GATA-1- and FOG-dependent Activation of Megakaryocytic αIIB Gene Expression
2000; Elsevier BV; Volume: 275; Issue: 44 Linguagem: Inglês
10.1074/jbc.m006017200
ISSN1083-351X
AutoresPeter Gaines, Justin N. Geiger, Geoff Knudsen, Dhaya Seshasayee, Don M. Wojchowski,
Tópico(s)Immunodeficiency and Autoimmune Disorders
ResumoFOG is a multitype zinc finger protein that is essential for megakaryopoiesis, binds to the amino-terminal finger of GATA-1, and modulates the transcription of GATA-1 target genes. Presently investigated are effects of FOG and GATA-1 on the transcription of the megakaryocytic integrin gene,αIIb. In GATA-1-deficient FDCER cells (in the presence of endogenous FOG), ectopically expressed GATA-1 activated transcription 3–10-fold both from αIIbtemplates and the endogenous αIIb gene. The increased expression of FOG increased reporter construct transcription 30-fold overall. Unexpectedly, αIIb gene transcription also was stimulated efficiently upon the ectopic expression in of FOG per se. This occurred in the absence of any detectable expression of GATA-1 and was observed in multiple independent sublines for both the endogenousαIIb gene and transfected constructs yet proved to depend largely upon conserved GATA elements 457 and 55 base pairs upstream from the transcriptional start site. In 293 cells, FOG plus GATA-1 but not FOG alone only moderately stimulatedαIIb transcription, and no direct interactions of FOG with the αIIb promoter were detectable. Thus, FOG acts in concert with GATA-1 to stimulateαIIb expression but also can act via a GATA-1-independent route, which is proposed to involve additional hematopoietic-restricted cofactors (possibly GATA-2). FOG is a multitype zinc finger protein that is essential for megakaryopoiesis, binds to the amino-terminal finger of GATA-1, and modulates the transcription of GATA-1 target genes. Presently investigated are effects of FOG and GATA-1 on the transcription of the megakaryocytic integrin gene,αIIb. In GATA-1-deficient FDCER cells (in the presence of endogenous FOG), ectopically expressed GATA-1 activated transcription 3–10-fold both from αIIbtemplates and the endogenous αIIb gene. The increased expression of FOG increased reporter construct transcription 30-fold overall. Unexpectedly, αIIb gene transcription also was stimulated efficiently upon the ectopic expression in of FOG per se. This occurred in the absence of any detectable expression of GATA-1 and was observed in multiple independent sublines for both the endogenousαIIb gene and transfected constructs yet proved to depend largely upon conserved GATA elements 457 and 55 base pairs upstream from the transcriptional start site. In 293 cells, FOG plus GATA-1 but not FOG alone only moderately stimulatedαIIb transcription, and no direct interactions of FOG with the αIIb promoter were detectable. Thus, FOG acts in concert with GATA-1 to stimulateαIIb expression but also can act via a GATA-1-independent route, which is proposed to involve additional hematopoietic-restricted cofactors (possibly GATA-2). embryonic stem kilobase pair(s) base pair(s) polymerase chain reaction secreted alkaline phosphatase murine αIIb The course of development of hematopoietic progenitor cells is dictated, in part, by the differential expression of lineage-specifying transcription factors. Lymphopoiesis, myelopoiesis, granulopoiesis, erythropoiesis, and megakaryopoiesis, for example, are known from gene disruption experiments to depend on the expression of Ikaros (1Cortes M. Wong E. Koipally J. Georgopoulos K. Curr. Opin. Immunol. 1999; 11: 167-171Crossref PubMed Scopus (128) Google Scholar), PU.1 (2Anderson K.L. Smith K.A. Conners K. McKercher S.R. Maki R.A. Torbett B.E. Blood. 1998; 91: 3702-3710Crossref PubMed Google Scholar), CCAAT/enhancer-binding protein-α (3Radomska H.S. Huettner C.S. 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Blood. 1993; 81: 3234-3241Crossref PubMed Google Scholar). GATA-1gene disruption in mice results in embryonic lethality due to anemia (4Pevny L. Simon M.C. Robertson E. Klein W.H. Tsai S.F. D'Agati V. Orkin S.H. Costantini F. Nature. 1991; 349: 257-260Crossref PubMed Scopus (1044) Google Scholar) and to an arrest in erythroid development at a late proerythroblast stage (17Fujiwara Y. Browne C.P. Cunniff K. Goff S.C. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12355-12358Crossref PubMed Scopus (619) Google Scholar). During megakaryopoiesis, important roles for GATA-1 have been illustrated by experiments wherein the targeted disruption of an upstream activating element in the GATA-1 gene results in an accumulation of early megakaryocytic progenitor cells and a deficiency in platelet production (18Shivdasani R.A. Fujiwara Y. McDevitt M.A. Orkin S.H. EMBO J. 1997; 16: 3965-3973Crossref PubMed Scopus (587) Google Scholar). FOG is a 110,000-kDa multitype zinc finger protein that was discovered in a yeast two-hybrid screen based on its ability to interact specifically with the amino-terminal zinc finger of GATA-1 (8Tsang 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). In FOG−/− mice (and in FOG−/−embryonic stem (ES)1 cells differentiated in vitro) (19Tsang A.P. Fujiwara Y. Hom D.B. Orkin S.H. Genes Dev. 1998; 12: 1176-1188Crossref PubMed Scopus (298) Google Scholar), erythropoiesis is blocked at a penultimate stage, while effects on megakaryopoiesis are more dramatic, and FOG−/− yolk sac and fetal liver cells give rise to few, if any, megakaryocytes (19Tsang A.P. Fujiwara Y. Hom D.B. Orkin S.H. Genes Dev. 1998; 12: 1176-1188Crossref PubMed Scopus (298) Google Scholar). This broad defect indicates that FOG expression is either essential for early commitment to this lineage and/or that FOG acts subsequently to promote the transcription of late megakaryocytic genes. Since FOG is a co-factor for GATA-1 (8Tsang 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) and since functional GATA elements occur within the promoters of most megakaryocytic genes studied to date (20Minami T. Tachibana K. Imanishi T. Doi T. Eur. J. Biochem. 1998; 258: 879-889Crossref PubMed Scopus (67) Google Scholar, 21Ravid K. Doi T. Beeler D.L. Kuter D.J. Rosenberg R.D. Mol. Cell. Biol. 1991; 11: 6116-6127Crossref PubMed Scopus (99) Google Scholar, 22Lemarchandel V. Ghysdael J. Mignotte V. Rahuel C. Romeo P.H. Mol. Cell. Biol. 1993; 13: 668-676Crossref PubMed Scopus (179) Google Scholar, 23Deveaux S. Filipe A. Lemarchandel V. Ghysdael J. Romeo P.H. Mignotte V. Blood. 1996; 87: 4678-4685Crossref PubMed Google Scholar, 24Hashimoto Y. Ware J. J. Biol. Chem. 1995; 270: 24532-24539Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 25Bastian L.S. Yagi M. Chan C. Roth G.J. J. Biol. Chem. 1996; 271: 18554-18560Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), FOG might act as an obligatory GATA-1 co-factor. However, GATA-1 mutants that fail to bind FOG have been shown to activate the expression of the EKLF, heme-regulated eIF-α-kinase, and FOG (26Crispino J.D. Lodish M.B. MacKay J.P. Orkin S.H. Mol. Cell. 1999; 3: 219-228Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) genes in GATA-1-deficient ES cells. Thus, GATA-1 and/or FOG also may act in combination with alternate co-factors to regulate erythromegakaryocytic gene expression. With regards to megakaryocytic genes, investigations of roles for FOG are limited to two studies to date. In 416B cells, ectopically expressed FOG and GATA-1 increased the frequency of cells expressing acetylcholinesterase (8Tsang 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), and in 3T3 fibroblasts expression of FOG plus GATA-1 significantly activated transcription from a 7-kb upstream region of the erythromegakaryocytic gene p45 NF-E2(8Tsang 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). To further determine how FOG might affect megakaryocytic gene expression, we presently have investigated whether FOG might regulate the expression of the megakaryocytic integrin subunit, αIIb. αIIb expression is restricted to megakaryocytes, platelets, and their progenitors (27Phillips D.R. Charo I.F. Parise L.V. Fitzgerald L.A. Blood. 1988; 71: 831-843Crossref PubMed Google Scholar) and, together with a more broadly expressed subunit β3, forms an integrin receptor that functions in platelet aggregation (28Plow E.F. Ginsberg M.H. Prog. Hemostasis Thromb. 1989; 9: 117-156PubMed Google Scholar). In the promoter domains of the rat and humanαIIb genes, upstream as well as TATA box-positioned GATA-1 elements previously have been shown to be important for transcription (22Lemarchandel V. Ghysdael J. Mignotte V. Rahuel C. Romeo P.H. Mol. Cell. Biol. 1993; 13: 668-676Crossref PubMed Scopus (179) Google Scholar, 29Martin F. Prandini M.H. Thevenon D. Marguerie G. Uzan G. J. Biol. Chem. 1993; 268: 21606-21612Abstract Full Text PDF PubMed Google Scholar, 30Block K. Ravid K. Phung Q.H. Poncz M. Blood. 1994; 84: 3385-3393Crossref PubMed Google Scholar). Flanking each of these two GATA elements are elements for Ets factor binding that likewise contribute to efficient transcription from the proximal promoters of the rat and human αIIb genes (28Plow E.F. Ginsberg M.H. Prog. Hemostasis Thromb. 1989; 9: 117-156PubMed Google Scholar, 30Block K. Ravid K. Phung Q.H. Poncz M. Blood. 1994; 84: 3385-3393Crossref PubMed Google Scholar, 31Prandini M.H. Martin F. Thevenon D. Uzan G. Blood. 1996; 88: 2062-2070Crossref PubMed Google Scholar). Together with a −14 bp element for Sp1 (32Block K. Shou Y. Poncz M. Blood. 1996; 88: 2071-2080Crossref PubMed Google Scholar), these elements (which lie within a 600-bp promoter domain) have been proposed to direct the lineage-specific expression of αIIb, and similarly distributed elements also occur within the promoters of several additional megakaryocytic-specific genes including the Tpo receptor (23Deveaux S. Filipe A. Lemarchandel V. Ghysdael J. Romeo P.H. Mignotte V. Blood. 1996; 87: 4678-4685Crossref PubMed Google Scholar), chemokine PF4 (20Minami T. Tachibana K. Imanishi T. Doi T. Eur. J. Biochem. 1998; 258: 879-889Crossref PubMed Scopus (67) Google Scholar), GPIbα (24Hashimoto Y. Ware J. J. Biol. Chem. 1995; 270: 24532-24539Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), andGPIX (25Bastian L.S. Yagi M. Chan C. Roth G.J. J. Biol. Chem. 1996; 271: 18554-18560Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) genes. The present investigation focuses on αIIb gene expression and provides evidence that FOG acts as an important positive regulator via both GATA-1-dependent and independent routes. pREP4-G1 was prepared by subcloning a wild-type murine GATA-1 cDNA (1.6-kbXbaI–NotI fragment) from pCINeoGATA-1 (33Seshasayee D. Gaines P. Wojchowski D.M. Mol. Cell. Biol. 1998; 18: 3278-3288Crossref PubMed Scopus (43) Google Scholar) to pREP4 (Invitrogen, Palo Alto, CA). For pA2PuroEts1, a wild-type murineets-1 cDNA (1.9-kb SmaI–BstXI fragment from pKS-Ets-1) (34Nye J.A. Petersen J.M. Gunther C.V. Jonsen M.D. Graves B.J. Genes Dev. 1992; 6: 975-990Crossref PubMed Scopus (311) Google Scholar) was blunt-ended, ligated toEcoRI adaptors, and cloned to pA2Puro (35Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (588) Google Scholar). Vectors pXMGATA-1, pCINeoGATA-1, pXMER, and pEFNeoFOG have been described previously (8Tsang 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, 33Seshasayee D. Gaines P. Wojchowski D.M. Mol. Cell. Biol. 1998; 18: 3278-3288Crossref PubMed Scopus (43) Google Scholar). FDCER-FOG cells and independent clonal lines were prepared via the stable co-electrotransfection of FDCW2 cells (36Dexter T.M. Garland J. Scott D. Scolnick E. Metcalf D. J. Exp. Med. 1980; 152: 1036-1047Crossref PubMed Scopus (534) Google Scholar) with 55 μg of pXM-190ER (37Quelle D.E. Wojchowski D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4801-4805Crossref PubMed Scopus (62) Google Scholar) plus 5 μg of pEFNeoFOG, stepwise selection in G418 (1 mg/ml) and erythropoietin (25 units/ml), and limiting dilution. FDCER, FDCER-G1, and FDCER-G1-pCG1 cell lines have been described previously (33Seshasayee D. Gaines P. Wojchowski D.M. Mol. Cell. Biol. 1998; 18: 3278-3288Crossref PubMed Scopus (43) Google Scholar). FDCER cell lines were maintained at 37 °C (5% CO2) in Opti-MEM I medium (Life Technologies, Inc.) supplemented with 8% fetal bovine serum, and 25 units of erythropoietin/ml. 293-G1, 293-FOG, and 293-Ets1 cells were prepared by transfecting 293 cells with pREP4-G1, pEFNeoFOG, and pA2PuroEts1, respectively. Transfections were performed using calcium phosphate (Life Technologies), 15 μg of expression vector DNA, and 5 μg of sheared and purified salmon sperm DNA. 293-G1 cells were selected in hygromycin B (75 μg/ml), 293-FOG cells were selected in G418 (1 mg/ml), and 293-Ets1 cells were selected in puromycin (0.8 mg/ml). For 293-G1-FOG cells, 293-G1 cells were transfected with pEFNeoFOG, and sublines expressing FOG and GATA-1 were isolated by selection in G418 plus hygromycin. For 293-Ets1-G1-FOG cells, 293-G1-FOG cells were transfected with pAPuroEts1 and selected in G418, hygromycin, and puromycin. 293 cells and derived cell lines were maintained in Opti-MEM I medium supplemented with 8% fetal bovine serum and PSF (penicillin at 100 units/ml, streptomycin at 100 μg/ml, and amphotericin B at 0.25 μg/ml). Clonal sublines were isolated by limiting dilution. From a genomic murineαIIb clone in λ phage, an extended promoter domain was isolated by PCR using the following primers and thermal cycle: 5′-CAG ATT CAG CCT TTC AGC AGC ACT-3′ (nucleotides −1016 to −993 upstream from the transcription start site) and 5′-CTT CCT TCT TCC CAA ACG TCC TAA AC-3′ (nucleotides +7 to +32); 94 °C for 1 min, 60 °C for 30 s, and 72 °C for 60 s. Amplified products (30 cycles) were cloned to pCR-Script (Stratagene, La Jolla, CA) and sequenced. pαIIb910-Luc was prepared by subcloning a 942-bp m-αIIb promoter domain (SacII–PstI fragment) to pGL2-BSBasic (i.e. pGL2-Basic (Promega, Madison, WI), modified to contain the polylinker region of the pBS-SK(+)) (38Seshasayee D. Geiger J.N. Gaines P. Wojchowski D.M. J. Biol. Chem. 2000; 275: 22969-22977Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). From pαIIb910-Luc, pαIIb545-Luc was prepared by PCR using the following primers and thermal cycle: 5′-TCG GGG TAC CAA TGC AAC TGG CTG AGG CTG C-3′ (nucleotides −545 to −524 plus a 5′ KpnI site) and 5′-CTT TCT TTA TGT TTT TGG CGT CTT CCA-3′ (within the luciferase coding region of pGL2-BSBasic); 94 °C for 1 min, 30 s at 60 °C, and 60 s at 72 °C. Products (30 cycles) were cloned to pCR-Script, and a 577-bp proximal promoter domain was cloned (KpnI–XhoI fragment) to pGL2-Basic. Mutation of −457 bp or −55 bp GATA elements (to CATA) in pαIIb545-Luc was by QuikChange mutagenesis (Stratagene) using the following primer pairs: pαIIb545-Δ5′G-Luc (−457 mutation), 5′-TGA CAG CCT CTG GTC TTA TGA GGG GAG AAC AGC TTG-3′ plus 5′-GCA AGC TGT TCT CCC CTC ATA AGA CCA GAG GCT GTC−3′; pαIIb545-Δ3′G-Luc (−55 mutation), 5′-CCA TGA GCT CCA GTC TCA TAA GCT GAA ACT TCC GG-3′ plus 5′-CCG GAA GTT TCA GCT TAT GAG ACT GGA GCT CAT GG-3′. For each construct, PCR (12 cycles) was at 94 °C for 1 min, 55 °C for 1 min, and 68 °C for 2 min. The double mutant construct pαIIb545-Δ5′Δ3′G-Luc was generated by mutating the −55 bp GATA-1 element in pαIIb545-Δ5′G-Luc. All products were sequenced using 3′ BigDye-labeled dideoxynucleotide triphosphates and an ABI PRISM 377 PCR Sequencer (PerkinElmer Life Sciences). Putative transcription factor binding elements were profiled using Sequence Interpretation Tools software (available on the World Wide Web). In transfections of FDCER and derived cell lines, exponentially growing cells were adjusted to 3 × 105 cells/ml and transferred to six-well plates (3 ml/well). For each single transfection, 12 μl of FuGENE-6 liposomes (Roche Molecular Biochemicals) were added to 88 μl of Opti-MEM I medium, and this mixture then was combined with 1.8 μg of reporter plasmid DNA plus 0.2 μg of pCMV-SEAP (Tropix, Bedford, MA). Complexes were incubated at 23 °C for 15 min and added to cells. At 24 h of culture, transfected cells were collected (200 ×g for 10 min), washed in PBS, and lysed in reporter lysis buffer (1% Triton X-100, 2 mm 1, 2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, 2 mm dithiothreitol, 10% glycerol, 25 mmTris phosphate, pH 7.8) (Promega). Cleared supernatants were assayed for protein concentration (BCA protein assay; Pierce) and for luciferase activity. To control for limited variability in transfection efficiencies, secreted alkaline phosphatase (SEAP) activities in culture medium were assayed (Phospha-light kit; Tropix, Bedford MA). The activities of reporter plasmids in 293 cell lines were assayed as follows. Cells (30% confluent, 100-mm dishes) were transfected using calcium phosphate (Life Technologies), 4.5 μg of reporter plasmid DNA, 0.2 μg of pCMV-βgal, and 15 μg of sheared and purified salmon sperm DNA. At 48 h of culture, transfected cells were collected (200 × g for 10 min), washed in phosphate-buffered saline (140 mm NaCl, 2.7 mmKCl, 1.8 mm KH2PO4, and 8.1 mm Na2HPO4, pH 7.2), and lysed in reporter lysis buffer. Cleared supernatants (10 min at 8000 ×g) were assayed for luciferase activities (luciferase assay reagent; Promega, Madison, WI) and for β-galactosidase activities (39Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 17.33-17.34Google Scholar). RNA was isolated from FDC and 293 cell lines using TRIzol reagent (Life Technologies). cDNA was synthesized using an oligo(dT) primer and Superscript II RNase H−reverse transcriptase (Life Technologies). GATA-1, m-αIIb, and HPRT cDNAs were amplified using the following primer pairs: 5′-CCG CAA GGC ATC TGG CAA A-3′ and 5′-CGG GAG GTA GAG GCA GGA-3′ for murine GATA-1 (40Baron M.H. Farrington S.M. Mol. Cell. Biol. 1994; 14: 3108-3114Crossref PubMed Scopus (25) Google Scholar); 5′-AGG CAG AGA AGA CTC CGG TA-3′ and 5′-TAC CGA ATA TCC CCG GTA AC-3′ for murine αIIb (41Shivdasani R.A. Rosenblatt M.F. Zucker-Franklin D. Jackson C.W. Hunt P. Saris C.J. Orkin S.H. Cell. 1995; 81: 695-704Abstract Full Text PDF PubMed Scopus (630) Google Scholar); and 5′-CAC AGG ACT AGA ACA CCT GC-3′ and 5′-GCT GGT GAA AAG GAC CTC T-3′ for HPRT (42Weiss M.J. Keller G. Orkin S.H. Genes Dev. 1994; 8: 1184-1197Crossref PubMed Scopus (480) Google Scholar). Cycles for each were 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C. 1 μCi of [α-32P]dATP (3000 Ci/nmol) was included in each reaction. Products were electrophoresed in 5% acrylamide gels and were analyzed by autoradiography and phosphorimaging. Polyadenylated RNA was isolated using Oligotex spin columns (Qiagen, Chatsworth, CA). RNA was electrophoresed in 1.2% agarose, 6% formaldehyde gels, blotted to Nytran (Schleicher & Schuell), and fixed (312-nm exposure for 3 min plus 1 h at 68 °C under vacuum). Probes were prepared by random priming (33Seshasayee D. Gaines P. Wojchowski D.M. Mol. Cell. Biol. 1998; 18: 3278-3288Crossref PubMed Scopus (43) Google Scholar) using 25 ng of the following cDNA fragments: 1.8-kbKpnI–NotI fragment of pXMGATA-1 (murine GATA-1) (43Tsai S.F. Martin D.I. Zon L.I., AD, D.A. Wong G.G. Orkin S.H. Nature. 1989; 339: 446-451Crossref PubMed Scopus (666) Google Scholar); 1.2-kb XbaI fragment of pBOS-EKLF (murine EKLF) (44Miller I.J. Bieker J.J. Mol. Cell. Biol. 1993; 13: 2776-2786Crossref PubMed Scopus (655) Google Scholar); 3.0-kb EcoRI fragment of pMT2ADA-hαIIb (human αIIb); 700-bp EcoRI fragment of pUC-GATA2 (5′ region of murine GATA-2); and 0.8-kb KpnI–XhoI fragment of pSP-GAPDH (murine glyceraldehyde-3-phosphate dehydrogenase).32P-Labeled probes were purified on Sephadex G-50 microcolumns (Amersham Pharmacia Biotech), and hybridizations were with 2 × 106 cpm of probe/ml in QuickHyb solution as described previously (33Seshasayee D. Gaines P. Wojchowski D.M. Mol. Cell. Biol. 1998; 18: 3278-3288Crossref PubMed Scopus (43) Google Scholar). For Western blotting, cells were washed in phosphate-buffered saline and lysed in 2.5% SDS, 0.1 mdithiothreitol, 7.5% glycerol, 8.75 mm Tris-Cl (pH 6.8) (100 μl/106 cells). An antibody to GATA-1 (N6; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used (1:300 dilution) and was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). In primary studies, roles for GATA-1 and FOG on endogenous αIIb gene transcription were tested via their stable expression in FDCW2-derived cell lines. Recently, our laboratory has shown that these cells do not express GATA-1 at detectable levels, yet support the ability of exogenous GATA-1 to (auto)activate the de novo expression of the endogenous GATA-1 gene (38Seshasayee D. Geiger J.N. Gaines P. Wojchowski D.M. J. Biol. Chem. 2000; 275: 22969-22977Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). As shown in Fig.1 A, Northern blot analyses of FDCER-GATA-1 cells revealed that exogenous GATA-1 expression also activated the expression of the endogenous αIIbgene. To confirm that this result was not a fortuitous clonal effect, αIIb transcript expression in FDCER-G1 clones c.10, c.9, and c.11 (i.e. three independent clones) was analyzed by32P-labeled reverse transcriptase-PCR (Fig. 1 B). In each clone, αIIb transcript expression was elevated severalfold due to the expression of exogenous GATA-1 (as compared directly with parental FDCER cells). Next, to test whether this effect was mediated by cis elements within the αIIb promoter, an extended upstream region of the murine αIIb gene was cloned, sequenced, and used to prepare promoter-luciferase reporter constructs. Within this approximately 1000-bp promoter region, elements at −457 bp and −55 bp exist together with flanking consensus Ets factor binding elements (at −508 to −501 and −44 to −37 bp) (Fig.2). Within the human and ratαIIb promoters (45Heidenreich R. Eisman R. Surrey S. Delgrosso K. Bennett J.S. Schwartz E. Poncz M. Biochemistry. 1990; 29: 1232-1244Crossref PubMed Scopus (149) Google Scholar), each of these elements are positionally conserved. Within the previously undescribed upstream region, no additional consensus elements for these or other possible transfactors were apparent. Extended and truncatedαIIb promoter-reporter constructs were prepared (i.e. pαIIb910-Luc and pαIIb545-Luc), and their activities first were assayed in FDCER-G1 cells versuscontrol parental FDCER cells (Fig. 3). Exogenous GATA-1 (in the presence of low to moderate levels of endogenous FOG; see below) stimulated transcription from pαIIb545-Luc and pαIIb910-Luc approximately 5.4- and 2.9-fold, respectively. Maximal rates of transcription from each construct in FDCER-G1 cells were comparable, but transcription from pαIIb910-Luc in parental FDCER cells was more pronounced. No effects of GATA-1 expression on low level transcription of the promoterless control template pGL2Basic were observed. For pαIIb545-Luc, essentially equivalent results were obtained in repeated transfections of independent clonal lines of FDCER-G1 cells (Fig. 3 B).Figure 3GATA-1 induction of m-αIIb gene expression in FDCER-G1 cells is mediated by a −545 bp proximal promoter domain. A, the test constructs pαIIb910-Luc and pαIIb545-Luc (upper panel) (and pGL2-Basic as a negative control) were transfected into FDCER and FDCER-G1 cells. Levels of luciferase activity then were assayed in triplicate. Plotted are mean activities ± S.D. Shown (in parenthesis) are -fold increases in transcriptional reporter activity due to the stable ectopic expression of GATA-1 in FDCER-G1 cells. Limited variability in transfection efficiencies was accounted for by co-transfection with pSEAP and the assay of secreted alkaline phosphatase activity.B, four independent clonal lines of FDCER-G1 cells also were transfected with the reporter construct pαIIb545-Luc and assayed for luciferase activity. Results illustrated in A andB are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In FDCER-G1 cells, possible effects of FOG on αIIb gene transcription next were tested by increasing FOG expression in these lines via stable transfection. In FDCER-G1-FOG, FDCER-FOG, FDCER-G1, and parental FDCER cells, Northern blotting first was used to assay levels of FOG and GATA-1 (as well as GATA-2) transcript expression (Fig.4). As a point of comparison, levels of these transcripts in erythroid B6SUt.EP cells (and lymphoid CTLL2-ER cells) were co-analyzed. FOG transcript levels in FDCER cells were appreciable yet below those observed in B6SUt.EP cells. In FDCER-G1 cells, the ectopic expression of GATA-1 interestingly led to an estimated 3-fold increase in FOG transcript levels. In contrast, levels of GATA-2 transcript expression in FDCER-G1 and FDCER-G1-FOG cells were diminished. With regard to αIIb expression, ectopic expression of FOG in FDCER-G1 cells proved to stimulate rates of αIIb transcription to levels at least 5-fold above levels in FDCER-G1 cells and 38-fold above levels in parental FDCER cells (Fig.5 A). This result also was observed in repeated independent experiments in FDCER-G1-FOG cell lines. Based on these results (and the knowledge that FOG does not affect GATA-1's DNA binding activity) (26Crispino J.D. Lodish M.B. MacKay J.P. Orkin S.H. Mol. Cell. 1999; 3: 219-228Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), it was predicted that levels of FOG in FDCER-G1 cells might limit αIIb expression. If so, further increases in ectopic GATA-1 expression in FDCER-G1 cells might squelch rather than enhance the activity of FOG-GATA-1 complexes. To test this prediction, FDCER-G1 cells were transfected stably with a second GATA-1 expression vector (pCINeoG1), and effects on transcription from m-αIIb reporter constructs were assayed. Increased expression of exogenous GATA-1 in FDCER-G1-pCG1 cells proved to inhibit transcription from pαIIb545-Luc (and pαIIb910-Luc) approximately 3-fold as compared with FDCER-G1 cells (Fig. 5 B). Results are representative of three independent experiments (and increased levels of GATA-1 expression in FDCER-G1-pCG1 cells have been documented previously) (33Seshasayee D. Gaines P. Wojchowski D.M. Mol. Cell. Biol. 1998; 18: 3278-3288Crossref PubMed Scopus (43) Google Scholar). This apparent squelching effect demonstrates that levels of GATA-1 in FDCER-G1 cells do not limit αIIb transcription and is at l
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