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Originally published In Press as doi:10.1074/jbc.M006017200 on August 3, 2000

J. Biol. Chem., Vol. 275, Issue 44, 34114-34121, November 3, 2000
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GATA-1- and FOG-dependent Activation of Megakaryocytic alpha IIB Gene Expression*

Peter GainesDagger , Justin N. GeigerDagger §, Geoff Knudsen§, Dhaya SeshasayeeDagger , and Don M. WojchowskiDagger ||

From the Departments of  Biochemistry and Molecular Biology and Dagger  Veterinary Science, Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication, July 7, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha IIb. In GATA-1-deficient FDCER cells (in the presence of endogenous FOG), ectopically expressed GATA-1 activated transcription 3-10-fold both from alpha IIb templates and the endogenous alpha IIb gene. The increased expression of FOG increased reporter construct transcription 30-fold overall. Unexpectedly, alpha IIb 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 endogenous alpha IIb 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 stimulated alpha IIb transcription, and no direct interactions of FOG with the alpha IIb promoter were detectable. Thus, FOG acts in concert with GATA-1 to stimulate alpha IIb expression but also can act via a GATA-1-independent route, which is proposed to involve additional hematopoietic-restricted cofactors (possibly GATA-2).


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1), PU.1 (2), CCAAT/enhancer-binding protein-alpha (3), and GATA-1 (4), respectively. In addition, the abilities of such factors to control rates of target gene expression can involve interactions with additional lineage-restricted co-regulators. This is illustrated by roles for distinct Ikaros-Helios-Aiolos complexes in specifying developmental fates of T cells (1, 5, 6), by the regulation of Oct factor activity by the B cell-specific coactivator OBF-1/Bob1/OCA-B, (7) and during erythropoiesis by the complexing of GATA-1 with FOG (friend of GATA-1) (8), C-terminal binding protein (9), and Tal1 plus Lmo2 (10, 11). GATA-1 is a Cys2/Cys2 zinc finger DNA-binding protein that binds preferentially to (A/T)GATA(A/G) elements via its carboxyl-terminal finger domain (12) and is expressed in erythrocytes, megakaryocytes, eosinophils, and mast cells (13-16). GATA-1 gene disruption in mice results in embryonic lethality due to anemia (4) and to an arrest in erythroid development at a late proerythroblast stage (17). 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 (18). 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 (8). In FOG-/- mice (and in FOG-/- embryonic stem (ES)1 cells differentiated in vitro) (19), 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 (19). 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 (8) and since functional GATA elements occur within the promoters of most megakaryocytic genes studied to date (20-25), 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-alpha -kinase, and FOG (26) 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 (8), 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 (8). 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, alpha IIb. alpha IIb expression is restricted to megakaryocytes, platelets, and their progenitors (27) and, together with a more broadly expressed subunit beta 3, forms an integrin receptor that functions in platelet aggregation (28). In the promoter domains of the rat and human alpha IIb genes, upstream as well as TATA box-positioned GATA-1 elements previously have been shown to be important for transcription (22, 29, 30). 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 alpha IIb genes (28, 30, 31). Together with a -14 bp element for Sp1 (32), these elements (which lie within a 600-bp promoter domain) have been proposed to direct the lineage-specific expression of alpha IIb, and similarly distributed elements also occur within the promoters of several additional megakaryocytic-specific genes including the Tpo receptor (23), chemokine PF4 (20), GPIbalpha (24), and GPIX (25) genes. The present investigation focuses on alpha IIb gene expression and provides evidence that FOG acts as an important positive regulator via both GATA-1-dependent and independent routes.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Expression Vectors-- pREP4-G1 was prepared by subcloning a wild-type murine GATA-1 cDNA (1.6-kb XbaI-NotI fragment) from pCINeoGATA-1 (33) to pREP4 (Invitrogen, Palo Alto, CA). For pA2PuroEts1, a wild-type murine ets-1 cDNA (1.9-kb SmaI-BstXI fragment from pKS-Ets-1) (34) was blunt-ended, ligated to EcoRI adaptors, and cloned to pA2Puro (35). Vectors pXMGATA-1, pCINeoGATA-1, pXMER, and pEFNeoFOG have been described previously (8, 33).

Cell Lines-- FDCER-FOG cells and independent clonal lines were prepared via the stable co-electrotransfection of FDCW2 cells (36) with 55 µg of pXM-190ER (37) 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 (33). 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.

Reporter Plasmids-- From a genomic murine alpha IIb clone in lambda  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. palpha IIb910-Luc was prepared by subcloning a 942-bp m-alpha IIb 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(+)) (38). From palpha IIb910-Luc, palpha IIb545-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 palpha IIb545-Luc was by QuikChange mutagenesis (Stratagene) using the following primer pairs: palpha IIb545-Delta 5'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'; palpha IIb545-Delta 3'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 palpha IIb545-Delta 5'Delta 3'G-Luc was generated by mutating the -55 bp GATA-1 element in palpha IIb545-Delta 5'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).

Transcriptional Reporter Assays-- 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 mM Tris 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-beta gal, 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 mM KCl, 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 beta -galactosidase activities (39).

RNA Isolation and Reverse Transcription-Polymerase Chain Reactions-- 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-alpha IIb, 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 (40); 5'-AGG CAG AGA AGA CTC CGG TA-3' and 5'-TAC CGA ATA TCC CCG GTA AC-3' for murine alpha IIb (41); and 5'-CAC AGG ACT AGA ACA CCT GC-3' and 5'-GCT GGT GAA AAG GAC CTC T-3' for HPRT (42). Cycles for each were 1 min at 94 °C, 1 min at 60 °C, and 2 min at 72 °C. 1 µCi of [alpha -32P]dATP (3000 Ci/nmol) was included in each reaction. Products were electrophoresed in 5% acrylamide gels and were analyzed by autoradiography and phosphorimaging.

Northern and Western Blotting-- 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 (33) using 25 ng of the following cDNA fragments: 1.8-kb KpnI-NotI fragment of pXMGATA-1 (murine GATA-1) (43); 1.2-kb XbaI fragment of pBOS-EKLF (murine EKLF) (44); 3.0-kb EcoRI fragment of pMT2ADA-halpha IIb (human alpha IIb); 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 (33). For Western blotting, cells were washed in phosphate-buffered saline and lysed in 2.5% SDS, 0.1 M dithiothreitol, 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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GATA-1-dependent Activation of alpha IIb Gene Transcription in FDCER Cell Lines-- In primary studies, roles for GATA-1 and FOG on endogenous alpha IIb 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 (38). As shown in Fig. 1A, Northern blot analyses of FDCER-GATA-1 cells revealed that exogenous GATA-1 expression also activated the expression of the endogenous alpha IIb gene. To confirm that this result was not a fortuitous clonal effect, alpha IIb transcript expression in FDCER-G1 clones c.10, c.9, and c.11 (i.e. three independent clones) was analyzed by 32P-labeled reverse transcriptase-PCR (Fig. 1B). In each clone, alpha IIb transcript expression was elevated severalfold due to the expression of exogenous GATA-1 (as compared directly with parental FDCER cells).


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  		Fig. 1.   Endogenous m-alpha IIb gene expression is activated upon the ectopic expression of GATA-1 in FDCER cells. Upper panel, shown in Northern blots are levels of GATA-1, m-alpha IIb, and FOG transcript expression in GATA-1-expressing FDCER-G1 cells versus parental myeloid FDCER cells. Lower panel, endogenous m-alpha IIb transcripts in three independent clonal lines of FDCER-G1 cells (versus parental FDCER cells) also were assayed by reverse transcriptase-PCR. Shown are PCR cycle numbers and the positions of amplified m-alpha IIb and HPRT products.

Next, to test whether this effect was mediated by cis elements within the alpha IIb promoter, an extended upstream region of the murine alpha IIb 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 rat alpha IIb promoters (45), 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 truncated alpha IIb promoter-reporter constructs were prepared (i.e. palpha IIb910-Luc and palpha IIb545-Luc), and their activities first were assayed in FDCER-G1 cells versus control parental FDCER cells (Fig. 3). Exogenous GATA-1 (in the presence of low to moderate levels of endogenous FOG; see below) stimulated transcription from palpha IIb545-Luc and palpha IIb910-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 palpha IIb910-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 palpha IIb545-Luc, essentially equivalent results were obtained in repeated transfections of independent clonal lines of FDCER-G1 cells (Fig. 3B).


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  		Fig. 2.   Features of the murine alpha IIb promoter. The 5' domain of m-alpha IIb was cloned, sequenced, and aligned with previously sequenced regions of the rat (r) and human (h) alpha IIb genes (45). Shown are consensus elements for GATA-1 (boxed) and Ets (bracketed), together with an E-box-like element (broken box). Differences from a previously reported sequence (58) are nucleotides C at position -534 and T at position -527 (previously assigned as T and C, respectively).


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  		Fig. 3.   GATA-1 induction of m-alpha IIb gene expression in FDCER-G1 cells is mediated by a -545 bp proximal promoter domain. A, the test constructs palpha IIb910-Luc and palpha IIb545-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 palpha IIb545-Luc and assayed for luciferase activity. Results illustrated in A and B are representative of three independent experiments.

FOG Amplifies GATA-1-dependent alpha IIb Gene Transcription in FDCER-G1 Cells-- In FDCER-G1 cells, possible effects of FOG on alpha IIb 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 alpha IIb expression, ectopic expression of FOG in FDCER-G1 cells proved to stimulate rates of alpha IIb transcription to levels at least 5-fold above levels in FDCER-G1 cells and 38-fold above levels in parental FDCER cells (Fig. 5A). 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) (26), it was predicted that levels of FOG in FDCER-G1 cells might limit alpha IIb 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-alpha IIb reporter constructs were assayed. Increased expression of exogenous GATA-1 in FDCER-G1-pCG1 cells proved to inhibit transcription from palpha IIb545-Luc (and palpha IIb910-Luc) approximately 3-fold as compared with FDCER-G1 cells (Fig. 5B). Results are representative of three independent experiments (and increased levels of GATA-1 expression in FDCER-G1-pCG1 cells have been documented previously) (33). This apparent squelching effect demonstrates that levels of GATA-1 in FDCER-G1 cells do not limit alpha IIb transcription and is at least consistent with the notion that, when overexpressed, GATA-1 instead may sequester an apparently limiting co-factor such as FOG.


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  		Fig. 4.   Northern blot analyses of FOG, GATA-1, and GATA-2 transcript levels in FDCER-G1 and FDCER-G1-FOG cells. Shown are FOG, GATA-1, and GATA-2 transcript levels in FDCER, FDCER-G1, FDCER-G1-FOG, and FDCER-FOG cells. In FDCER-G1 cells, FOG transcript expression was increased due to the ectopic expression of GATA-1, while levels of GATA-2 transcripts were diminished. In FDCER cells transfected stably with pEFNeoFOG (i.e. FDCER-FOG cells), levels of GATA-2 transcripts were unaffected. Also analyzed were CTLL2ER cells (a cytotoxic T-cell line ectopically expressing the EpoR) and erythroid B6SUt.EP cells (as negative and positive control cell lines, respectively).


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  		Fig. 5.   Rates of GATA-1-induced transcription at the m-alpha IIb promoter in FDCER-G1 cells are limited by FOG. A, FDCER-G1 cells were transfected stably with a FOG expression vector (pEFNeoFOG), and the ability of clonal (cF) and polyclonal (PC) FDCER-G1-FOG cells (versus parental FDCER and FDCER-G1 cells) to support transcription from palpha IIb545-Luc was assayed. Plotted are mean activities ± S.D. for triplicate transfections. B, also illustrated is an inhibition of palpha IIb545-Luc and palpha IIb910-Luc transcription due to the overexpression of GATA-1 in FDCER-G1-pCG1 cells. Results in each panel are representative of three independent experiments.

FOG Activation of alpha IIb Gene Transcription via a GATA-1-independent Route-- In control experiments, FOG per se also was expressed in FDCER cells, and levels of alpha IIb gene transcription were assayed. Initially, this was tested using palpha IIb545-Luc. Somewhat unexpectedly, the expression of FOG at levels 2-3-fold above endogenous levels (see above; Fig. 4) increased rates of palpha IIb545 transcription in FDCER-FOG cells to levels essentially equivalent to those supported by GATA-1 in FDCER-G1 cells (Fig. 6A). This was observed in clonal as well as in polyclonal FDCER-FOG cell lines and suggested that FOG might promote alpha IIb gene transcription in the absence of GATA-1. To critically test this possibility, 32P-labeled reverse transcriptase-PCR was used to assay endogenous GATA-1 and alpha IIb transcript levels (Fig. 6B). In FDCER-FOG cells, no GATA-1 transcripts were detected. However, levels of endogenous alpha IIb gene expression in all clones tested were increased to levels approximating those induced by GATA-1 in FDCER-G1 cells.


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  		Fig. 6.   Exogenous FOG stimulates transcription from the m-alpha IIb proximal promoter and endogenous gene in the absence of detectable levels of GATA-1. A, FDCER cells were transfected with pEFNeoFOG (plus pA2Puro), and sublines expressing FOG at elevated levels were isolated. The reporter construct palpha IIb545-Luc (together with pSEAP) then were transfected into three independent clones of FDCER-FOG cells (and in parallel FDCER and FDCER-G1 cells), and transcriptional activities were assayed. Plotted are the means ± S.D. of triplicate transfections normalized against SEAP activities. Results are representative of three independent experiments. B, in FDCER cells, FDCER-G1 cells ectopically expressing GATA-1 and FDCER-FOG cells ectopically expressing FOG, reverse transcriptase-PCR also was used to analyze levels of m-alpha IIb and GATA-1 transcript expression. cDNAs were amplified for the number of cycles shown, and HPRT primers were included in all reactions as an internal control.

The extent to which FOG-stimulated transcription of the m-alpha IIb gene depended upon intact -457 bp and/or -55 bp TATA box position GATA elements next was tested. First, roles for these elements in supporting palpha IIb545-Luc transcription in FDCER-G1 cells versus parental FDCER cells were examined. One, the other, or both GATA elements were mutated to the nonfunctional sequence CATA (46), and activities of the derived constructs palpha IIb545-Delta 5'G-Luc, palpha IIb545-Delta 3'G-Luc and palpha IIb545-Delta 5'Delta 3'G-Luc were assayed. Mutation of the -457 bp GATA element inhibited transcription from the m-alpha IIb promoter in FDCER-G1 cells 5.5-fold (20,900- to 3800-unit decrease); mutation of the TATA box-positioned -55 bp GATA element inhibited transcription 2.7-fold (20,900- to 7800-unit decrease); and the mutation of both GATA elements inhibited transcription 9.1-fold (20,900- to 2300-unit decrease) (Fig. 7A, upper panel). Similar results were observed in all independent clonal lines of FDCER-G1 cells tested.2 These effects on m-alpha IIb gene transcription of disrupting -457 and -55 bp GATA elements are similar to effects observed previously for analogous mutations of the rat alpha IIb promoter as assayed in transfected rat marrow cells (30) and suggest that these two GATA elements and their associated trans-factors act in a somewhat more than additive fashion to promote m-alpha IIb gene transcription. Effects of mutating GATA elements on FOG-stimulated m-alpha IIb transcription next were tested in FDCER-G1-FOG cells (Fig. 7A, lower panel). Consistent with data in Fig. 5, the increased expression of FOG in these cells stimulated transcription from the wild-type palpha IIb545-Luc construct 4-fold above levels in FDCER-G1 cells (and 42-fold above levels in FDCER cells). Mutation of the -457 bp GATA element inhibited transcription 7.3-fold (84,200- to 11,600-unit decrease), while mutation of the TATA box-positioned -55 bp GATA element inhibited transcription 5.4-fold (84,200- to 15,700-unit decrease). Thus, in the presence of increased levels of FOG, both GATA elements appear to contribute to the efficient m-alpha IIb transcription. Disruption of both GATA elements inhibited palpha IIb545-Luc (Delta 5'Delta 3'G) transcription in FDCER-G1-FOG cells 12.0-fold (84,200- to 7000-unit decrease) but interestingly did not inhibit transcription to the background levels observed for palpha IIb545-Delta 5'Delta 3'G-Luc in FDCER cells. This result, together with the observed ability of FOG in the absence of GATA-1 to activate m-alpha IIb gene transcription in FDCER-FOG cells, raised the possibility that FOG might also stimulate m-alpha IIb gene expression via GATA-1-independent mechanisms. To further test this possibility, transcription from the mutant construct palpha IIb545-Delta 5'Delta 3'G-Luc was assayed in FDCER-FOG cells (Fig. 7B). Residual transcription from palpha IIb545-Delta 5'Delta 3'G-Luc in these GATA-1-deficient cells was somewhat higher than in FDCER cells, again suggesting that FOG might stimulate m-alpha IIb gene expression at least to a limited extent via a GATA element-independent route.


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  		Fig. 7.   GATA-1- and FOG-induced transcription at the m-alpha IIb promoter depends (in part) on upstream and TATA-positioned GATA elements. To test the extent to which FOG-stimulated transcription depends on intact GATA elements at -457 and -55 bp within the m-alpha IIb promoter, palpha IIb545-Delta G5'-Luc (mutated -457 element), palpha IIb545-Delta G3'-Luc (mutated -55 element), and palpha IIb545-Delta G5'Delta G3'-Luc (both elements mutated to CATA) were constructed, and their activities in FDCER, FDCER-G1, FDCER-G1-FOG, and FDCER-FOG cell lines were assayed. A, upper panel, illustrated is the GATA-1-dependent activation of wild type and mutant palpha IIb545-Luc constructs in FDCER versus FDCER-G1 cells. Mean luciferase activities ± S.D. of triplicate transfections are shown. Shown in parentheses are -fold increases of activity in FDCER-G1 cells above FDCER cells transfected in parallel with the same construct. A, lower panel, illustrated is the FOG-enhanced activation of wild type and mutant palpha IIb544-Luc constructs in FDCER-G1-FOG cells. For each construct, shown in parentheses, are -fold increases in activity as compared with levels in FDCER cells (see upper panel). The hatched area in the wild-type histogram represents increases in m-alpha IIb transcription in FDCER-G1-FOG cells above levels in FDCER-G1 cells. Limited variability in transfection efficiencies was controlled for based on secreted alkaline phosphatase activities. Results in each panel are representative of three independent experiments. B, the reporter constructs palpha IIb545-Luc (upper panel) and palpha IIb545-Delta 5'GDelta 3'G-Luc (lower panel) (together with pSEAP) were transfected into FDCER, FDCER-G1, and FDCER-FOG cells, and transcriptional activities were assayed. Shown are -fold increases in luciferase activities of the palpha IIb545-Delta 5'GDelta 3'G-Luc construct in FDCER-G1 or FDCER-FOG cells as compared with those in FDCER cells. Plotted are the means ± S.D. of triplicate transfections normalized against SEAP activities. Results in each panel are representative of three independent experiments.

Finally, to test whether transcription from alpha IIb proximal promoter constructs would be stimulated efficiently by ectopically expressed FOG, GATA-1, and/or Ets-1 in nonhematopoietic cells, these factors first were expressed stably in 293 fibroblasts to yield 293-G1, 293-FOG, 293-G1-FOG, and 293-G1-FOG Ets-1 cells. Transfections with palpha IIb545-Luc then were performed, and activities were assayed in triplicate (with pCMV-beta Gal as a co-reporter). As shown in Fig. 8A (upper panel), ectopically expressed GATA-1 per se only slightly increased rates of palpha IIb545-Luc transcription in 293-G1 cells (approximately 2-fold above parental 293 cells), while ectopically expressed FOG (in 293-FOG cells) per se had no detectable positive effect. In combination, however, these factors in 293-G1-FOG cells reproducibly stimulated transcription from the m-alpha IIb gene proximal promoter approximately 6-fold above levels in parental 293 cells. Ectopically expressed Ets-1, in contrast, did not significantly stimulate m-alpha IIb transcription in this reconstituted system in the absence or presence of GATA-1 (or GATA-1 plus FOG) (Fig. 8A, lower panel). Data shown are representative of three independent experiments in which essentially equivalent effects of these trans-factors on transcription from the m-alpha IIb proximal promoter were observed and similar activities were observed for palpha IIb910-Luc.2 In advance, Western and Northern blotting were used to identify matched sublines in which expression levels were highly comparable (Fig. 8B). These results demonstrate the positive co-action of FOG and GATA-1 in fibroblastic cells. These effects, however, were blunted as compared with those in FDCER-G1-FOG cells, and this is at least consistent with possible roles for alternate hematopoietic factors in activating the alpha IIb gene.


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  		Fig. 8.   FOG and GATA-1-dependent transcription of the m-alpha IIb promoter in 293 fibroblasts. A, 293 cells were transfected stably with expression vectors for GATA-1 (pREP4GATA-1), FOG (pEFNeoFOG), and/or mEts1 (pAPuroEts-1), and the following cell lines stably expressing these factors (separately or in combination) were isolated: 293, 293-G1, 293-FOG, 293-G1-FOG, 293-Ets1, 293 Ets1-G1, and 293-Ets1-G1-FOG cells. The ability of each subline to support transcription from palpha IIb545-Luc then was assayed. pCMVbeta gal was co-transfected, and samples were normalized for beta -galactosidase activity to correct for limited variability in transfection efficiencies. Plotted are the activities (mean relative light units ± S.D.) of triplicate transfections. Shown in parentheses are -fold increases in luciferase activity supported by the specified transcription factors. B, levels of GATA-1, FOG, and/or Ets-1 expression in the above 293 cells and derived cell lines were assayed by Western blotting (for GATA-1; upper panel) or by Northern blotting (for FOG and Ets-1; lower panels). Equivalence in RNA loading was confirmed by hybridization to a 32P-labeled glyceraldehyde-3-phosphate dehydrogenase probe (GAPDH).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As introduced above, the disrupted expression of FOG in mice blocks the formation of megakaryocytes and erythrocytes. As shown initially in studies by Crispino et al. (26), however, FOG appears to be dispensable for the activation of at least certain GATA-1 target genes and has been proposed to act differentially with GATA-1 at distinct subsets of erythroid and megakaryocytic genes (19, 26). In addition, a FOG homologue in Xenopus recently has been discovered and demonstrated through ectopic expression and explant assays to repress the transcription of at least certain erythroid genes (possibly via interactions with C-terminal binding protein) (47). In separate studies, FOG also has been shown to inhibit GATA-1-dependent transcription from the eosinophil- and basophil-specific gene, eosinophil major basic protein (48). These reports suggest that FOG does not act simply as a GATA-1 co-activator and that its activity depends upon not only lineage but also promoter contexts. Despite FOG's essential role in megakaryopoiesis (19), it also is noted that studies of its effects on megakaryocytic genes are limited to date to the demonstrated ability of FOG to stimulate transcription from a 7000-bp promoter domain of the erythromegakaryocytic p45 NFE2 gene in transiently transfected 3T3 fibroblasts (8). Such considerations prompted the present investigations of roles for FOG (and GATA-1) in alpha IIb gene expression.

As a hematopoietic cell line that is GATA-1-deficient and expresses endogenous FOG at moderate levels, FDCER cells proved to be an advantageous model in which to test dosage effects of these (co)factors on alpha IIb gene expression. With regard to GATA-1 effects per se, with the exception of the observation in chicken HD50M myeloblastic cells that exogenous GATA-1 can promote the outgrowth of thromboblastic-like cells (including a subline that stained with an antibody thought to be specific for avian alpha II/beta 3 integrins) (49), the present study is the first to demonstrate GATA-1-dependent activation of endogenous alpha IIb gene expression. Consistent with the results of previous experiments, promoter-reporter transfection experiments in FDCER-G1 cells showed this to depend upon GATA elements positioned at -457 and -55 bp within the proximal alpha IIb promoter. In several additional megakaryocytic genes including mpl (23), GPIbalpha (24), GP-1X (25), and PF4 (20), GATA elements likewise occur within 90 bp of transcription start sites and have been proposed to substitute for canonical TATA boxes by binding to a multisubunit TFIID complex, which may contain GATA-1, an Ets factor, Sp1, and (based on the present findings) possibly FOG. In the present studies, however, this -55 GATA element contributed meaningfully to GATA-1-stimulated (and FOG-stimulated) alpha IIb transcription yet proved to be somewhat less important than a -457 bp element. Flanking each of these GATA elements are sites for the binding of one or more Ets family transcription factors, and these sites also have been demonstrated to support transcription at the human and rat alpha IIb proximal promoters (22, 30-32, 50). In FDCER and FDCER-G1 cells, Northern blot analyses of Fli-1, Spi-1, and Ets-1 transcripts revealed each to be expressed at appreciable levels, and in FDCER-G1 lines Ets-1 levels were increased approximately 2-fold.2 Interestingly, Ets-1 (and Ets-2) recently has been shown to bind to C-terminal binding protein/p300 (51, 52), and based on the ability of GATA-1 to bind a nonequivalent region of C-terminal binding protein/p300 (9, 53), it is possible that FOG might also tether at least indirectly to one or another Ets factor.

More remarkable are, first, the overall 30-40-fold increase in levels of alpha IIb promoter transcription stimulated by ectopic co-expression of GATA-1 plus FOG in FDCER-G1-FOG cell lines and, second, the ability of FOG to activate alpha IIb gene transcription in GATA-1-deficient FDCER cells. Increases in alpha IIb transcription due to exogenous FOG in FDCER-G1-FOG cells are suggested to reflect FOG's role as a limiting factor in GATA-1-dependent alpha IIb gene activation, and direct interactions between these co-factors are the most straightforward to propose as a mechanism underlying observed effects on alpha IIb transcription. However, opportunities also exist for GATA-1 and possibly FOG to modulate by secondary routes the expression of other potential regulators of alpha IIb gene expression. The case for direct action mechanisms is underlined by the apparent ability of exogenous GATA-1 to squelch alpha IIb transcription when expressed at elevated levels in FDCER-G1-pCG1 cells (see Fig. 5B) and by the major dependence of FOG activity in FDCER-G1-FOG cells on the intactness of -457 and -55 bp GATA elements. Nonetheless, several observations also are consistent with alternate mechanisms of FOG action in addition to those mediated by interactions with GATA-1. These include FOG's ability to activate alpha IIb transcription in the apparent absence of GATA-1; the residual activity exerted by FOG toward a palpha IIb545-Delta 5'Delta 3'G-Luc template in FDCER-G1, FDCER-G1-FOG, and FDCER-FOG cells (see Fig. 8); and the comparably limited ability of FOG plus GATA-1 to activate alpha IIb transcription in 293 cells in the absence of additional hematopoietic factors (other than Ets-1). FDCER cells normally express FOG at readily detectable levels (see Fig. 4). Thus, the moderate ectopic increase in FOG expression, while not predicted (in the absence of GATA-1) to significantly affect m-alpha IIb gene expression, proved to stimulate the transcription of palpha IIb545-Luc and the endogenous m-alpha IIb gene in FDCER-FOG cells at levels comparable with those supported in FDCER-G1 cells by exogenous GATA-1. Recently, GATA-2 has been shown to be capable of binding via its amino-terminal zinc finger to FOG (8, 54) and is known to possess DNA binding properties highly similar to those of GATA-1 (55, 56). Also, GATA-2 is expressed at appreciable levels in FDCER cell lines (see Fig. 4), and it therefore presently is speculated that FOG activation of m-alpha IIb expression in FDCER-FOG cells might be facilitated by its partnering with GATA-2. This raises questions as to whether FOG might also interact with or otherwise regulate GATA-2 in other cells, including immature hematopoietic cells, which require high level GATA-2 expression for their early development (57). Consistent with this notion, Deconinck et al. recently have hypothesized that proliferation of hematopoietic progenitor cells in Xenopus might involve effects of FOG on GATA-2 expression (47). In this context, the down-regulation of GATA-2 and up-regulation of FOG due to GATA-1 expression in FDCER-G1 cells (see Fig. 4) is again noted. Finally, it also is possible that a presently identified E-box-like element immediately 3' to the -457 bp GATA element in the murine and human alpha IIb promoters (see Fig. 2) might also recruit FOG-GATA-1 (and/or FOG-GATA-2) complexes via its potential to bind Tal1-Lmo2 complexes (11). Each of the above possible architectures is consistent with recently mapped interactions among these transcription factors (9, 11, 53), and in future experiments, it should be of interest to discover which of these architectures might provide for the selective high level expression of alpha IIb in megakaryocytic but not erythroid cells (each of which are believed to express all of the above-mentioned factors).

    ACKNOWLEDGEMENTS

We thank Dr. Stuart Orkin for pEFNeoFOG and pUC-GATA2, Dr. Barbara Graves for pKS-Ets-1, and Dr. Mortimer Poncz for the genomic murine alpha IIb clone and pMT2ADA-halpha IIb.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by National Institutes of Health Grants R01 HL44491 (to D. M. W.) and F32 HL09749 (to P. G.).

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: 115 William L. Henning Bldg., Pennsylvania State University, University Park, PA 16802. Tel.: 814-865-0657; Fax: 814-863-6140; E-mail: dmw1@psu.edu.

Published, JBC Papers in Press, August 3, 2000, DOI 10.1074/jbc.M006017200

2 P. Gaines, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: ES, embryonic stem; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; SEAP, secreted alkaline phosphatase; m-alpha IIb, murine alpha IIb.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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