Blood online
Home About Blood Authors Subscriptions Permission Advertising Public Access contact us
 

 
Advanced
Current Issue
First Edition
Future Articles
Archives
Submit to Blood
Search
American Society of Hematology
Meeting Abstracts
Email Alerts
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamaguchi, Y.
Right arrow Articles by Suda, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamaguchi, Y.
Right arrow Articles by Suda, T.
Related Collections
Right arrow Phagocytes
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

arrow to previous article Previous Article  |  Table of Contents  |  Next Article next article arrow

Blood, Vol. 94 No. 4 (August 15), 1999: pp. 1429-1439

C/EBPbeta and GATA-1 Synergistically Regulate Activity of the Eosinophil Granule Major Basic Protein Promoter: Implication for C/EBPbeta Activity in Eosinophil Gene Expression

By Yuji Yamaguchi, Hitoshi Nishio, Kenji Kishi, Steven J. Ackerman, and Toshio Suda

From the Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan; the Department of Hematology and Rheumatology, Tokai University School of Medicine, Isehara, Japan; and the Department of Biochemistry and Molecular Biology, University of Illinois at Chicago, Chicago, IL.


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBPalpha mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPbeta expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPbeta protein could bind to the potential C/EBP-binding site (bp -90 to -82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPbeta and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPbeta and GATA-1 can stimulate the MBP promoter in the C/EBPbeta and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPbeta and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPbeta or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPbeta . Expression of FOG (<UNL>F</UNL>riend <UNL>o</UNL>f <UNL>G</UNL>ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPbeta synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.
© 1999 by The American Society of Hematology.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

DIFFERENTIATION OF eosinophils from myeloid progenitors is regulated by several different cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5.1-3 Of these cytokines, IL-5 is eosinophil-lineage specific and plays a principal role in the terminal differentiation of eosinophils and development of eosinophilia.3-6 However, the molecular basis for the commitment of hematopoietic stem cells to the eosinophil lineage remains to be elucidated. We have been characterizing the regulatory regions of genes encoding eosinophil-specific proteins, including eosinophil peroxidase (EPO),7 Charcot-Leyden crystal (CLC) protein,8 and eosinophil major basic protein (MBP),9 to identify transcription factors involved in regulating the commitment and terminal differentiation of myeloid progenitors to the eosinophil lineage. In analyses of the MBP promoter region, we have identified a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription, providing the first evidence for a GATA-regulated gene in the eosinophil lineage.9 Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in a positively acting region of the MBP promoter.9 The C/EBP family belongs to the larger family of basic leucine zipper (bZip) transcription factors. To date, 6 members have been cloned and characterized, including C/EBPalpha ,10,11 C/EBPbeta ,12-15 C/EBPgamma ,16 C/EBPdelta ,17-20 C/EBPepsilon ,21,22 and CHOP.23 C/EBP proteins have been shown to regulate a number of hepatic10,14,24,25 and adipocyte17,26,27 genes. Expression of C/EBP proteins in the hematopoietic system, particularly C/EBPalpha , C/EBPbeta , and C/EBPepsilon , is limited to myeloid cells, suggesting that they play an important role in the differentiation of myeloid cells.21,22,28-37 Recent studies from other laboratories have shown that PU.1 (Spi-1) and the C/EBP family, in particular C/EBPalpha , may serve as master regulators of myeloid development, in part through their regulation of lineage-specific growth factor receptor genes, including macrophage colony-stimulating factor receptor (M-CSFR),29,30 granulocyte colony-stimulating factor receptor (G-CSFR),31,32 and GM-CSF receptor alpha  (GM-CSFRalpha ).38 In addition, in the avian hematopoietic system, it has been reported that NF-M, the chicken homolog of C/EBPbeta , induces eosinophil differentiation in a multipotent progenitor cell line transformed by the Myb-Ets oncoprotein.39 However, the target promoter(s) of eosinophil-associated genes for the C/EBP transcription factor family remains unknown.

We have recently demonstrated that GATA-1 is one of the transcription factors involved in regulating expression of eosinophil genes during the differentiation of myeloid progenitors to the eosinophil lineage.9,40 However, GATA-1 plays a crucial role in the differentiation of hematopoietic progenitors to the erythroid and megakaryocyte lineages.41-43 Differences in the role of GATA-1 in eosinophil versus erythroid/megakaryocyte development and gene expression remain to be elucidated.

We report here that C/EBPbeta can physically interact with GATA-1 and synergistically transactivate the MBP P2 promoter with GATA-1. In addition, we provide evidence that <UNL>F</UNL>riend <UNL>o</UNL>f <UNL>G</UNL>ATA (FOG), a cofactor for GATA-binding proteins, may act as a negative cofactor in the eosinophil lineage, unlike its role as a positive cofactor for erythroid and megakaryocyte gene expression.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cultures.   The eosinophil-committed subline of the HL-60 promyelocytic leukemic cell line, HL-60-C15 (a gift of Dr Steven Fishkoff),44 and T-lymphocytic Jurkat cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS) and 2 mmol/L L-glutamine and passaged twice weekly. Another eosinophil cell line, HT93A cells, which are able to differentiate towards the eosinophil and neutrophil lineages in response to retinoic acid (RA), was also maintained in RPMI 1640 supplemented with 8% FBS, 2 mmol/L L-glutamine, 5 × 10-5M2-mercaptoethanol (2ME), and 1 mmol/L sodium pyruvate.45

Northern blot analysis.   Total RNA was prepared from HT93A cells stimulated with 2 µmol/L RA by TRizol Reagent (GIBCO BRL, Rockville, MD). Poly (A)+ mRNA was then separated by Oligotex-dT30 (Takara Shuzo, Kyoto, Japan). The poly (A)+ mRNA was transferred to Hybond-N nylon membranes (Amersham International plc, Buckinghamshire, UK) and the blots were probed sequentially with full-length cDNAs for MBP,46 gp91phox,47 neutrophil elastase,48 GATA-1,41 GATA-2,49 GATA-3,50 C/EBPalpha ,51 C/EBPbeta ,12 PU.1 (Spi-1),52 and beta -actin. Hybridization with the random-primed DNA probes was performed at 42°C in 50% formamide, 6× SSC, 0.2% Ficoll-polyvinylpyrrolidone (PVP), and 0.1% sodium dodecyl sulfate (SDS). Filters were washed twice in 2× SSC with 0.2% SDS at 53°C for 30 minutes and twice in 0.2× SSC with 0.2% SDS at 55°C for 30 minutes. Autoradiography was performed at -80°C with Kodak XAR-5 film (Eastman Kodak, Rochester, NY).

Plasmids for transient transfections.   The promoterless luciferase plasmid pXP2 containing the sequence between positions bp -117 to +47 of MBP gene P2 promoter region9 was used for all promoter studies.

Expression vectors for human GATA-1 and human C/EBPbeta under control of the elongation factor-1alpha promoter, pEF-GATA1 and pEF-C/EBPbeta , were prepared by cloning the human GATA-1 or C/EBPbeta cDNAs inserted into pEF-BSSHII, a modified version of the expression vector pEF-BOS.53

Transient transfections.   DNA was prepared and cells were transfected by electroporation as previously described using 1.5 × 107 cells in 500 µL with minor modifications.7 Briefly, the HL-60-C15 cells were electroporated at 280 V, 960 µF, and the Jurkat cells were electroporated at 250 V, 960 µF, respectively, conditions previously optimized for these lines.7-9 Luciferase activity in cell lysates was measured as relative light units (RLU) using a Lumat LB9501 luminometer (Berthold GmbH & Co KG, Bad Wildbad, Germany). Cell extracts were prepared in 500 µL of 1% Triton X-100, 25 mmol/L Gly-gly, 15 mmol/L MgSO4, 4 mmol/L EGTA, and 1 mmol/L dithiothreitol (DTT), and 100-µL aliquots of these extracts (equivalent to 1.5 × 106 cells) were analyzed for luciferase activity. Cotransfection with a cytomegalovirus (CMV)-beta galactosidase (Gal) plasmid (CMV-beta Gal) was used for normalization of transfection efficiency among the different cell lines, different plasmid DNA preparations, and individual transfection experiments. beta -Galactosidase activity was measured by a beta -galactosidase enzyme assay kit (Promega Co, Madison, WI). Individual transfection experiments were repeated at least 3 times, and the results are shown as mean RLU per milliunit of beta -galactosidase per milliliter (±SEM).

Electrophoretic mobility shift assay (EMSA).   COS cells transfected with the GATA-1 expression vector, pEF-GATA-1, were harvested 24 hours after electroporation with this plasmid. Nuclear extracts were prepared as described,54 and protein concentrations were determined using the Bradford assay (Bio-Rad, Richmond, CA). Purification of recombinant C/EBPbeta protein was performed as described previously.55 Human C/EBPbeta cDNA was subcloned into the maltose-binding protein (MBP) expression plasmid, pMALc-2 (New England Biolabs, Boston, MA), yielding the plasmid designated pMBP-C/EBPbeta . This plasmid was transferred to the Escherichia coli strain PS1, and the transformed cells were grown and harvested after induction with 0.3 mmol/L isopropyl-1-thio-beta -galactopyranoside for 3 hours at 37°C. The MBP-C/EBPbeta fusion protein was then purified by amylose resin affinity chromatography according to the manufacturer's instructions and was diluted with a buffer containing 25 mmol/L HEPES/KOH, pH 7.6, 40 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10% glycerol, and 1 mg/mL bovine serum albumin (BSA) for further use in a gel-shift assay. Nuclear extracts or recombinant proteins were incubated with radiolabeled oligonucleotides (0.1 to 1 ng) and then subjected to electrophoresis as described previously.9 Reactions were electrophoresed at 14 V/cm on 6% polyacrylamide gels cast in 0.5× TBE at 4°C.

EMSAs were performed with the following oligonucleotides (mutated sites are underlined): (A) MBP WT (-93/-58 bp) (5'-AAGTGATGAAATGGTCCTTATCAGCCTTGCTATCTC-3'); (B) MBP mut GATA (5'-AAGTGATGAAATGGTCCGGCGACGCCTTGCTATCTC-3'); (C) MBP mut C/EBP (5'-AAGGTATTACCGGGTTCCTTATCAGCCTTGCTATCTC-3'); and (D) MBP mut GATA/C/EBP (5'-AAGGTATTACCGGGTTCCGGCGACGCCTTGCTATCTC-3').

GST pull-down assays with [35S] methionine-labeled proteins.   DNA was transcribed and translated in vitro using the TNT T7-coupled reticulocyte lysate system (Promega), following the instructions of the manufacturer. The reticulocyte lysate containing the [35S]-labeled protein was then incubated with GST or GST-hGATA-1 in a buffer containing 50 mmol/L Tris-HCl (pH 7.8), 150 mmol/L KCl, 0.1% (vol/vol) Nonidet P-40, 0.1 % (vol/vol) Triton X-100, 5 mmol/L MgCl2, 0.5 mmol/L EDTA, 10% (vol/vol) glycerol, 50 µmol/L ZnCl2, 0.1 mmol/L sodium orthovanadate, 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF), leupeptin (5 µg/mL), aprotinin (10 µg/mL), and pepstatin A (5 µg/mL) in a total volume of 500 µL at 4°C for overnight. The resin was subsequently washed 4 times with 1 mL of binding buffer. Bound proteins were released in an SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and then analyzed by electrophoresis under denaturing conditions and autoradiography.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of a C/EBP consensus site in the functional promoter region of the MBP gene.   There are 2 promoters, P1 and P2, in the MBP gene. The distal promoter, which exists 5' upstream to exon 1 and drives expression of the 1.6-kb transcript, and the proximal promoter, which exists 5' upstream to exon 9 and drives expression of the 1.0-kb transcript, have been designated the P1 and P2 promoters, respectively.56 We previously reported the specificity of P2 promoter for the eosinophil lineage and identified a positively acting region in the bp -117 to -67 segment of the MBP P2 gene.9 In addition, we showed that GATA-1 could bind to a GATA consensus site, bp -76 to -71, in this region and could transactivate the MBP P2 promoter.9 As shown in Fig 1, we have identified a C/EBP consensus binding site at bp -90 to -82, 6 bp upstream of the functional GATA-binding site in this promoter.


View larger version (7K):
[in this window]
[in a new window]
  		Fig 1. The positive acting cis-elements of MBP P2 promoter. Sequence of the MBP gene between bp -108 and -70, showing the GATA consensus site (bp -76 to -71) and C/EBP consensus site (bp -90 to -82).

Induction of C/EBPbeta mRNA expression in a cell line capable of differentiating towards both the eosinophil and neutrophil lineages.   We have established a cell line capable of differentiating towards the eosinophil and neutrophil lineages in response to RA, HT93A.45 As shown by Northern blot analysis (Fig 2), no mRNA expression for MBP, gp91phox, and neutrophil elastase, which are markers for eosinophil and neutrophil lineages, GATA-1, or PU.1 (Spi-1), was observed in untreated HT93A cells (Fig 2, lane 1). This result shows that the uninduced HT93A cell line does not express genes characteristic of the eosinophil and neutrophil lineages. For the GATA-binding proteins, GATA-1 expression was induced 3 days after induction of HT93A cells with RA, whereas GATA-2 was expressed constitutively before and during the eosinophilic and neutrophilic differentiation of the cell line, consistent with our previous studies.9,40 In contrast, expression of GATA-3, which plays an important role in lymphoid development, was markedly downregulated during the eosinophilic and neutrophilic differentiation of HT93A cells. For the C/EBPs, expression of C/EBPalpha decreased somewhat after 7 days of culture with RA, whereas the expression of C/EBPbeta markedly increased from day 1 to 7 during differentiation towards the eosinophil and neutrophil lineages. The expression of PU.1 (Spi-1), an ETS-domain transcription factor essential for the development of myeloid and B-lymphoid cells, also increased considerably along with the eosinophilic and neutrophilic differentiation of HT93A cells. These results suggest that GATA-1, C/EBPbeta , and PU.1 (Spi-1) play an important role in eosinophilic and neutrophilic differentiation.


View larger version (35K):
[in this window]
[in a new window]
  		Fig 2. Northern blot analysis for MBP, gp91phox, neutrophil elastase, GATA-1, GATA-2, GATA-3, C/EBPalpha , C/EBPbeta , PU.1 (Spi-1), and beta -actin mRNA expression in HT93A cells treated with RA. Lane 1, untreated HT93A cell; lanes 2 through 5, HT93A cells treated with 2 µmol/L RA for 1, 3, 5, and 7 days, respectively. Each lane contained 4 µg of poly(A)+ RNA. The blot was probed sequentially with each cDNA probe and the beta -actin probe to control for equivalent loading.

C/EBPbeta binds to the C/EBP consensus site in the MBP P2 promoter region.   Northern blot analysis in HT93A cells treated with RA indicated that C/EBPbeta was induced during the eosinophilic and neutrophilic differentiation. Müller et al39 have reported that NF-M, the chicken homolog of C/EBPbeta , induces eosinophil differentiation in a multipotent progenitor cell line transformed by the Myb-Ets oncoprotein. Furthermore, we have recently shown that MBP promoter activity is increased 10-fold by cotransfecting the plasmids expressing C/EBPalpha or C/EBPdelta , whereas cotransfection with the C/EBPbeta expression vector produced a 20-fold increase.9 From the above-noted findings, it was suggested that C/EBPbeta might be involved in the regulation of MBP gene expression. Accordingly, to determine whether C/EBPbeta binds to the potential C/EBP-binding site, bp -90 to -82, in the MBP P2 promoter region, EMSAs were performed (Fig 3). The gel-shift analysis was performed using a purified recombinant C/EBPbeta protein, maltose-binding protein (MBP)-C/EBPbeta , and nuclear extracts from COS7 cells transiently transfected with the GATA-1 expression vector, as described previously.9 Full-length murine C/EBPbeta fused to the maltose-binding protein (MBP) was expressed in E coli and then purified by amylose resin affinity chromatography.55 A DNA probe spanning bp -93 to -58 of the MBP promoter formed a specific protein-DNA complex when incubated with MBP-C/EBPbeta (Fig 3, lane 2), but not with maltose-binding protein alone (Fig 3, lane 1). The complex formed with the specific probe was inhibited by an excess amount of the wild-type (competitor DNA A) and GATA site-mutated (competitor DNA B) oligonucleotides (Fig 3, lanes 3 and 4) but not by an oligonucleotide mutated in only the C/EBP consensus site (competitor DNA C) or an oligonucleotide mutated in both sites (competitor DNA D; Fig 3, lanes 5 and 6). An EMSA with nuclear extracts from COS7 cells transfected with a GATA-1 expression vector and oligonucleotide spanning the MBP promoter from position bp -93 to -58 showed the formation of a specific GATA-1 complex (Fig 3, lane 7). Incubation of both GATA-1 and C/EBPbeta proteins with the labeled probe showed 3 specific complexes, GATA-1 alone, C/EBPbeta alone, and combined GATA-1 and C/EBPbeta (Fig 3, lane 8). The C/EBPbeta -DNA complex band was competed by unlabeled oligonucleotides A and B (Fig 3, lanes 9 and 10), but not by unlabeled oligonucleotides C and D containing a mutated C/EBP site (Fig 3, lanes 11 and 12). The GATA-1-DNA complex band was also competed by oligonucleotides A and C (Fig 3, lanes 9 and 11), but not by oligonucleotides B and D (Fig 3, lanes 10 and 12). To confirm the results shown in Fig 3, we performed the EMSA with various labeled oligonucleotide probes (Fig 4). When incubated with labeled wild-type oligonucleotide (probe A) and MBP-C/EBPbeta or GATA-1 protein, the probe spanning position bp -93 to -58 (probe A) yielded 1 DNA-protein complex with C/EBPbeta or GATA-1 protein (Fig 4, lanes 2 and 3). Incubation of both C/EBPbeta and GATA-1 proteins with the wild-type probe (probe A) formed 3 specific bands, GATA-1, C/EBPbeta , and GATA-1/CEBPbeta complexes (Fig 4, lane 4). In contrast, incubation of both proteins with the GATA-site mutated probe (probe B) or C/EBP-site mutated probe (probe C) did not yield the 3 complexes (Fig 4, lanes 5 and 6). Also, the incubation of both proteins with C/EBP and GATA site-mutated probes formed no DNA-protein complex (Fig 4, lane 7). These results demonstrate that C/EBPbeta binds to the C/EBP consensus site, bp -90 to -82, and that both C/EBPbeta and GATA-1 can bind to the C/EBP- and GATA-binding sites simultaneously.


View larger version (43K):
[in this window]
[in a new window]
  		Fig 3. Binding of recombinant C/EBPbeta fusion protein and GATA-1 protein to the MBP promoter. A double-stranded MBP promoter oligonucleotide extending from bp -93 to -58 was end-labeled with [gamma -32P] ATP and incubated with 1 µg of double-stranded poly(dI-dC) in the presence of 1 µg maltose-binding protein (MBP)-C/EBPbeta fusion protein (lanes 2 through 6 and 8 through 12) and 8 µg nuclear protein from COS7 cells that were transiently transfected with a GATA-1 expression vector (lanes 7 through 12). Unlabeled double-stranded competitor oligonucleotides, shown schematically in the lower panel, were added at a 100-fold molar excess over the labeled probe oligonucleotide, MBP bp -93 to -58 (competitor A; lanes 3 and 9), mutated GATA-consensus site oligonucleotide (competitor B; lanes 4 and 10), mutated C/EBP-consensus site oligonucleotide (competitor C; lanes 5 and 11), and mutated GATA-1 and C/EBP-consensus site oligonucleotide (competitor D; lanes 6 and 12).



View larger version (35K):
[in this window]
[in a new window]
  		Fig 4. Demonstration of C/EBPbeta and GATA-1 binding to the MBP promoter by gel-shift assay. Double-stranded oligonucleotides (lower panel) containing MBP promoter sequence from bp -93 to -58 (probe A), with mutated GATA-consensus site (probe B), mutated C/EBP-consensus site (probe C), and mutated GATA- and C/EBP-consensus sites (probe D) were end-labeled with [gamma -32P] ATP and incubated with 1 µg double-stranded poly (dI-dC) in the presence of 1 µg purified maltose-binding protein (MBP)-C/EBPbeta fusion protein (lanes 2 and 4 through 7) and 8 µg nuclear proteins from COS7 cells that were transiently transfected with a GATA-1 expression vector (lanes 3 through 7).

C/EBPbeta transactivates the MBP promoter synergistically with GATA-1.   Our gel-shift analysis showed that C/EBPbeta can bind to the C/EBP consensus site of the MBP promoter. Accordingly, to determine whether the C/EBP-binding site is functional, we analyzed the ability of C/EBPbeta and GATA-1 either individually or in combination to transactivate the MBP promoter in the C/EBPbeta and GATA-1 negative Jurkat cell line. Cotransfection with the GATA-1 or C/EBPbeta expression vector produced a 5-fold increase in MBP promoter activity (Fig 5). When both C/EBPbeta and GATA-1 expression vectors were transfected together, there was a 5-fold increase in MBP promoter activity above that obtained with these vectors individually (Fig 5). To obtain more detailed information on the relative function of the GATA- versus C/EBP-binding sites, we constructed mutant reporter plasmids in which the GATA- and/or the C/EBP-binding sites were disrupted by multiple nucleotide sequence substitutions (Fig 6A and B). As shown in Fig 6A, the constructs with the mutated C/EBP- and/or GATA-binding sites decreased the promoter activity compared with those shown in the wild-type construct. When GATA-1 and C/EBPbeta expression vectors were added together with a wild-type construct, there was a 60-fold induction of MBP promoter activity (Fig 6B). When C/EBPbeta or GATA-1 expression vector was added individually with constructs containing mutated GATA- or C/EBP-binding sites, a 4-fold induction of MBP promoter activity could be detected, confirming a synergistic effect of GATA-1 and C/EBPbeta for MBP promoter activity (Fig 6B). On the other hand, when both factors, GATA-1 and C/EBPbeta , were used together with the constructs containing mutated GATA- or C/EBP-binding sites, MBP promoter activity was induced by 30-fold or 50-fold, respectively (Fig 6B). When GATA-1 and C/EBPbeta expression vectors were added simultaneously together with the construct containing mutations in both the GATA- and C/EBP-binding sites, the promoter activity was the same as that obtained with these vectors individually for the construct of both sites' mutants (data not shown). This suggests that GATA-1 and C/EBPbeta may interact physically. However, the mechanisms by which C/EBPbeta synergistically transactivates the MBP promoter in association with GATA-1 remain to be determined.


View larger version (14K):
[in this window]
[in a new window]
  		Fig 5. C/EBPbeta and GATA-1 synergistically transactivate the MBP P2 promoter. Transactivation of the MBP promoter (bp -117/pXP2-MBP) in Jurkat cells, in which neither GATA-1 nor C/EBPbeta transcription factors are expressed. The T-lymphocytic Jurkat cell line was transfected by the electroporation method with 5 µg of the MBP promoter construct (bp -117/pXP2-MBP) along with the following expression constructs: pXP2-MBP + pEF-BOS, pXP2-MBP (control) and 4 µg of pEF-BOS (the control plasmid containing the elongation factor promoter without cDNAs); pXP2-MBP + pEF-GATA1, 2 µg of pEF-GATA-1, and 2 µg of pEF-BOS; pXP2-MBP + pEF-C/EBPbeta ; 2 µg of pEF-C/EBPbeta and 2 µg of pEF-BOS, pXP2-MBP + pEF-GATA-1 + pEF-C/EBPbeta ; and 2 µg of pEF-GATA-1 and 2 µg of pEF-C/EBPbeta . Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of a cotransfected beta -galactosidase expression vector (CMV-beta Gal). Data are shown as the mean of 3 independent experiments (±SEM).



View larger version (32K):
[in this window]
[in a new window]
  		Fig 6. Mutations of the GATA- or C/EBP-binding sites diminish MBP promoter activity (A) and mutations of the GATA- or C/EBP-sites does not prevent synergy (B). Jurkat cells were cotransfected with pXP2-MBP containing wild-type GATA-1 and C/EBP-sites, pXP2-MBP containing a mutated GATA-binding site, and/or pXP2-MBP containing a mutated C/EBP-binding site, along with 2 µg of pEF-GATA-1 and/or pEF-C/EBPbeta expression vectors. The error bar represents the SEM for 3 independent experiments. Luciferase activity was normalized to beta -galactosidase activity from a cotransfected CMV-beta Gal plasmid.

Physical interaction between GATA-1 and C/EBPbeta .   Because the data in Fig 6B suggested that GATA-1 may be able to bind C/EBPbeta through protein-protein interactions, we proceeded to test this possibility directly in vitro. 35S-labeled C/EBPbeta produced by translation in vitro was allowed to bind to hGATA-1 fused to GST or GST alone and was immobilized onto a glutathione-Sepharose matrix, after which the bound proteins were analyzed by SDS-PAGE. GATA-1 associated clearly with C/EBPbeta in vitro, and as much as 15% of the input protein was recovered as complexes with GATA-1 (Fig 7). However, C/EBPbeta did not adhere to GST resin devoid of GATA-1. Thus, there is a physical interaction between GATA-1 and C/EBPbeta .


View larger version (25K):
[in this window]
[in a new window]
  		Fig 7. Physical interaction between C/EBPbeta and GATA-1 in vitro. 35S-labeled full-length C/EBPbeta was synthesized by translation in vitro and incubated with GST alone (lane 2) or GST-hGATA-1 (lane 3) adsorbed to glutathione-Sepharose, after which the matrix was washed and bound proteins were analyzed as described in Materials and Methods. Lane 1 represents 15% of the amount of labeled protein incubated with matrices.

FOG decreases the ability of GATA-1 to transactivate the MBP promoter.   The GATA-1 protein has 2 zinc fingers, a carboxyl-terminal zinc finger (C-f) and an amino-terminal zinc finger (N-f). The C-f of GATA-1 provides the essential DNA binding domain necessary for GATA-1 binding to GATA motifs, whereas the N-f of GATA-1 is not necessary for DNA binding. Recently, Tsang et al57 identified a novel, multitype zinc finger protein, FOG, that binds to the N-f of GATA-1 and acts as a cofactor for GATA-binding proteins. To determine the effect of FOG on GATA-1 transactivation of the MBP promoter, we analyzed the expression of FOG mRNA in HT93A cells capable of differentiating towards eosinophil and neutrophil lineages. As shown in Fig 8A, the transcript for FOG was rapidly downregulated during the RA-induced eosinophilic and neutrophilic differentiation of the HT93A cell line. It has been shown that FOG and GATA-1 synergistically activate transcription of an erythroid- and megakaryocyte-associated p45NF-E2 gene promoter.57 We addressed whether FOG can synergize with GATA-1 to activate transcription of the eosinophil-specific MBP P2 promoter. A fixed amount of GATA-1 and increasing amounts of FOG were transiently coexpressed in Jurkat cells together with pXP2-MBP. Surprisingly, expression of FOG decreased the transactivation activity of GATA-1 for the MBP P2 promoter in a dose-dependent manner (Fig 8B). To examine the effect of the pMT2 vector used to express FOG on GATA-1 transactivation of the MBP promoter, a constant amount (8 µg) of pMT2 plus pMT2-FOG was used in the transactivation study. As shown in Fig 8C, a pMT2 vector did not affect the negative regulatory role of FOG for GATA-1 transactivation of the MBP promoter. In addition, to analyze the specificity of the inhibitory effect of FOG on GATA-1 transactivation of the MBP promoter, a fixed amount of FOG-unrelated expression vector, pEF-C/EBPbeta , and increasing amounts of FOG expression vectors were coexpressed in Jurkat cells together with the construct containing mutated GATA-binding site. As shown in Fig 8D, expression of FOG did not affect the transactivation activity of C/EBPbeta for the MBP P2 promoter, indicating the specificity of the inhibitory effect of FOG for GATA-1-dependent transactivation of the MBP promoter. These results suggest an inhibitory role of FOG for GATA-1-regulated genes in the eosinophil lineage, unlike that of FOG activity in the erythroid and megakaryocyte lineages.



View larger version (44K):
[in this window]
[in a new window]
  		Fig 8. FOG acts as a negative (inhibitory) cofactor for GATA-1 transactivation of the MBP promoter. (A) Northern blot analysis of mRNA for FOG in uninduced HT93A cells (lane 1) and HT93A cells induced with RA for 1, 3, 5, and 7 days (lanes 2 through 5, respectively). Control hybridization with beta -actin cDNA is shown in the lower panel. (B) FOG inhibits transactivation of the MBP promoter by GATA-1. Jurkat cells were transiently transfected with 5 µg of pXP2-MBP, 2 µg of pEF-GATA-1, increasing amounts of pMT2-FOG as shown, and 1 µg of beta -galactosidase expression vector (pCMV-beta Gal). Data are shown as the mean of 3 independent experiments (±SEM). (C) Jurkat cells were transfected by the electroporation method with 2 µg of the pXP2-MBP containing wild-type GATA- and C/EBP-binding sites along with the following expression vectors: 4 µg of pEF-BOS, 2 µg of pEF-GATA-1 plus 8 µg of pMT2, 2 µg of pEF-GATA-1 plus 7 µg of pMT2 plus 1 µg of pMT2-FOG, 2 µg of pEF-GATA-1 plus 6 µg of pMT2 plus 2 µg of pMT2-FOG, 2 µg of pEF-GATA-1 plus 4 µg of pMT2 plus 4 µg of pMT2-FOG, and 2 µg of pEF-GATA-1 plus 8 µg of pMT2-FOG. pBluescriptIIKS(-) plasmids were added to maintain total DNA constant at 22 µg. Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of cotransfected beta -galactosidase expression vector (pCMV-beta Gal). (D) Jurkat cells were transfected with 5 µg of the pXP2-MBP containing a mutated GATA-binding site (pXP2-MBP mutGATA) along with the following expression vectors: 2 µg of pEF-BOS, 2 µg of pEF-C/EBPbeta , 2 µg of pEF-C/EBPbeta plus 7 µg of pMT2 plus 1 µg of pMT2-FOG, 2 µg of pEF-C/EBPbeta plus 6 µg of pMT2 plus 2 µg of pMT2-FOG, 2 µg of pEF-C/EBPbeta plus 4 µg of pMT2 plus 4 µg of pMT2-FOG, and 2 µg of pEF-C/EBPbeta plus 8 µg of pMT2-FOG. pBluescriptIIKS(-) plasmids were added to maintain total DNA constant at 22 µg. Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of a cotransfected beta -galactosidase expression vector (pCMV-beta Gal). Data are shown as the mean of 3 independent experiments (±SEM).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously reported that GATA-1 can independently transactivate the MBP P2 promoter, but that GATA-2 has the capacity to compete for GATA-1 binding and to inhibit GATA-1 transactivation.9 Furthermore, we have also shown that C/EBP family members, in particular C/EBPalpha , C/EBPbeta , and C/EBPdelta , are able to transactivate the MBP promoter individually.9 In the present study, we have shown that C/EBPbeta can bind to the C/EBP consensus site (bp -90 to -82) of the MBP P2 promoter and can transactivate MBP promoter activity synergistically with GATA-1. In the hematopoietic system, C/EBPalpha , -beta , and -epsilon play an important role in myeloid differentiation. Scott et al28 have reported that the expression of C/EBPalpha predominates in immature myelomonocytic cells and that C/EBPbeta expression predominates in terminally differentiated cells, consistent with our Northern blot analysis of RA-induced HT93A cells. C/EBPalpha is crucial to the activity of the G-CSFR promoter,31 and C/EBPalpha knock out (KO) mice have impaired G-CSFR expression and signaling through this lineage-specific cytokine receptor.32 Expression of C/EBPbeta was found to parallel the differentiation of myeloid cells to macrophages, and C/EBPbeta KO mice display impaired macrophage function.58 Recently, Chih et al37 have shown that C/EBPepsilon shows a restricted pattern of expression in cells of the myeloid lineage during their differentiation toward granulocytes. In particular, they suggest that C/EBPepsilon plays an important role in the differentiation of granulocytic progenitors to myeloblasts/promyelocytes.37 In this study, we have not analyzed the effect of C/EBPepsilon on eosinophilic differentiation, but C/EBPepsilon null mice have been shown to have impaired eosinophil development.59 Further studies on the role of C/EBPepsilon in the eosinophilic differentiation are required. Although these studies indicate that C/EBP family members are closely associated with the differentiation of myeloid cells, the C/EBP transcription factors that play key roles in the differentiation of eosinophil lineage are not known. We found that C/EBPbeta expression increased along with the eosinophilic and neutrophilic differentiation of HT93A cell line and that C/EBPalpha mRNA was downregulated in these cells (Fig 2). This result suggested that C/EBPbeta played an important role in the eosinophilic and neutrophilic differentiation. Additionally, granulocyte-committed cells express little C/EBPalpha compared with C/EBPbeta , whereas in more primitive myeloid progenitors, only C/EBPalpha is present.28 These findings suggest that C/EBPbeta plays a more important role in the terminal differentiation of the eosinophil lineage than C/EBPalpha .

Cell-type specific gene expression often involves interaction with different transcription factors. In particular, C/EBP family members can form homodimers or heterodimers with each other and also interact with other transcription factors such as the myb oncogene.39 Attempts to demonstrate direct interactions between two transcription factors by gel-shift analysis, eg, C/EBPbeta and the glucocorticoid receptor for the rat alpha 1-acid glycoprotein gene60 and C/EBPalpha and PU.1 for the murine neutrophil elastase gene,61 have been performed without success. Williams et al62 have shown that C/EBPbeta contains a negative regulatory region composed of 2 elements, RD1 and RD2. They suggested that the inactive form of C/EBPbeta adopts a tightly folded conformation that masks the activation and DNA-binding domains located at the N-and C-termini, respectively, and that activation of C/EBPbeta requires some event, which causes the protein to unfold and unmask the activation and DNA-binding domains.

Transcription factors activate promoter activity by interacting with the basal transcriptional machinery. The CREB-binding protein (CBP) has been suggested to bridge c-Myb and NF-M, the chicken homolog of C/EBPbeta .63 Recently, it has been reported that CBP acts as a coactivator of GATA-1.64 These observations suggest that CBP functions as a bridging protein between C/EBPbeta , GATA-1, and the basal transcriptional complex in the MBP promoter. It has been reported that FOG acts as a cofactor for GATA-binding proteins and that FOG and GATA-1 synergize in activating transcription of the erythroid/megakaryocytic-expressed p45 NF-E2 gene.57 However, in our study, FOG acted as a negative cofactor for transcription of the eosinophil-specific MBP P2 promoter. FOG is expressed in both erythroblasts and megakaryocytes, but not in mast cells.57 In our experiment, the expression of FOG mRNA was downregulated during the RA-induced eosinophilic and neutrophilic differentiation of the HT93A cells. These findings suggest that FOG acts as a negative regulator for the eosinophil, neutrophil, or mast cell lineages, in contrast to its positive regulatory function for the erythroid and megakaryocytic lineages. One of the mechanisms by which FOG decreases the transactivation activity of GATA-1 for the MBP P2 promoter may be to block the interaction between GATA-1 and the basal transcriptional complex in the MBP gene.

To date, it has been reported that C/EBPbeta has the capacity to act synergistically to transactivate promoters with a number of other factors, including NF-kappa B,65 c-Jun,66 glucocorticoid receptor,60 and v-Myb.67 In the present study, physical interaction between GATA-1 and C/EBPbeta was demonstrated by a GST pull-down assay. It has been reported that C/EBPbeta can bind to the c-fos serum response element binding protein (SRE BP),68 NF-kappa B,69 glucocorticoid receptor,60 and AML1.30 However, to date, there are no reports of a physical interaction between GATA-1 and C/EBPbeta . Our report is the first to describe both a physical interaction and cooperativity between GATA-1 and C/EBPbeta . However, these data are not definitive. We have to perform the coimmunoprecipitation experiment using extracts from eosinophilic cell lines for demonstrating that endogenous GATA-1 and C/EBPbeta interact in vivo. The extent of the enhancement of C/EBPbeta -GATA-1-mediated transcription for the eosinophil-specific MBP P2 promoter by cotransfected CBP was less than that of GATA-1-dependent transcription for the erythroid-specific EKLF promoter by cotransfected CBP (data not shown). The role of FOG and CBP in the eosinophil lineage may be different from that in the erythroid/megakaryocytic lineages, with the difference in commitment towards eosinophilic versus erythroid/megakaryocytic differentiation due to the differential functions of these cofactors.

It has been shown that GATA-1 and PU.1, an ets transcription factor, stimulate activity of the IL-4 intronic enhancer70 and that both GATA-1 and the ets family transcription factors increase promoter activity of the c-mpl (the thrombopoietin receptor) gene.71 In addition, it was reported that GATA-1 could convert myeloid cells in part to eosinophils in Myb-Ets-transformed chicken myeloblasts.72 Recently, McNagny et al73 have reported that low levels of GATA-1 stimulated the eosinophil-specific EOS47 promoter moderately in the presence of Ets-1, c-Myb, and C/EBPalpha in the chicken hematopoietic system. Also, Nerlov et al74 have shown that chicken C/EBPalpha and C/EBPbeta can induce both myeloid and eosinophil lineage commitment. However, as far as we know, it has not been previously reported that the GATA-1 and C/EBP transcription factors have the capacity to act coordinately or interactively to stimulate promoter activity in a synergistic fashion. Our results provide the first evidence that GATA-1 and C/EBPbeta have the capacity to act synergistically to affect the transcriptional activity of an eosinophil-specific granule protein gene.


    ACKNOWLEDGMENT

The authors thank Dr Michio Nakamura, Dr Ronald G. Crystal, Dr Masayuki Yamamoto, Dr Kleanthis G. Xanthopoulos, Dr Akira Shizuo, Dr Francoise Moreau-Gachelin, and Stuart H. Orkin for providing the gp91phox; neutrophil elastase; GATA-1, GATA-2, and GATA-3; C/EBPalpha ; C/EBPbeta ; PU.1 (Spi-1) cDNAs; and a FOG expression vector, respectively. We are also grateful to Dr Masaki Takiguchi for providing the MBP-C/EBPbeta fusion protein.


    FOOTNOTES

Submitted July 30, 1998; accepted April 22, 1999.

Supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan (to Y.Y.) and the National Institutes of Health, NIAID, Grant No. AI33043 (to S.J.A.).

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

Address reprint requests to Yuji Yamaguchi, MD, Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto, 860 Japan; e-mail: yujiya{at}gpo.kumamoto-u.ac.jp.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Clutterbuck EJ, Hirst EM, Sanderson CJ: Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: Comparison and interaction with IL-1, IL-3, IL-6, and GMCSF. Blood 73:1504, 1989[Abstract/Free Full Text]

2. Silberstein DS, David JR: The regulation of human eosinophil function by cytokines. Immunol Today 8:380, 1987

3. Yamaguchi Y, Suda T, Suda J, Eguchi M, Miura Y, Harada N, Tominaga A, Takatsu K: Purified interleukin 5 suppoprts the terminal differentiation and proliferation of murine eosinophilic precursors. J Exp Med 167:43, 1988[Abstract/Free Full Text]

4. Yamaguchi Y, Matsui T, Kasahara T, Etoh S, Tominaga A, Takatsu K, Miura Y, Suda T: In vivo changes of hemopoietic progenitors and the expression of the interleukin 5 gene in eosinophilic mice infected with Toxocara canis. Exp Hematol 18:1152, 1990[Medline] [Order article via Infotrieve]

5. Yamaguchi Y, Suda T, Shiozaki Y, Miura Y, Hitoshi Y, Tominaga A, Takatsu K, Kasahara T: Role of IL-5 in IL-2-induced eosinophilia. In vivo and in vitro expression of IL-5 mRNA by IL-2. J Immunol 145:873, 1990[Abstract]

6. Kopf M, Bronbacher F, Hodgkin PD, Ramsay AJ, Milbourne EA, Dai WJ, Ovington KS, Behm CA, Kohler G, Young IG, Matthaei KI: IL-5-deficient mice have a developmental defect in CD5+ B-1 cells and lack eosinophilia but have normal antibody and cytotoxic T cell responses. Immunity 4:15, 1996[Medline] [Order article via Infotrieve]

7. Yamaguchi Y, Zhang D-E, Sun Z, Albee EA, Nagata S, Tenen DG, Ackerman SJ: Functional characterization of the promoter for the gene encoding human eosinophil peroxidase. J Biol Chem 269:19410, 1994[Abstract/Free Full Text]

8. Gomolin HI, Yamaguchi Y, Paulpillai AV, Dvorak LA, Ackerman SJ, Tenen DG: Human eosinophil Charcot-Leyden crystal protein: Cloning and characterization of a lysophospholipase gene promoter. Blood 82:1868, 1993[Abstract/Free Full Text]

9. Yamaguchi Y, Ackerman SJ, Minegishi N, Takiguchi M, Yamamoto M, Suda T: Mechanisms of transcription in eosinophils: GATA-1, but not GATA-2, transactivates the promoter of the eosinophil granule major basic protein gene. Blood 91:3447, 1998[Abstract/Free Full Text]

10. Landschulz WH, Johnson PF, Adashi EY, Graves BJ, McKnight SL: Isolation of a recombinant copy of the gene encoding C/EBP. Genes Dev 2:786, 1988[Abstract/Free Full Text]

11. Xanthopoulos KG, Mirkovitch J, Decker T, Kuo CF, Darnell JE Jr: Cell-specific transcriptional control of the mouse DNA-binding protein mC/EBP. Proc Natl Acad Sci USA 86:4117, 1989[Abstract/Free Full Text]

12. Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T: A nuclear factor for IL-6 expression is a member of a C/EBP family. EMBO J 9:1897, 1990[Medline] [Order article via Infotrieve]

13. Chang CJ, Chen TT, Lei HY, Chen DS, Lee SC: Molecular cloning of a transcription factor, AGP/EBP, that belongs to members of the C/EBP family. Mol Cell Biol 10:6642, 1990[Abstract/Free Full Text]

14. Descombes P, Chojkier M, Lichtsteiner S, Falvey E, Schibler U: LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev 4:1541, 1990[Abstract/Free Full Text]

15. Poli V, Mancini FP, Cortese R: IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell 63:643, 1990[Medline] [Order article via Infotrieve]

16. Roman C, Platero JS, Shuman JD, Calame K: Ig/EBP-1: A ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev 4:1404, 1990[Abstract/Free Full Text]

17. Cao Z, Umek RM, McKnight SL: Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev 5:1538, 1991[Abstract/Free Full Text]

18. Kageyama R, Sasai Y, Nakanishi S: Molecular characterization of transcription factors that bind to the cAMP responsive region of the substance P precursor gene. J Biol Chem 266:15525, 1991[Abstract/Free Full Text]

19. Williams SC, Cantwell CA, Johnson PA: A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev 5:1553, 1991[Abstract/Free Full Text]

20. Kinoshita S, Akira S, Kishimoto T: A member of the C/EBP family, NF-IL6beta , forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci USA 87:1473, 1992

21. Antonson P, Stellan B, Yamanaka R, Xanthopoulos KG: A novel human CCAAT/enhancer binding protein gene, C/EBPepsilon , is expressed in cells of lymphoid and myeloid lineages and is localized on chromosome 14q11.2 clone to the T-cell receptor alpha /delta locus. Genomics 35:30, 1996[Medline] [Order article via Infotrieve]

22. Chumakov AM, Grillier I, Chumakova E, Chih D, Slater J, Koeffler HP: Cloning of the novel human myeloid-specific C/EBP transcription factor (C/EBPepsilon ). Mol Cell Biol 17:1375, 1997[Abstract]

23. Ron D, Habener JF: Chop, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev 6:439, 1992[Abstract/Free Full Text]

24. van Ooji C, Snyder RC, Paeper BW, Duester G: Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein. Mol Cell Biol 12:3023, 1992[Abstract/Free Full Text]

25. Nishiyori A, Tashiro H, Kimura A, Akagi K, Yamamura K, Mori M, Takiguchi M: Determination of tissue specificity of the enhancer by combinatorial operation of tissue-enriched transcription factors: Both HNF-4 and C/EBPbeta are required for liver-specific activity of the ornithine transcarbamylase enhancer. J Biol Chem 269:1323, 1994[Abstract/Free Full Text]

26. Manchado C, Yubero P, Vinas O, Iglesias R, Villavroya F, Mampel T, Giralt M: CCAAT/binding proteins alpha  and beta  in human adipose tissue: Evidence for a tissue-specific pattern of expression during development. Biochem J 302:695, 1994

27. Tanaka T, Yoshida N, Kishimoto T, Akira S: Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. EMBO J 16:7432, 1997[Medline] [Order article via Infotrieve]

28. Scott LM, Civin CI, Rorth P, Friedman AD: A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells. Blood 80:1725, 1992[Abstract/Free Full Text]

29. Zhang DE, Fujioka KI, Hetherington CJ, Shapiro LH, Chen HM, Look AT, Tenen DG: Identification of a region which directs monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1). Mol Cell Biol 14:8085, 1994[Abstract/Free Full Text]

30. Zhang DE, Hetherington CJ, Meyers S, Rhoades KL, Chen HM, Hiebert SW, Tenen DG: CCAAT enhancer binding protein (C/EBP) and AML1 (CBFalpha 2) synergistically activate the M-CSF receptor promoter. Mol Cell Biol 16:1231, 1996[Abstract]

31. Smith LT, Hohaus S, Gonzalez DA, Dziennis SE, Tenen DG: PU.1 (Spi-1) and C/EBPalpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood 88:1234, 1996[Abstract/Free Full Text]

32. Zhang P, Zhang D-E, Wang N, Heltherington CJ, Darlington GJ, Tenen DG: Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice. Proc Natl Acad Sci USA 94:569, 1997[Abstract/Free Full Text]

33. Natsuka S, Akira S, Nishio Y, Hashiomoto S, Sugita T, Isshiki H, Kishimoto T: Macrophage differentiation-specifc expression of NF-IL6, a transcription factor interleukin-6. Blood 79:460, 1992[Abstract/Free Full Text]

34. Katz S, Kowenzleutz E, Müller C, Meese K, Ness SA, Leutz A: The NF-M transcription factor is related to C/EBP-beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. EMBO J 12:1321, 1993[Medline] [Order article via Infotrieve]

35. Ness SA, Kowenzleuz E, Casini T, Graf T, Leutz A: Myb and NF-M-combinatorial activators of myeloid genes in heterologous cell types. Genes Dev 7:749, 1993[Abstract/Free Full Text]

36. Burk O, Mink S, Ringwald M, Klempnauer KH: Synergistic activation of the chicken mim-1 gene by v-myb and C/EBP transcription factors. EMBO J 12:2027, 1993[Medline] [Order article via Infotrieve]

37. Chih DY, Chumakov AM, Park DJ, Silla AG, Koeffler HP: Modulation of mRNA expression of a novel human myeloid-selective CCAAT/enhancer binding protein gene (C/EBPepsilon ). Blood 90:2987, 1997[Abstract/Free Full Text]

38. Hohaus S, Petrovick MS, Voso MT, Sun Z, Zhang D-E, Tenen DG: PU.1 (Spi-1) and C/EBPalpha regulate the expression of the granulocyte-macrophage colony-stimulating factor receptor alpha  gene. Mol Cell Biol 15:5830, 1995[Abstract]

39. Müller C, Kowenz-Leutz E, Grieser-Ade S, Graf T, Leutz A: NF-M (chicken C/EBPbeta ) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line. EMBO J 14:6127, 1995[Medline] [Order article via Infotrieve]

40. Zon LI, Yamaguchi Y, Yee K, Albee EA, Kimura A, Bennett JC, Orkin SH, Ackerman SJ: Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: Potential role in gene transcription. Blood 81:3234, 1993[Abstract/Free Full Text]

41. Yamamoto M, Ko LJ, Leonard MW, Beug H, Orkin SH, Engel JD: Activity and tissue-specific expression of the transcription factor NF-E1 multigene family. Genes Dev 4:1650, 1990[Abstract/Free Full Text]

42. Pevny L, Simon MC, Robertson E, Klein WH, Tsai SF, D'Agati V, Orkin SH, Constantini F: Erythroid differentiation in chimeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature 349:257, 1991[Medline] [Order article via Infotrieve]

43. Yamaguchi Y, Zon LI, Ackerman SJ, Yamamoto M, Suda T: Forced GATA-1 expression in the murine myeloid cell line M1: Induction of c-mpl expression and megakaryocytic/erythroid differentiation. Blood 91:450, 1998[Abstract/Free Full Text]

44. Fishkoff SA: Graded increase in probability of eosinophilic differentiation of HL-60 promyelocytic leukemic cells induced by culture under alkaline conditions. Leuk Res 12:679, 1988[Medline] [Order article via Infotrieve]

45. Kishi K, Toba K, Azegami T, Tsukada N, Uesugi Y, Masuko M, Niwano H, Hashimoto S, Sakaue M, Furukawa T, Koike T, Takahashi H, Maekawa T, Abe T, Aizawa Y: Hematopoietic cytokine dependent differentiation to eosinophils and neutrophils in newly established acute promyelocytic leukemia cell line with t(15; 17). Exp Hematol 26:135, 1998[Medline] [Order article via Infotrieve]

46. Barker RL, Gleich GJ, Pease LR: Acid precursor revealed in human eosinophil granule major basic protein cDNA. J Exp Med 168:1493, 1988[Abstract/Free Full Text]

47. Bjorgvinsdottir H, Zhen L, Dinauer MC: Cloning of murine gp91phox cDNA and functional expression in a human X-linked chronic granulomatous disease cell line. Blood 87:2005, 1996[Abstract/Free Full Text]

48. Takahashi H, Nukiwa T, Basset P, Crystal RG: Myelomonocytic cell lineage expression of the neutrophil elastase gene. J Biol Chem 263:456, 1989

49. Dorfman DM, Wilson DB, Bruns GA, Orkin SH: Human transcription factor GATA-2. Evidence for regulation of preproendothelin-1 gene expression in endothelial cells. J Biol Chem 267:1279, 1992[Abstract/Free Full Text]

50. Ho I-C, Vorhees P, Marin N, Oakley BK, Tsai S-F, Orkin SH, Leiden JM: Human GATA-3: A lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha  gene. EMBO J 10:1187, 1991[Medline] [Order article via Infotrieve]

51. Antonson P, Xanthopoulos KG: Molecular cloning, sequence, and expression patterns of the human gene encoding CCAAT/enhancer binding protein alpha  (C/EBPalpha ). Biochem Biophys Res Commun 215:106, 1995[Medline] [Order article via Infotrieve]

52. Ray D, Culine S, Tavitian A, Moreau-Gachelin F: The human homologue of the putative proto-oncogene Spi-1: Characterization and expression in tumors. Oncogene 5:663, 1990[Medline] [Order article via Infotrieve]

53. Mizushima S, Nagata S: pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res 18:5322, 1990[Free Full Text]

54. Andrews NC, Faller DV: A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cell. Nucleic Acids Res 19:2499, 1991[Free Full Text]

55. Chowdhury S, Gotoh T, Mori M, Takiguchi M: CCAAT/enhancer-binding protein beta  (C/EBPbeta ) binds and activitates while hepatocyte nuclear factor-4 (HNF-4) does not bind but represses the liver-type arginase promoter. Eur J Biochem 236:500, 1996[Medline] [Order article via Infotrieve]

56. Li M-S, Sun Li, Satoh T, Fisher LM, Spry CJF: Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from promoters. Biochem J 305:921, 1995

57. Tsang AP, Visvader JE, Turner CA, Fujiwara Y, Yu C, Weiss MJ, Crossley M, Orkin SH: FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation. Cell 90:109, 1997[Medline] [Order article via Infotrieve]

58. Tanaka T, Akira S, Yoshida K, Umemoto M, Yoneda T, Shirafuji N, Fujiwara H, Suematsu S, Yoshida N, Kishimoto T: Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages. Cell 80:353, 1995[Medline] [Order article via Infotrieve]

59. Yamanaka R, Barlow C, Lekstrom-Himes J, Castillo LH, Liu PP, Eckhaus M, Decker T, Wynshaw-Boris A, Xanthopoulos KG: Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice. Proc Natl Acad Sci USA 94:13187, 1997[Abstract/Free Full Text]

60. Nishio Y, Isshiki H, Kishimoto T, Akira S: A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction. Mol Cell Biol 13:1854, 1993[Abstract/Free Full Text]

61. Oelgeschlager M, Nuchprayoon I, Luscher B, Friedman AD: C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter. Mol Cell Biol 16:4717, 1996[Abstract]

62. Williams SC, Baer M, Dillner AJ, Johnson PF: CRP2 (C/EBPbeta ) contains a bipartite regulatory domain that controls transcriptional activation, DNA binding and cell specificity. EMBO J 14:3170, 1995[Medline] [Order article via Infotrieve]

63. Oelgeschlager M, Janknecht R, Krieg J, Schreek S, Luscher B: Interaction of the co-activator CBP with Myb proteins: effects on Myb-specific transactivation and on the cooperativity with NF-M. EMBO J 15:2771, 1996[Medline] [Order article via Infotrieve]

64. Blobel GA, Nakajima T, Eckner R, Montminy M, Orkin SH: CREB-binding protein cooperates with transcription factor GATA-1 and is required for erythroid differentiation. Proc Natl Acad Sci USA 95:2061, 1998[Abstract/Free Full Text]

65. LeClair KP, Blanar MA, Sharp PA: The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor. Proc Natl Acad Sci USA 89:8145, 1992[Abstract/Free Full Text]

66. Hsu W, Kerppola PL, Chen T, Curran T, Chen-Kiang S: Fos and Jun repress transcription activation by NF-IL6 through association at the basic zipper region. Mol Cell Biol 14:268, 1994[Abstract/Free Full Text]

67. Mink S, Kerber U, Klempnauer K-H: Interaction of C/EBPbeta and v-Myb is required for synergistic activation of the mim-1 gene. Mol Cell Biol 16:1316, 1996[Abstract]

68. Sealy L, Malone D, Pawlak M: Regulation of the cfos serum response element by C/EBPbeta . Mol Cell Biol 17:1744, 1997[Abstract]

69. Stein B, Cogswell PC, Baldwin AS: Functional and physical associations between NF-kB and C/EBP family members: A Rel domain-bZip interaction. Mol Cell Biol 13:3964, 1993[Abstract/Free Full Text]

70. Henkel G, Brown MA: PU.1 and GATA: Components of a mast cell-specific interleukin 4 intronic enhancer. Proc Natl Acad Sci USA 91:7737, 1994[Abstract/Free Full Text]

71. Deveaux S, Filipe A, Lemarchandel V, Ghysdael J, Remeo P-H, Mignotte V: Analysis of the thrombopoietin receptor (MPL) promoter implicates GATA and ets proteins in the coregulation of megakaryocyte-specific genes. Blood 87:4678, 1996[Abstract/Free Full Text]

72. Kulessa H, Frampton J, Graf T: GATA-1 reprograms avian myelomonocytic cell lines into eosinophils, thromboblasts, and erythroblasts. Genes Dev 9:1250, 1995[Abstract/Free Full Text]

73. McNagny KM, Sieweke MH, Doderlein G, Graf T, Nerlov C: Regulation of eosinophil-specific gene expression by a C/EBP-Ets complex and GATA-1. EMBO J 17:3669, 1998[Medline] [Order article via Infotrieve]

74. Nerlov C, McNagny KM, Doderlein G, Kowemz-Leutz E, Graf T: Distinct C/EBP functions are required for eosinophil lineage commitment and maturation. Genes Dev 12:2413, 1998[Abstract/Free Full Text]

© 1999 by The American Society of Hematology.
 
0006-4971/99/9404-0014$3.00/0

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Z. Qiu, K. D. Dyer, Z. Xie, M. Radinger, and H. F. Rosenberg
GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene
J. Biol. Chem., May 8, 2009; 284(19): 13099 - 13109.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
S. Koschmieder, B. Halmos, E. Levantini, and D. G. Tenen
Dysregulation of the C/EBP{alpha} Differentiation Pathway in Human Cancer
J. Clin. Oncol., February 1, 2009; 27(4): 619 - 628.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. Milanovic, G. Terszowski, D. Struck, O. Liesenfeld, and D. Carstanjen
IFN Consensus Sequence Binding Protein (Icsbp) Is Critical for Eosinophil Development
J. Immunol., October 1, 2008; 181(7): 5045 - 5053.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
I.-M. Kim, Y. Zhou, S. Ramakrishna, D. E. Hughes, J. Solway, R. H. Costa, and V. V. Kalinichenko
Functional Characterization of Evolutionarily Conserved DNA Regions in Forkhead Box F1 Gene Locus
J. Biol. Chem., November 11, 2005; 280(45): 37908 - 37916.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Bystrom, T. A. Wynn, J. B. Domachowske, and H. F. Rosenberg
Gene microarray analysis reveals interleukin-5-dependent transcriptional targets in mouse bone marrow
Blood, February 1, 2004; 103(3): 868 - 877.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. F. Gombart, S. H. Kwok, K. L. Anderson, Y. Yamaguchi, B. E. Torbett, and H. P. Koeffler
Regulation of neutrophil and eosinophil secondary granule gene expression by transcription factors C/EBPepsilon and PU.1
Blood, April 15, 2003; 101(8): 3265 - 3273.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Du, M. J. Stankiewicz, Y. Liu, Q. Xi, J. E. Schmitz, J. A. Lekstrom-Himes, and S. J. Ackerman
Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPepsilon Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein
J. Biol. Chem., November 1, 2002; 277(45): 43481 - 43494.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. J. Tremblay, F. Hamel, and R. S. Viger
Protein Kinase A-Dependent Cooperation between GATA and CCAAT/Enhancer-Binding Protein Transcription Factors Regulates Steroidogenic Acute Regulatory Protein Promoter Activity
Endocrinology, October 1, 2002; 143(10): 3935 - 3945.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
K. McNagny and T. Graf
Making Eosinophils Through Subtle Shifts in Transcription Factor Expression
J. Exp. Med., June 3, 2002; 195(11): F43 - F47.
[Full Text] [PDF]

Home page
 JEMHome page
R. Hirasawa, R. Shimizu, S. Takahashi, M. Osawa, S. Takayanagi, Y. Kato, M. Onodera, N. Minegishi, M. Yamamoto, K. Fukao, et al.
Essential and Instructive Roles of GATA Factors in Eosinophil Development
J. Exp. Med., June 3, 2002; 195(11): 1379 - 1386.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Kurata, H.-J. Lee, T. McClanahan, R. L. Coffman, A. O'Garra, and N. Arai
Friend of GATA Is Expressed in Naive Th Cells and Functions As a Repressor of GATA-3-Mediated Th2 Cell Development
J. Immunol., May 1, 2002; 168(9): 4538 - 4545.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
T. Graf
Differentiation plasticity of hematopoietic cells
Blood, May 1, 2002; 99(9): 3089 - 3101.
[Full Text] [PDF]

Home page
 JEMHome page
A. Iwama, M. Osawa, R. Hirasawa, N. Uchiyama, S. Kaneko, M. Onodera, K. Shibuya, A. Shibuya, C. Vinson, D. G. Tenen, et al.
Reciprocal Roles for CCAAT/Enhancer Binding Protein (C/EBP) and PU.1 Transcription Factors in Langerhans Cell Commitment
J. Exp. Med., February 25, 2002; 195(5): 547 - 558.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
E. Querfurth, M. Schuster, H. Kulessa, J. D. Crispino, G. Döderlein, S. H. Orkin, T. Graf, and C. Nerlov
Antagonism between C/EBPbeta and FOG in eosinophil lineage commitment of multipotent hematopoietic progenitors
Genes & Dev., October 1, 2000; 14(19): 2515 - 2525.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
Z. Yang, N. Wara-aswapati, C. Chen, J. Tsukada, and P. E. Auron
NF-IL6 (C/EBPbeta ) Vigorously Activates il1b Gene Expression via a Spi-1 (PU.1) Protein-Protein Tether
J. Biol. Chem., July 7, 2000; 275(28): 21272 - 21277.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Gaines, J. N. Geiger, G. Knudsen, D. Seshasayee, and D. M. Wojchowski
GATA-1- and FOG-dependent Activation of Megakaryocytic alpha IIB Gene Expression
J. Biol. Chem., October 27, 2000; 275(44): 34114 - 34121.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamaguchi, Y.
Right arrow Articles by Suda, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamaguchi, Y.
Right arrow Articles by Suda, T.
Related Collections
Right arrow Phagocytes
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

click for free articles
home about blood authors subscriptions permissions advertising public access contact us
  Copyright © 1999 by American Society of Hematology         Online ISSN: 1528-0020