubMed Google Scholar). The expression of egr-1, like that of other immediate early genes, is governed by preexisting regulatory proteins that are posttranslationally modified and thus activated upon receptor stimulation. The precise signaling events that mediate expression ofegr-1 in response to G-CSF in proliferative responses have not been elucidated. Identification of this pathway may provide insights into possible mechanisms that lead to the development of leukemogenesis. It has been suggested that G-CSF selectively activates distinct early growth response genes through different Janus kinase-STAT proteins. For example, G-CSF stimulation of the early genes OSM, IRF-1, and egr-1 is dependent on STAT5 activation, whereas activation of c-fos is STAT5-independent (21.Tian S.-S. Tapley P. Sincich C. Stein R.B. Rosen J. Lamb P. Blood. 1996; 88: 4435-4444Crossref PubMed Google Scholar). Although STAT5 protein expression is induced in response to G-CSF, that STAT5 DNA recognition element has not been identified in the murineegr-1 promoter (21.Tian S.-S. Tapley P. Sincich C. Stein R.B. Rosen J. Lamb P. Blood. 1996; 88: 4435-4444Crossref PubMed Google Scholar). Signaling pathways that control egr-1 expression and myeloid cell proliferation have been examined for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 (22.Sakamoto K.M. Fraser J.K. Lee H.-J.J. Lehman E. Gasson J. Mol. Cell. Biol. 1994; 14: 5975-5985Crossref PubMed Scopus (92) Google Scholar). The receptors for GM-CSF and IL-3, like G-CSF receptor, are also members of the cytokine receptor superfamily and regulate early myeloid development. GM-CSF- and IL-3-induced signals converge upon the cAMP response element in theegr-1 promoter in the factor-dependent human myeloid leukemic TF-1 cell line (22.Sakamoto K.M. Fraser J.K. Lee H.-J.J. Lehman E. Gasson J. Mol. Cell. Biol. 1994; 14: 5975-5985Crossref PubMed Scopus (92) Google Scholar). egr-1 promoter sequences mediating G-CSF-induced egr-1 expression and myeloid proliferation, including the egr-1 promoter sites responsive to G-CSF, appear to be distinct from those involved in GM-CSF and IL-3 signaling, due to the different proteins activated by these proteins. The goal of our study was to define G-CSF-responsive sequences of theegr-1 promoter and to identify interacting proteins in NFS60 cells. We show that egr-1 is rapidly and transiently expressed in NFS60 cells stimulated with G-CSF and that this activation occurs independently of protein synthesis. Transient transfections with recombinant egr-1 promoter constructs in NFS60 cells demonstrated that the CArG box and the ETS protein binding site (EBS) are required for maximal transcriptional activation of egr-1in response to G-CSF. Electromobility gel shift assays (EMSAs) showed that SRF and Fli-1 bind the CArG and EBS between nucleotides (nt) –418 and –391 in the egr-1 promoter, respectively. Our experiments suggest that serum response element (SRE)-binding proteins may associate as a quaternary complex to maximally activateegr-1 transcription. Thus, signaling pathways activated by G-CSF may be distinct from those activated by GM-CSF or IL-3, suggesting a potential mechanism for specificity between growth factors that regulate myelopoiesis. The murine myeloid leukemic factor-dependent NFS60 cell line was cultured in 1× RPMI medium containing 10% fetal calf serum, penicillin (100 units/ml)-streptomycin (1 mg/ml) at a ratio of 1 unit/ml to 1 mg/ml,l-glutamine (2 mm), and gentamicin (10 mg/ml). Cells were maintained on IL-3 WEHI-3 conditioned media (1:100) in tissue culture flasks at 37 °C. Cells were serum- and factor-starved for 18 h and then stimulated with G-CSF for 0, 30, 60, 90, and 120 min. Cells were also stimulated with 12-O-tetradecanoylphorbol-13-acetate (50 ng/ml) for 60 min (positive control) or diluent (0.02% BSA in phosphate-buffered saline). To demonstrate protein expression in the absence of protein synthesis, cells were stimulated with G-CSF (10 nm) or tetradecanoyl phorbol acetate (50 ng/ml) in combination with the protein synthesis inhibitor cycloheximide for 30 min. The cells were harvested, and total RNA was extracted by the Nonidet P-40 method (23.Kumar A. Lindberg U. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 681-685Crossref PubMed Scopus (92) Google Scholar). Twenty micrograms of extracted RNA from each sample was separated on a 1% formaldehyde gel and transferred to a nylon membrane. The membrane was hybridized with a [32P]dCTP-labeled 1.3EcoRI fragment of murine egr-1/tis8 or with a [32P]dCTP-labeled 500-base pair actin fragment as control. A –600 nt HinfI fragment of the egr-1 promoter (nt –606 to –7 of the putative transcription start site) was gel-purified from the full-length human egr-1 genomic clone (24.Sakamoto K. Bardeleben C. Yates K. Raines M. Golde D. Gasson J. Oncogene. 1991; 6: 867-871PubMed Google Scholar). Construction of chloramphenicol acetyltransferase (CAT) reporter plasmids containing the full-length or various deletions of the –600 egr-1fragment (–480, –235, –180, –116, and –56 nt) has been described (25.Sakamoto K. Nimer S. Rosenblatt J. Gasson J. Oncogene. 1992; 7: 2125-2130PubMed Google Scholar). A –418 and –387 nt egr-1 fragment was amplified from the p-480 CAT plasmid by polymerase chain reaction. A reverse primer flanking the egr-1 promoter at –10 nt, containing aXbaI site (5′-GC[TCTAGA]GCCGGATCCGCCTCTATTTGAAGG-3′), was used in combination with a forward polymerase chain reaction primer flanking the egr-1 promoter at –418 nt, containing aHindIII site (5′-ACC[AAGCTT]C(CCGGAAT)G(CCATATAAGG)A(CAGGAAG)-3′). A reverse primer flanking the egr-1 promoter at –7 nt, containing aXbaI site (5′-GC[TCTAGA]GCCCCGGAT-3′), was used with a forward polymerase chain reaction primer flanking the egr-1promoter at –387 nt, containing a PstI site (5′-GAGT[CTGC AG]CTGGAACAAC CCTTA-3′). The polymerase chain reaction-amplifiedegr-1 fragments were digested with XbaI andHindIII or PstI and then gel-purified and directionally subcloned into the pCAT vector (Promega Corp., Madison, WI). The SRE from nt –418 to –391 of the human egr-1 promoter (SRE-1) was synthesized as a single element, with HindIII and XbaI sites created at the 5′ and 3′ ends, respectively (5′-AGCTTCGGA(CCGGAAT)G(CCATATAAGG)A(GCAGGAAG)GATCCT-3′). In addition, mutated SRE-1 were synthesized with the 5′ and 3′HindIII and XbaI ends, including CArGm(5′-AGCTTCGGA(CCGGAAT)G(CCA g AT ct GG)A(GCAGGAAG)GATCCT-3′), LmR (5′-AGCTTCGGA(CC tt AAT)G(CCATATAAGG)A(GCAGGAAG)GATCCT-3′), LRm(5′-AGCTTCGGA(CCGGAAT)G(CCATATAAGG)A(GCA tt AAG)GATCCT-3′), and LmRm(5′-AGCTTCGGA(CC tt AAT)G(CCATATAAGG)A(GCA tt AAG)GATCCT-3′). The oligonucleotides were annealed and ligated into theHindIII and XbaI sites in the pTE2 vector. Transient co-transfections of constructs into NFS60 cells were performed by electroporation (Bio-Rad) at 250 V, 960 F. Cells were serum- and growth factor-starved for 18 h in RPMI-0.5% BSA. Twenty million cells were transfected with 20 μg of the specified egr-1promoter/CAT construct or 20 μg of the specified minimal promoter pTE2/CAT construct and 5 μg of the pCMV-β-galactosidase plasmid (pCMV-βgal) (internal control). Transfected cells were resuspended in RPMI-0.5% BSA and stimulated with G-CSF (10 nm) or diluent control (0.02% BSA in phosphate-buffered saline) for 3 h. The cells were harvested; half the lysates were assayed for CAT activity and the other half for βgal activity. The CAT assay was used to measure egr-1 promoter activity and was performed as described previously (22.Sakamoto K.M. Fraser J.K. Lee H.-J.J. Lehman E. Gasson J. Mol. Cell. Biol. 1994; 14: 5975-5985Crossref PubMed Scopus (92) Google Scholar). The amount of acetylated and unacetylated [14C]chloramphenicol was determined by thin-layer chromatography and quantified by liquid scintillation counting. The βgal assay (Promega) was used as internal control for transfection efficiency. Corrected fold stimulation was determined by dividing the percentage of acetylation of the G-CSF-stimulated cells by unstimulated (diluent) cells. Statistical analysis was performed using the JMP In program (SAS Institute Inc.). The probes used for EMSA experiments included theegr-1 SRE-1 sequence (forward,5′-AGCTTGCGAC[CCGGAAAT]G[CCATATAAGG]A[GCAGGAAG]GATCCCCT-3′; reverse, 5′-CTAGAGGGGATC[CTTCCTGC]T[CCTTATATGG]C[ATTTCCGG]GTCGCA-3′), the left EBS sequence (forward, 5′-AC[CCGGAAAT]GC-3′; reverse, 5′-GC[ATTTCCGG]GT), the right EBS sequences (forward, 5′-AGCTTGA[GCAGGAAG]GAT-3′; reverse, 5′-CTAGATC[CTTCCTGC]TCA-3′), control CArG consensus element (forward, 5′-GGATGT[CCATATTAGG]ACATCT-3′; reverse, 5′-AGATGT[CCTAATATGG]ACATCC), or mutant SRE-1 sequences, including CArGm (forward, 5′-AGCTTGCGAC[CCGGAAAT]G[CCA g AT ct GG] A[GCAGGAAG]GATCCT-3′; reverse, 5′-CTAGAGGGGATC[CTTCCTGC]T [CC ag AT c TGG]C[ATTTCCGG]GTCGCA-3′), LmR (forward, 5′-AGCTTGCGAC[CC tt AAAT]G[CCATATAAGG]A[GCAGGAAG]GATCCT-3′; reverse, 5′-CTAGAGGGGATC[CTTCCTGC]T[CCTTATATGG]C[ATTT aa GG]GTCGCA-3′), and LRm (forward, 5′-AGCTTGCGAC[CCGGAAAT]G[CCATATAAGG] A[GCA tt AAG]GATCCT-3′; reverse, 5′-CTAGAGGGGATC[CTT aa TGC]T [CCTTATATGG]C[ATTTCCGG]GTCGCA-3′), and LmRm(forward, 5′-AGCTTGCGAC[CC tt AAAT]G[CCATATAAGG]A[GCA tt AAG]GATCCT-3′; reverse, 5′-CTAGAGGGGATC[CTT aa TGC]T[CCTTATATGG]C[ATTT aa GG] GTCGCA-3′). Complimentary single-stranded oligonucleotides were synthesized, annealed, and end-labeled using [γ-32P]dATP and T4 polynucleotide kinase. Labeled probe was purified with Nuctrap Push Columns (Stratagene Cloning Systems, La Jolla, CA). Nuclear extracts were prepared by the modified Dignam method (26.Fraser J. Guerra J. Nguyen C. Indes J. Gasson J. Nimer S. Mol. Cell. Biol. 1994; 14: 2213-2221Crossref PubMed Scopus (17) Google Scholar) from unstimulated (diluent) or G-CSF-stimulated NFS60 cells for 30 min. Protein concentrations were determined by the Bradford assay with Pierce protein assay reagents. Nuclear extracts (15–20 μg) were incubated with 0.1 μg of labeled probe in the presence of 1 μg of poly(dI-dC):(dI-dC) and 5 μg of BSA in 20 μl of gel shift buffer (20 mm Tris-HCl (pH 7.4), 50 mm NaCl, 1 mm EDTA, 10 mm MgCl2, 25% (v/v) glycerol) for 30 min on ice. Competitor oligonucleotides or antibodies were preincubated with the nuclear extracts for 30 min on ice prior to the addition of probe. Competitor oligonucleotides, including the nonspecific sequence, 69ΔALL, or wild-type or three mutant forms of the SRE-1 sequence, were added in 100- or 200-fold molar excess. Fli-1 antibody (2, 4, or 8 μg; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used in supershift assays. Polyclonal rabbit IgG (Sigma) was used as the control antiserum (1.1 ng/μl). After incubation periods, the samples were loaded onto a 4% polyacrylamide gel and run at 100 V in 0.4× TBE. Gels were dried and exposed to film at −70 °C. We demonstrate that NFS60 cells are a growth factor-dependent myeloid cell line (data not shown). Furthermore, NFS60 cells proliferate in response to G-CSF in a dose-dependent manner (27.Metcalf D. Leukemia. 1989; 3: 349-355PubMed Google Scholar, 28.Gascan H. Moreau J. Jacques Y. Soulillou J. Lymphokine Res. 1989; 8: 79-84PubMed Google Scholar). Therefore, NFS60 cells provide a good model in which to examine G-CSF-induced proliferative signals. Rapid and transient expression of egr-1 has been previously shown to occur in response to GM-CSF in myeloid leukemic TF-1 cells (22.Sakamoto K.M. Fraser J.K. Lee H.-J.J. Lehman E. Gasson J. Mol. Cell. Biol. 1994; 14: 5975-5985Crossref PubMed Scopus (92) Google Scholar). To demonstrate egr-1 expression in NFS60 cells in response to G-CSF, Northern blot analysis was performed. Northern blot analysis with RNA from NFS60 cells stimulated with G-CSF for 0, 30, 60, 90, and 120 min demonstrated a rapid and transient induction of egr-1 (Fig. 1). Expression of egr-1 was not induced in diluent-treated cells (0 min). Accumulation of egr-1 RNA was observed within 30 min and was no longer observed at 60 min following G-CSF treatment. Stimulation of cells for 60 min with G-CSF or 12-O-tetradecanoylphorbol-13-acetate in the presence of the protein synthesis inhibitor cycloheximide resulted in induction ofegr-1. 12-O-tetradecanoylphorbol-13-acetate has been previously shown to induce egr-1 expression within 60 min of cell treatment (8.Varnum B.C. Lim R.W. Kujubu D.A. Luner S.J. Kaufman S.E. Greenberger J.S. Gasson J.C. Herschman H. Mol. Cell. Biol. 1989; 9: 3580-3583Crossref PubMed Scopus (64) Google Scholar). These results were confirmed in two independent experiments. Our results demonstrate that egr-1expression occurs rapidly and transiently in cells stimulated with G-CSF and that induction of egr-1 occurs independently of protein synthesis. To identify the egr-1promoter sequences that are responsive to G-CSF signaling, NFS60 cells were transiently transfected with egr-1 promoter constructs. Serum- and growth factor-starved NFS60 cells were transfected with constructs containing –600, –480, –387, –235, –180, –116, or –56 nt of the human egr-1 promoter (Fig.2) and pCMV-βgal for measurement of transfection efficiency. We previously showed that the –56 nt region of egr-1contains minimal activity that is equal to the pCAT empty vector (22.Sakamoto K.M. Fraser J.K. Lee H.-J.J. Lehman E. Gasson J. Mol. Cell. Biol. 1994; 14: 5975-5985Crossref PubMed Scopus (92) Google Scholar). We have therefore used p-56 CAT as the vector control in our experiments. In response to G-CSF, the –600 and –480 ntegr-1 constructs demonstrated maximal stimulation (Fig. 2) at 9.0-fold activity relative to p-56 CAT control vector. Deletion of nucleotides between –480 and –387 resulted in a 6-fold decrease in transcriptional activation (p = 0.0024; Fig. 2), and further deletion to nt –116 did not reduce this activity. In diluent-treated cells, the constructs had basal activities that were not statistically significantly different from that of the control vector (data not shown). All transfections were performed in duplicate or triplicate, representing an average of three to seven experiments. These results indicate that the region between –480 and –387 nt of the egr-1 promoter contains critical sequences required for maximal transcriptional activation of egr-1. The region between nt –480 and –387 contains a single SRE, with a central CArG box flanked by EBSs, which we have called SRE-1 (Fig.3 A). Transfections with a construct containing –418 nt of the egr-1 promoter (Fig.3 A) showed similar activity levels with the p-480 nt construct (data not shown). These results suggested that the SRE, between nt –418 and –391 (SRE-1), may contain a critical transcription factor binding site regulating G-CSF-induced transcription of egr-1. Previous studies have identified SRF binding to the CArG box of SREs in the promoters of immediate early genes (29.Treisman R. Cell. 1986; 46: 567-574Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 30.Norman C. Runswick M. Pollock R. Treisman R. Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (709) Google Scholar, 31.Christy B. Nathans D. Mol. Cell. Biol. 1989; 9: 4889-4895Crossref PubMed Scopus (147) Google Scholar, 32.Latinkic B. Lau L. J. Biol. Chem. 1994; 269: 23163-23170Abstract Full Text PDF PubMed Google Scholar). To determine whether endogenous SRE-binding proteins in nuclear extracts from NFS60 cells interact with the wild-type SRE-1 sequence between nt –418 and –391 of the egr-1 promoter (Fig. 3 A), EMSAs were performed. Nuclear extracts prepared from diluent- or G-CSF-treated NFS60 cells were incubated with the SRE-1 oligonucleotide probe with an excess of unlabeled specific or nonspecific competitor and then analyzed on the same SDS-polyacrylamide gel. The diluent- and G-CSF-treated nuclear extracts produced four and five gel shift bands or complexes, designated D1–D4 and G1–G5, respectively (Fig. 3 B, lanes 1 and 3). Band specificity was demonstrated by competition of the bands with a 200m excess of specific unlabeled probe sequence (Fig.3 B, lanes 2 and 4) and by the lack of competition with an excess of unlabeled nonspecific sequence (Fig. 3 B, lanes 1 and 3). The two slowest migrating bands in the diluent-treated extracts, D1 and D2 (Fig. 3 B, lane 1), were only evident when an increased amount of protein was used, and band D1 migrated differently from band G1 in the G-CSF-stimulated extracts (Fig. 3 B, lanes 1 and3). These results suggest that bands/complexes 1–5 represent proteins that specifically bind the SRE-1 sequence and that potentially different or biochemically modified (i.e.phosphorylated) SRE-binding proteins bind to SRE-1 in G-CSF-treated compared with diluent-treated extracts. To determine whether SRF binds the SRE-1 in theegr-1 promoter, we performed EMSA supershift experiments with the SRF antibody. Addition of SRF antibody resulted in a supershift of bands 1 and 2 (Fig. 3 C, lanes 4–6), whereas addition of IgG control did not produce any change in the gel shift pattern (Fig. 3 C, lane 7). The control probe containing the CArG sequence (Santa Cruz Biotechnology Inc.) resulted in a supershifted band that co-migrates with the supershifted band seen with the SRE-1 probe (Fig. 3 C, lane 11). Furthermore, an excess of unlabeled CArG oligonucleotide alone specifically competed bands 1 and 2 (Fig. 3 C, lane 3), suggesting that these bands are SRF-containing complexes. Addition of the SRF antibody in EMSAs using diluent-treated extracts also resulted in a supershifted band pattern (data not shown). These results were confirmed in two separate experiments. SRF binding to the SRE has been shown to be enhanced upon SRF phosphorylation (33.Janknecht R. Hipskind R. Houthaeve T. Nordheim A. Stunnenberg H. EMBO J. 1992; 11: 1045-1054Crossref PubMed Scopus (108) Google Scholar, 34.Misra R. Rivera V. Wang J. Fan P. Greenberg M. Mol. Cell. Biol. 1991; 11: 4545-4554Crossref PubMed Scopus (73) Google Scholar), yet it has also been shown to bind the SRE constitutively (35.Herrera R. Shaw P. Nordheim A. Nature. 1989; 340: 68-70Crossref PubMed Scopus (251) Google Scholar, 36.Sheng M. Dougan S. McFadden G. Greenberg M. Mol. Cell. Biol. 1988; 7: 2787-2796Crossref Scopus (297) Google Scholar, 37.Fisch T. Prywer R. Roeder R. Mol. Cell. Biol. 1987; 7: 3490-3502Crossref PubMed Scopus (210) Google Scholar, 38.Gilman M. Genes Dev. 1988; 2: 394-402Crossref PubMed Scopus (164) Google Scholar). Our data indicate that SRF binds the SRE-1, and the supershift band corresponds with the band observed with SRF binding to the CArG probe. Thus, these results indicate that SRF binds the CArG sequence of SRE-1 in G-CSF-treated nuclear extracts. A previous report has demonstrated that Fli-1 is one of the ETS proteins that recognizes the EBS of SRE-1 in theegr-1 promoter (39.Watson D.K. Robinson L. Hodge D.R. Kola I. Papas T.S. Seth A. Oncogene. 1997; 14 (211): 213Crossref PubMed Scopus (100) Google Scholar). To identify ETS proteins in NFS60 nuclear extracts that bind the EBS of SRE-1, EMSA experiments were performed using antibodies to various ETS proteins. Addition of Fli-1 antibody to G-CSF-stimulated extracts resulted in the disappearance of band 1 and the formation of two supershifted bands, 1A and 1B (Fig.4, lanes 1–3). This suggests that band 1A represents a complex composed of both SRF and Fli-1 bound to the SRE-1 probe. Band 1B may represent a complex of SRF and Fli-1 with additional proteins. Addition of Fli-1 antibody in EMSAs using diluent-treated extracts resulted in a very weak supershift band pattern under certain conditions (data not shown). Addition of antibodies to other ETS proteins, including Elk-1, Sap1a/b, PU.1, Elf-1, ETS-1/2, ERG-1/2, and PEA3, did not produce a change in the gel shift pattern in either diluent- or G-CSF-treated extracts (data not shown). Our data suggest that in addition to SRF, Fli-1 also binds the SRE-1 in the egr-1 promoter in G-CSF-stimulated extracts. Ternary complexes composed of SRF and ETS protein family members have been shown to regulate the expression of many immediate early gene SREs (32.Latinkic B. Lau L. J. Biol. Chem. 1994; 269: 23163-23170Abstract Full Text PDF PubMed Google Scholar, 40.Shaw P. Schroter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 41.Hill C. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar, 42.Hipskind R. Buscher D. Nordheim A. Baccarini M. Genes Dev. 1994; 1994: 1803-1816Crossref Scopus (108) Google Scholar, 43.Hipskind R. Baccarini M. Nordheim A. Mol. Cell. Biol. 1994; 14: 6219-6231Crossref PubMed Scopus (137) Google Scholar, 44.Chung K. Gomes I. Wang D. Lau L. Rosner M. Mol. Cell. Biol. 1998; 18: 2272-2281Crossref PubMed Scopus (63) Google Scholar, 45.Latinkiac B. Zeremski M. Lau L. Nucleic Acids Res. 1996; 24: 1345-1351Crossref PubMed Scopus (43) Google Scholar, 46.Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (534) Google Scholar, 47.Clarkson R. Shang C. Levitt L. Howard T. Waters M. Mol. Endocrinol. 1999; 13: 619-631Crossref PubMed Scopus (61) Google Scholar) and may also regulate G-CSF-induced egr-1 expression. To determine whether the SRE-1 sequence is sufficient to induce egr-1 transcriptional activation in response to G-CSF, NFS60 cells were transiently transfected with a construct containing a synthetic oligonucleotide representing a single human SRE-1 in the pTE2 vector that contains a heterologous TK promoter and the CAT gene. pTE2 constructs with mutations within the SRE-1 were also prepared (Fig.5 A). The mutant SRE-1 constructs included a 3-base substitution within the CArG core consensus binding sequence (CArGm/BglII) and a GG to TT substitution within the ETS consensus binding site (GGA) at the left (LmR), the right (LRm), or both the left and right (LmRm) EBSs of SRE-1. Upon G-CSF treatment, the pTE2 SRE-1 wild-type construct demonstrated a 3.5-fold induction compared with the empty vector (p < 0.001; Fig. 5 B). Upon G-CSF stimulation, the pTE2-CArGm/ BglII, -LmR, -LRm, and -LmRm constructs behaved similarly to vector control, but all demonstrated reduced induction levels as compared with pTE2 SRE-1 (p = 0.0132, 0.0381, 0.0022, and 0.0056, respectively; Fig. 5 B). In diluent-treated cells, the basal activity of the pTE2-CArGm/ BglII, -LmR, -LRm, and -LmRm constructs demonstrated percentage of acetylation values similar to the pTE2 empty vector, ranging from 1.5 to 0.82%, respectively (data not shown). These experiments were repeated 3–11 times and were performed in triplicate. The CArG box mutation (pTE2-CArGm/ BglII) also efficiently inhibited competition of the SRF/SRE gel shift band (gel shift band 2) in EMSA experiments (Fig. 5 C, lanes 2 and 3). Mutations within the EBS core consensus sequence at the 5′ (LmR), 3′ (LRm), or both 5′ and 3′ EBS (LmRm) were previously shown to inhibit ETS protein binding (39.Watson D.K. Robi" @default.
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• W1991298889 title "Granulocyte Colony-stimulating Factor Induces egr-1Up-regulation through Interaction of Serum Response Element-binding Proteins" @default.
• W1991298889 cites W1426173733 @default.
• W1991298889 cites W1490733930 @default.
• W1991298889 cites W1494401713 @default.
• W1991298889 cites W1509136795 @default.
• W1991298889 cites W1525502277 @default.
• W1991298889 cites W1536698642 @default.
• W1991298889 cites W1539871462 @default.
• W1991298889 cites W1551086083 @default.
• W1991298889 cites W1648689484 @default.
• W1991298889 cites W1780245091 @default.
• W1991298889 cites W1964203341 @default.
• W1991298889 cites W1986309376 @default.
• W1991298889 cites W1993004730 @default.
• W1991298889 cites W1995801870 @default.
• W1991298889 cites W1998202437 @default.
• W1991298889 cites W1999157780 @default.
• W1991298889 cites W1999823156 @default.
• W1991298889 cites W2000082154 @default.
• W1991298889 cites W2002282120 @default.
• W1991298889 cites W2005150284 @default.
• W1991298889 cites W2005942661 @default.
• W1991298889 cites W2012959255 @default.
• W1991298889 cites W2014188686 @default.
• W1991298889 cites W2014350166 @default.
• W1991298889 cites W2020672093 @default.
• W1991298889 cites W2021253208 @default.
• W1991298889 cites W2027719624 @default.
• W1991298889 cites W2046630742 @default.
• W1991298889 cites W2051628416 @default.
• W1991298889 cites W2059796872 @default.
• W1991298889 cites W2060851546 @default.
• W1991298889 cites W2072907224 @default.
• W1991298889 cites W2080184511 @default.
• W1991298889 cites W2083129294 @default.
• W1991298889 cites W2090585013 @default.
• W1991298889 cites W2111204942 @default.
• W1991298889 cites W2117100531 @default.
• W1991298889 cites W2117231990 @default.
• W1991298889 cites W2123084648 @default.
• W1991298889 cites W2131713324 @default.
• W1991298889 cites W2142122628 @default.
• W1991298889 cites W2144042281 @default.
• W1991298889 cites W2144766409 @default.
• W1991298889 cites W2156692094 @default.
• W1991298889 cites W2161314639 @default.
• W1991298889 cites W2169385878 @default.
• W1991298889 cites W2262297515 @default.
• W1991298889 cites W4252438878 @default.
• W1991298889 cites W90944528 @default.
• W1991298889 cites W912507531 @default.
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