HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 45, 43481-43494, November 8, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/45/43481    most recent M204777200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Du, J. Articles by Ackerman, S. J. Search for Related Content PubMed PubMed Citation Articles by Du, J. Articles by Ackerman, S. J. Social Bookmarking                      What's this?

Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBP Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein^*

Jian Du, Monika J. Stankiewicz, Yang Liu, Qing Xi, Jonathan E. Schmitz, Julie A. Lekstrom-Himes^§, and Steven J. Ackerman^¶

From the  Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612 and ^§ Laboratory of Host Defenses, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, May 16, 2002, and in revised form, July 24, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein  (C/EBP) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBP isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBP²⁷ isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBP²⁷ are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBP^32/30 and ²⁷ physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU.1, and C/EBP isoforms in eosinophil gene transcription that include GATA-1/PU.1 synergy and repressor activity for C/EBP²⁷.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hematopoietic development is regulated in part by the combinatorial actions of transcription factors that coordinate temporal and lineage-specific patterns of gene expression. The transcription factors GATA-1, PU.1, and members of the CCAAT enhancer binding protein (C/EBP)¹ family (C/EBP and C/EBP in particular) are essential for the commitment and/or terminal differentiation of myeloid progenitors to the eosinophil lineage (1-9). A direct interaction and functional antagonism between GATA-1 and PU.1 have been reported recently by several groups (10-12). PU.1 has also been shown to directly or indirectly interact with multiple transcription factors including C/EBP, C/EBP (NF-IL6, CRP3), c-Jun, c-Myb, AML1, and NF-B (13-17).

GATA-1, a zinc finger family member, has been shown to be essential for virtually all aspects of erythroid and megakaryocyte gene transcription and lineage development (18-20). We have shown that eosinophil-committed promyelocytic cell lines express GATA-1-3 mRNA in an inducible fashion, and peripheral blood eosinophils of mixed developmental maturity isolated from patients with the hypereosinophilic syndrome also express GATA-1 mRNA (21). Studies in the avian system using myb-ets-transformed myeloid progenitors have suggested that an intermediate level of GATA-1 expression in the context of C/EBP and PU.1 is critical for eosinophil lineage-specific gene expression and differentiation (1, 2, 7, 12, 22). As well, GATA-1 expression is slowly down-regulated during eosinophilopoiesis in the avian system (3, 7). However, the GATA-1 null mutation in the mouse has not been evaluated for defects in eosinophil development.

PU.1, an ets family member, is selectively expressed in B lymphocytes, granulocytes, and monocytes and is required for the development of these hematopoietic lineages (23-28). During the proliferation and differentiation of multipotential lymphoid-myeloid progenitors, PU.1 is expressed at all stages of granulocyte development, both during the early onset of expression of the GM-CSF and G-CSF receptors in myeloid progenitors (27-30), and during the more terminal differentiation steps that include expression of the CD11b, CD18, and FcRI receptors (31-35). PU.1 expression increases gradually through the myelocytic stages of hematopoietic development and then remains relatively constant through terminal differentiation (26, 36). Importantly, graded expression levels of PU.1 in the mouse have been shown to specify distinct cell lineage fates in hematopoietic development, with low levels of PU.1 specifying lymphocytic and high levels specifying monocytic differentiation (37). Moreover, disruption of the PU.1 gene affects multiple hematopoietic lineages, including defects in granulocyte terminal differentiation, resulting in the absence of functionally mature neutrophils (24, 25, 28, 38) and eosinophils² (39). Thus, PU.1 is a critical regulator of hematopoietic gene transcription that possesses multiple and varied roles at different stages of both lymphoid and myeloid development.

The CCAAT/enhancer-binding proteins (C/EBPs), members of leucine zipper transcription factor family, include six members to date, of which four (C/EBP, -, -, and -) are expressed in the myeloid lineages and play key roles in hematopoiesis and/or functional maturation of these cells, and two (C/EBP and -) lack transactivation domains and act as dominant negative repressors of transcription (40, 41). C/EBP is expressed in a variety of tissues with the highest expression levels in adipose and liver (42, 43). C/EBP plays a critical role in the terminal differentiation of hepatocytes, lipid storage, and early granulocyte development (14, 15, 42, 44-48). The null mutation of C/EBP in mice results in profound abnormalities in glucose metabolism and granulocyte (both neutrophil and eosinophil) development, including impaired G-CSF receptor signaling (15, 16, 49-52). C/EBP, originally identified as a mediator of IL-6 signaling (53, 54), is critical for lipid storage, Th1 immune responses, and macrophage function (55, 56), but targeted disruption of the C/EBP gene had no effect on hematopoietic development.

C/EBP, the most recently identified C/EBP family member, is expressed almost exclusively in the myeloid lineages and is mostly restricted to the later stages of differentiation from the promyelocyte to mature granulocyte (57). Disruption of the C/EBP gene blocks the terminal differentiation of granulocytes, including impaired production of eosinophils (4) and decreased, but not loss, expression of a number of eosinophil secondary (specific) granule protein genes (see Fig. 10).³ A frameshift mutation in the C/EBP gene has been implicated recently (58, 59) as the primary defect specifying neutrophil-specific granule deficiency. Four isoforms of C/EBP (32, 30, 27, and 14 kDa), generated by alternative promoter usage, mRNA splicing, and different translation start sites have been identified (60-62). Structural and functional analyses have shown that full-length C/EBP³² (32-kDa isoform) contains both transcriptional activation and repression domains (61-63). The C/EBP²⁷ isoform contains DNA binding, dimerization, transactivation, and possible repression domains, whereas C/EBP¹⁴ is the only isoform that lacks a transactivation domain. Essentially nothing is known about the differential expression of the C/EBP²⁷ and ¹⁴ isoforms during hematopoietic development nor their activities or specific roles in myeloid gene expression. Although C/EBP family proteins generally bind DNA as either homo- or heterodimers (64), dimer formation was not detected for C/EBP³² expressed recombinantly in either bacteria or mammalian cells (65).

Functional and physical interactions between C/EBP and GATA-1 were first identified in vitro through our studies of the transcriptional regulation of the gene encoding the human eosinophil granule major basic protein (MBP) (9), in which we demonstrated synergy between these factors in the transactivation of the MBP promoter. Regulation of an eosinophil-specific gene (EOS47) by the combinatorial actions of C/EBP, ets-1, and GATA-1 has also been reported for the avian system (2, 3). In the present study, we have further characterized the eosinophil-specific MBP-P2 promoter, identifying novel combinatorial interactions among GATA-1, PU.1, and the C/EBP²⁷ isoform that result in either synergistic activation or transcriptional repression. Human MBP, one of the principal mediators of inflammation and tissue damage in eosinophil-associated allergic responses, is expressed by eosinophils (66), and to a lesser extent by basophils (67), and a proform of MBP is elaborated by placental trophoblasts during gestation (68, 69). Two different MBP transcripts are regulated by alternative splicing from two distinct promoters (P1 and P2) located 34 kb apart (70). Eosinophils and basophils transcribe principally from the P2 promoter, whereas the P1 promoter predominates in placental cells (8, 70).

We identified previously a major functional region for the MBP-P2 promoter between bp 117 and 67 (8, 9). In the present studies, a more extensive consensus binding site analysis showed that the bp 117 to +1 region of the promoter contains not one but two adjacent GATA-binding sites, two C/EBP sites, and two previously unidentified PU.1 sites, suggesting that the combinatorial activities of all three of these factors are involved in MBP gene transcription. Because PU.1 has been reported to antagonize GATA-1-mediated erythroid gene expression and development (10-12), and we have shown that GATA-1 is a key regulator of the MBP gene, we utilized the MBP-P2 promoter as a model to characterize further the combinatorial interactions and roles of GATA-1, PU.1, and C/EBP in the eosinophil lineage. Consensus sites in the functional region of the MBP-P2 promoter were evaluated for their ability to bind GATA, PU.1, or C/EBP family members using nuclear extracts from the MBP-expressing eosinophil myelocyte cell line, AML14.3D10, and mature peripheral blood eosinophils. The functional relevance of these binding sites and transcription factor interactions were evaluated using mutagenesis, transactivation analyses in CV-1 cells, and transient transfection analyses in AML14.3D10 eosinophils. Co-immunoprecipitation (co-IP) experiments were performed to confirm the in vivo physical interactions of GATA-1, PU.1, and the various C/EBP isoforms in eosinophil cell lines and mature peripheral blood eosinophils.

Our results show that GATA-1, PU.1, and the various C/EBP isoforms are differentially expressed, and physically and functionally interact during human eosinophil development to activate and then repress eosinophil gene transcription. In contrast to the functional antagonism reported for PU.1 and GATA-1 for activation of the M-CSFR promoter and various erythroid genes, we show that these factors cooperate synergistically in the activation of the eosinophil MBP-P2 promoter. Importantly, we demonstrate that PU.1 expression levels relative to GATA-1 determine whether the GATA-1/PU.1 interaction leads to synergy for GATA-1-regulated eosinophil target genes such as MBP. Moreover, we identify a novel role for the C/EBP²⁷ isoform as a repressor of GATA-1-mediated transactivation and GATA-1/PU.1 synergy. Finally, co-IP analyses demonstrate for the first time that the C/EBP^32/30 and C/EBP²⁷ isoforms physically interact with both PU.1 and GATA-1 in vivo, in eosinophils. Our findings demonstrate key combinatorial interactions of GATA-1, PU.1, and C/EBP isoforms that may mediate either synergy or antagonism (repression) of granulocyte (eosinophil) gene transcription during myeloid development and terminal differentiation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Primary Cells and Cell Lines-- Human blood eosinophils were isolated from a unit (475 ml) of blood obtained from normal, non-allergic, healthy donors. The use of normal human subjects as blood donors was in full compliance with all federal guidelines and was approved by the University of Illinois Institutional Review Board. Eosinophil isolation and purification were performed as described previously (71) using a Miltenyi Biotec SuperMacs immunomagnetic sorting kit and apparatus according to manufacturer's specifications. Eosinophil preparations of >99% purity were routinely obtained and utilized for these studies. The human AML14.3D10 cell line is a fully differentiated eosinophil myelocyte line that contains eosinophil secondary (specific) granules and displays many characteristics of mature peripheral blood eosinophils. These include expression of the secondary granule cationic proteins major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil cationic protein (72, 73). AML14.3D10 cells can be induced to express chemokine receptors (e.g. for eotaxins) (74) and express GM-CSF which drives their proliferation, differentiation, and survival in culture (75). The AML14 parental cell line is a human myeloid leukemia-derived myeloblast committed to the eosinophil lineage. AML14 and AML14.3D10 cells were maintained in RPMI 1640 supplemented with 8% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 5 × 10⁵ M -mercaptoethanol without any antibiotics. Cells were passaged every 3-4 days and maintained at a concentration between 3 × 10⁵ and 1 × 10⁶ cells/ml. CV-1 cells were maintained by passage twice weekly in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (10 units/ml), and streptomycin (10 µg/ml) and were used for transfection at 60-70% confluence.

Whole Cell Lysates and Nuclear Extract-- AML14.3D10 eosinophils were lysed in a lysis buffer containing 0.5-1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 200 µM sodium orthovanadate, 50 mM Hepes, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1.5 mM magnesium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin (76), supplemented with protease inhibitor mixture tablets (Roche Molecular Biochemicals) for 1 h at 4 °C. Whole cell lysates were collected by centrifugation at 14,000 × g for 20 min at 4 °C to remove cell debris, and lysates were stored at 80 °C. Nuclear extracts were prepared by the method of Dignam et al. (77) with minor modifications, including the use of protease inhibitor mixture tablets (Roche Molecular Biochemicals) and the addition of phenylmethylsulfonyl fluoride (0.5 mM) and diisopropylfluorophosphate (1 mM) to the resuspension and lysis buffers (78, 79). The protein concentration of nuclear or whole cell extracts was determined by the BCA method (Pierce), and extracts were aliquoted and stored at 80 °C.

DNA Constructs and Mutagenesis-- The bp 117MBP-P2-pXP2 luciferase reporter gene construct was described in detail previously (8, 9). Mutagenesis PCR was performed to create mutations in the PU.1, GATA-1, or C/EBP consensus sites that block transcription factor binding using the QuickChange^TM PCR kit (Stratagene, CA). Dr. Daniel Tenen kindly provided the M-CSFR promoter construct (M-CSFR-pXP2). The expression vector PU.1-pECE contains the mouse cDNA for PU.1 and was a gift from Dr. R. Maki (23). The GATA-1-pXM expression vector contains the murine GATA-1 cDNA and was kindly provided by Dr. L. Zon (80, 81). Both C/EBP and -, in the pMSV expression vector, were kindly provided by Dr. A. Friedman, and the various C/EBP isoforms in the pcDNA3 expression vector (pc32, pc27, and pc14) were kindly provided by Dr. K. Xanthopoulos. All DNA constructs used in transfection and transactivation experiments were prepared by alkaline lysis maxi-preparation followed by CsCl₂ purification as described previously (79, 82).

Western Blotting and Co-immunoprecipitations-- Western blotting was performed as described previously (71, 76) using either whole cell lysates or nuclear extracts from blood eosinophils, AML14.3D10 eosinophil myelocytes, and undifferentiated AML14 parental cells. HL-60 and K562 cell lines were used as positive controls as appropriate to the transcription factors being analyzed. Twenty µg of each extract was separated by SDS-PAGE and electroblotted onto Immobilon-P transfer membranes (Millipore, MA) following standard procedures. Antibodies against GATA-1 (M-20), PU.1 (T-21 and N-19), and C/EBP (C-20) and negative control antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All antibodies were non-cross-reactive with other members of their respective transcription factor families. In particular, the antibodies to PU.1 do not recognize other ets family proteins. The blots were analyzed using an enhanced chemiluminescence kit (Pierce) following the manufacturer's recommended procedure. Immunoprecipitation (IP) and co-IP were carried out using total cell lysates. Antibodies used for IP and co-IP were the same as used for Western blotting. IP and co-IP were performed essentially as described previously (71, 76). Briefly, the whole cell lysate from 2 to 4 × 10⁷ AML14.3D10 cells was precleared with 1-2 µg of normal rabbit, goat, or rat affinity-purified IgG antibodies plus protein A- or G-Sepharose (Amersham Biosciences) with rotation at 4 °C for 2.5 h. Antibodies were added to the precleared whole cell lysates for 2 h of rotation at 4 °C. Non-immune purified rabbit, rat, and goat immunoglobulin (Santa Cruz Biotechnology) were utilized as negative controls. Protein A- or protein G-Sepharose was added to the lysates for another 2 h at 4 °C. Immunoprecipitates were washed 4 times with 0.1% Triton X-100 lysis buffer and analyzed by Western blotting.

Electrophoretic Mobility Shift Assays (EMSA)-- Oligonucleotides containing the GATA, PU.1, and C/EBP consensus sites in the MBP-P2 promoter were synthesized and purified by Integrated DNA Technologies, Inc. (Coralville, Iowa). Double-stranded probes were end-labeled with [-³²P]ATP (PerkinElmer Life Sciences) and purified on 15% polyacrylamide gels as described previously (79). Probe sequences were as follows: dual GATA sites (5'-GTCCTTATCAGCCTTGCTATCTCCCT-3'), PU.1 site 1 (5'-CCCTGGGGGAAGTTCCTCCAAGGCCT-3'), PU.1 site 2 (5'-AAGTCTTTGTGAGAGGAAGCAAAGAA-3'), C/EBP site (5'-GAAGTGATGAAATGGTCC-3'), C/EBP and GATA-1 sites (5'-GAAGTGATGAAATGGTCCTTATCAGCCT-3'). EMSAs were performed as described previously (71). The same antibodies used for Western blotting and co-IP were also used for supershift analyses but were supershift grade (Santa Cruz Biotechnology). The antibodies to PU.1 (T-21 and N-19) were specifically chosen because they are non-cross-reactive with other ets family member proteins.

Transactivation and Transient Transfection Assays-- Transactivation experiments were performed using the DMRIE-C reagent (Invitrogen) for transfections according to the manufacturer's protocol. CV-1 cells (60-80% confluent in 6-well plates) were transfected with 0.5 µg of the MBP-P2-pXP2 wild-type or mutant promoter constructs in the presence or absence of expression vectors for PU.1 (0.001-0.1 µg), GATA-1 (0.2 µg), and/or C/EBP and -, or ³², ²⁷, or ¹⁴ isoforms (0.2 µg). Because GATA-1 may be an activator at low levels or a repressor at high levels of expression as reported for the avian Eos-47 eosinophil promoter (2, 7), the amount of the GATA-1 expression vector added (0.2 µg) to co-transactivations with PU.1 was first optimized by dose-response titration in order to provide maximum transactivation of the MBP-P2-pXP2 promoter construct. The empty vectors corresponding to each of the transcription factors were used to normalize the total DNA content of each reaction. Transfection efficiency was normalized by the addition of 0.2 µg of the pRL-CMV (Renilla luciferase) control reporter. Promoter activities were determined using the Dual-luciferase Reporter Assay System (Promega) 48 h after transfection. Luciferase activities were measured as relative light units using an EG & G Berthold Lumat LB 1507 Luminometer as described previously (71). Results are reported as the mean ± S.D. (fold induction over control) for at least three independent transactivation experiments. Transient transfections of the AML14 parental and AML14.3D10 eosinophil cell lines were performed by electroporation as described previously (71, 79). Briefly, 10 µg of each DNA construct and 1.5 × 10⁷ cells were used in each reaction. Promoter activities (relative light units) were measured 6 h after electroporation, and transfection efficiency was normalized using the pRL-CMV vector as above in the dual luciferase assay. Results are reported as the mean ± S.D. (fold induction over control) for at least three independent experiments.

Semi-quantitative Reverse Transcriptase-PCR-- Total RNA was prepared from fetal liver or bone marrow cells of mice with targeted disruptions of the C/EBP (50, 52) and C/EBP (4) genes using standard purification methods as described previously (4, 83). C/EBP null mice were first stimulated for 4.5 or 24 h with 4% thioglycolate broth by intraperitoneal injection prior to harvesting the bone marrow as described previously (4). The RNA was electrophoresed on 1% formaldehyde agarose gels, and 18 S and 28 S bands were visualized by ethidium bromide staining to assess the quality. Three µg of total RNA was reverse-transcribed with random hexadeoxynucleotide primers using a First-strand cDNA Synthesis kit (Amersham Biosciences) according to the manufacturer's protocol. Calculations were based on the assumption that 100% of the RNA was reverse-transcribed to cDNA. The PCR primer pairs for cDNA amplification of murine MBP were forward 5'-CCAAGGAAGAGGACACAACAAGTC-3' and reverse 5'-TTGACCCTGGTTGATTCCCC-3'; GAPDH forward 5'-CCATGGAGAAGGCTGGGG-3' and reverse 5'-CAAAGTTGTCATGGATGACC-3'. BLAST search analysis of these primers indicated they were specific for the genes of interest. The signal from amplification of the GAPDH cDNA (mRNA) was used first as a control to normalize the amount of cDNA input for subsequent PCR amplification of each sample. The PCR mix contained 10-50 ng of cDNA, 10 pmol of each primer, 200 µM dNTPs, 0.3 µl of 10 mCi/ml [-³²P]dCTP (PerkinElmer Life Sciences), 0.5 µl of 100 mg/µl of bovine serum albumin, 10× PCR buffer, and 0.5 ml (2.5 units) of Taq DNA polymerase from Roche Molecular Biochemicals. The total reaction volume was 50 µl. The number of PCR cycles determined to yield a linear, quantitative signal for MBP and GAPDH were 30 and 21, respectively. Each reaction cycle consisted of 50 s at 94 °C for DNA denaturation, 45 s at the annealing temperature for the primer pair (55 °C and 53 °C for mMBP1 and GAPDH, respectively), and 45 s at 72 °C for extension. Following PCR amplification, 5 µl of PCR buffer was analyzed by electrophoresis on 6% native polyacrylamide gels.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PU.1, GATA-1, and C/EBP Isoforms Are Differentially Expressed in Eosinophilic Cell Lines and Mature Blood Eosinophils and Bind to the Their Consensus Sites in the Functional Region of the MBP-P2 Promoter-- We identified a number of additional GATA-1 and C/EBP consensus sites, as well as two PU.1 consensus sites not characterized previously in the MBP-P2 promoter, using subsequence homology searches of the bp 117 to +1 functionally active region (Fig. 1). This is the region for which we had shown previously the most 5' GATA-1 and C/EBP sites to be necessary for MBP promoter activity in eosinophilic cell lines using deletional and mutagenesis analyses (8, 9). We first determined the expression patterns for GATA-1, PU.1, and C/EBP in AML14 eosinophilic cell lines and mature peripheral blood eosinophils by Western blotting (Fig. 2), and their binding activity for the multiple consensus sites was identified in the MBP-P2 promoter by gel shifts (Fig. 3). Expression levels for GATA-1, PU.1, and the various C/EBP isoforms was compared for AML14 parental myeloblasts, AML14.3D10 eosinophil myelocytes, and peripheral blood eosinophils from normal donors, covering the range of differentiation stages from undifferentiated progenitors to terminally differentiated mature eosinophils. GATA-1 was well expressed both for undifferentiated AML14 parental cells and differentiated AML14.3D10 eosinophilic myelocytes (Fig. 2A, lanes 1 and 2) but was undetectable in normal peripheral blood eosinophils (Fig. 2A, lane 3) from two different donors (2nd donor not shown). K562 cells, which express abundant endogenous GATA-1 protein, were used as the positive control (Fig. 2A, lane 4). This finding suggests that GATA-1 expression is significantly down-regulated during eosinophil terminal differentiation, supporting prior results in the avian system (2, 7). PU.1 was also expressed by both AML14 parental myeloblasts and AML14.3D10 eosinophil myelocytes with AML14 < AML14.3D10 (Fig. 2B, lanes 1 and 2) but not detectable in mature blood eosinophils and K562 cells (Fig. 2B, lanes 3 and 4). However, much longer exposure times revealed weak expression in both blood eosinophils and K562 cells (not shown). This finding indicates a significant reduction in PU.1 expression as eosinophils terminally differentiate. All four isoforms of C/EBP including C/EBP^32/30, C/EBP²⁷, and C/EBP¹⁴ were well expressed in AML14.3D10 eosinophilic myelocytes (Fig. 2C, lane 2), comparable with HL-60 promyelocytes (lane 4), but expression was lower in the AML14 myeloblast line (Fig. 2C, lane 1). Importantly, only the C/EBP¹⁴ isoform is abundantly expressed in terminally differentiated blood eosinophils (Fig. 2C, lane 3), a finding confirmed using two different blood donors (Fig. 2C, lanes 5 and 6). The high level expression of C/EBP¹⁴ in blood eosinophils was initially detected as a broad smear on Western blots using equal protein loading for all nuclear extracts (Fig. 2C, lane 3). We therefore reduced protein loading by a factor of 10 to show the C/EBP¹⁴ signal as a single 14-kDa band in the blood eosinophils from two different normal subjects (Fig. 2C, lanes 5 and 6). Further reductions in protein loading eliminated all isoforms of C/EBP expressed by AML14.3D10 cells but still showed a sharp single band for the C/EBP¹⁴ isoform expressed by authentic blood eosinophils (not shown), indicating very high level expression of C/EBP¹⁴ in the mature eosinophil. These findings demonstrate that GATA-1, PU.1, and C/EBP are all well expressed in the AML14.3D10 eosinophil myelocyte cell line, which is therefore an ideal model for functional studies of the roles of GATA-1, PU.1, and C/EBP isoforms in vivo and in vitro. The differential expression of the C/EBP isoforms in developing eosinophil progenitors versus the mature, terminally differentiated cell may reflect their specific roles as activators and then repressors of eosinophil gene expression during granulocyte development (see below and Fig. 11).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Schematic of the functional 5'-upstream region of the MBP-P2 promoter. Binding sites for three principal transcription factors have been identified in the most functional bp 117 to +1 region of the MBP-P2 promoter by nucleic acid subsequence analysis. These include two GATA, two C/EBP, and two PU.1 consensus sites as indicated (top schematic). Mutations in the GATA-1, C/EBP, and PU.1 sites were generated as indicated in the pXP2 luciferase reporter vector for transactivation (Fig. 7A) and transfection (Fig. 7B) experiments.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Differential expression of PU.1, GATA-1, and C/EBP isoforms in eosinophil progenitor and myelocytic cell lines compared with mature peripheral blood eosinophils. Western blot analysis of the levels of PU.1, GATA-1, and C/EBP isoforms using nuclear extracts from AML14 (eosinophil myeloblast) and AML4.3D10 (eosinophil myelocyte) cell lines and mature peripheral blood eosinophils (>99% purity) from two different normal subjects. Western blots were probed with GATA-1 antibody (A), PU.1 antibody (B), and C/EBP antibody (C). K562 nuclear extract was used as a positive control for GATA-1 and PU.1. HL-60 nuclear extract was used as a positive control for C/EBP expression.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   PU.1, GATA-1, and C/EBP in AML14.3D10 eosinophil nuclear extracts bind to their consensus sites in the MBP-P2 promoter. A, GATA-1 binds to the dual GATA sites in the MBP-P2 promoter. The gel shift assay used the dual GATA consensus site in the MBP-P2 promoter as probe. Two specific complexes formed with nuclear extract from AML14.3D10 eosinophilic myelocytes (lane 2). These complexes were completely inhibited by the addition of unlabeled self-competitors (lane 3) and partially inhibited by an oligonucleotide containing only a single GATA site (lanes 4 and 5) but not by a dual GATA site mutation (lane 6). The protein-DNA complexes were completely supershifted by the addition of antibody to GATA-1 (lane 7) but not by antibodies to PU.1 or the C/EBPs (, , and ) (lanes 8-11). B, PU.1 binds to both PU.1 consensus sites in the MBP-P2 promoter. The gel shift was performed using the second PU.1 consensus site of the MBP-P2 promoter as probe. Two protein-DNA complexes formed with nuclear extract of AML14.3D10 cells as indicated by the arrows (lane 2). Although both complexes were completely inhibited by the addition of unlabeled probe (the 2nd PU.1 site) (lane 3), only the higher mobility complex (labeled PU.1) was specifically eliminated by the addition of competitor containing the first PU.1 site (lane 4) but not by other transcription factor-binding sites (lanes 5-7). This PU.1 complex was also eliminated by the addition of two different antibodies to PU.1 (lanes 8 and 9) but not by antibodies to GATA-1, C/EBP, -, or - (lanes 10-13). C, C/EBP and C/EBP both bind to the first (most 5') C/EBP consensus site in the MBP-P2 promoter. The gel shift was performed using both C/EBP consensus sites of the MBP-P2 promoter as probes. However, no binding was detected using the 2nd C/EBP site. Two specific complexes formed with the first C/EBP consensus site using nuclear extract of AML14.3D10 eosinophils (lane 1, bands  and ). Complex formation was completely inhibited by the addition of unlabeled competitors including self or the C/EBP consensus site of the M-CSFR promoter (lanes 2 and 4, respectively) but not by a C/EBP binding site mutation (lane 3). Antibody to C/EBP completely removed and supershifted complex  and partially removed complex (lane 8). The addition of antibody to C/EBP partially removed complex (lane 6). Supershifted bands (S and S) were detected by addition of both C/EBP and C/EBP antibodies (lanes 6 and 8) but not by antibodies to C/EBP and C/EBP (lanes 5 and 7).

The bp 117 to +1 functional region of the MBP-P2 promoter possesses two consensus sites each for GATA-1, PU.1, and the C/EBPs (Fig. 1). To assess their ability to bind these factors, we performed gel and antibody supershift analyses using nuclear extracts from the AML14.3D10 eosinophil myelocyte cell line and [-³²P]ATP-end-labeled oligonucleotides containing the GATA-1, PU.1, and C/EBP consensus sites of interest (Fig. 3). GATA-1 binds to the two tandem GATA sites in the promoter (Fig. 3A). Two specific protein-DNA-binding complexes formed with the AML14.3D10 nuclear extract using the dual GATA site probe (Fig. 3A, lane 2). Complex formation was completely inhibited by the addition of a 100-fold molar excess of the unlabeled dual GATA site oligonucleotide (lane 3), partially inhibited by oligonucleotides containing only the first or second GATA site (lanes 4 and 5) but not inhibited by oligonucleotides containing a dual GATA site mutation (lane 6). The GATA-binding complexes were completely supershifted by an antibody to GATA-1 (lane 7) but not by antibodies to PU.1 or the various C/EBPs (lanes 8-11). These results demonstrate that GATA-1 binds to both GATA consensus sites comprising the dual GATA-1 site of the MBP-P2 promoter, with maximum binding to the dual site as compared with the single sites (data not shown).

For the PU.1 consensus sites, two major protein-DNA complexes formed with nuclear extract from AML14.3D10 cells using the second PU.1 consensus site as a probe (Fig. 3B, lane 2). Formation of these complexes was completely inhibited by the addition of a 100-fold molar excess of the unlabeled probe (lane 3) but only the higher mobility complex (PU.1) was removed by competition with both the first (not shown) and second PU.1 sites (lane 4), indicating the specificity of this complex. This PU.1 complex was selectively blocked by the addition of two different antibodies to PU.1 (lanes 8 and 9) but not by antibodies against GATA-1 or the C/EBPs (lanes 10-13). These results demonstrate that both PU.1 consensus sites in the MBP promoter have the capacity to bind PU.1 expressed by AML14.3D10 eosinophils. For the C/EBP consensus sites in the MBP promoter, both C/EBP and C/EBP in AML14.3D10 eosinophil nuclear extracts formed specific protein-DNA complexes with the first C/EBP site (Fig. 3C). Complex formation was completely inhibited by the addition of a 100-fold molar excess of an unlabeled C/EBP consensus site oligonucleotide matching the MBP-P2 or M-CSFR promoters (lanes 2 and 4) but not by an oligonucleotide containing a C/EBP site mutation (lane 3). One of the DNA-binding complexes was completely supershifted by the addition of antibody to C/EBP (lane 8). A second, higher mobility binding complex was partially removed by the addition of antibody to C/EBP or C/EBP. No C/EBP or C/EBP complexes were detected using AML14.3D10 eosinophil nuclear extracts (lanes 5 and 7), whereas in equivalent gel shifts using AML14 parental cell nuclear extracts, only C/EBP complexes could be identified by antibody supershift (not shown). The second C/EBP consensus site in the promoter did not form any complexes, nor did it inhibit complex formation with the first C/EBP site (not shown). These results indicate that C/EBP and C/EBP homodimers, as well as C/EBP-C/EBP heterodimers, present in AML14.3D10 eosinophil nuclear extracts bind to the first C/EBP site in the MBP-P2 promoter; this is the C/EBP site we previously showed to be functionally transactivated by C/EBP (9) and in the present study by C/EBP as well (see Fig. 9A).

Synergy Versus Antagonism between GATA-1 and PU.1-- As noted above, a functional antagonism between GATA-1 and PU.1 has been reported for the transcriptional regulation of myeloid genes such as the M-CSFR (11), for which the promoter possesses a functional PU.1 site but no GATA sites, and GATA-1 antagonizes PU.1 transactivating activity. Because the MBP-P2 promoter possesses both GATA-1- and PU.1-binding sites, and the MBP-P2 promoter is strongly transactivated by GATA-1, we sought to determine whether PU.1 would similarly antagonize GATA-1 activity in our system. Transactivation experiments were performed in CV-1 cells by transfection of the bp 117MBP-P2 luciferase reporter construct with GATA-1 and/or PU.1 expression vectors (Fig. 4A). Because GATA-1 has been shown previously (2, 7) to function as an activator at low concentrations and repressor at high concentrations for an avian eosinophil promoter EOS47, we first performed dose-response titrations of the GATA-1 expression vector to determine the optimal concentration for maximal GATA-1 transactivation of the MBP-P2-pXP2 reporter construct (data not shown); this optimal amount was used for all subsequent co-transactivation assays. A PU.1-regulated M-CSFR promoter construct, used previously to demonstrate GATA-1 antagonism of PU.1 transactivation (11), was also transfected into CV-1 cells along with the same GATA-1 and/or PU.1 expression vectors in these experiments for comparison (Fig. 4B). Contrary to the anticipated PU.1 antagonism of GATA-1, our results clearly show that MBP-P2 promoter activity was induced ~20-fold by GATA-1 alone and ~60-fold by GATA-1 plus PU.1, with PU.1 alone being essentially inactive (Fig. 4A).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Synergy versus antagonism of GATA-1 and PU.1. A, GATA-1 and PU.1 synergistically transactivate the MBP-P2 promoter. CV-1 cells were transfected with 0.5 µg of the MBP-P2-pXP2 luciferase construct, 0.2 µg of GATA-1-pXM, and 0.01 µg of PU.1-pECE expression vectors. Promoter activity was measured 48 h after transfection and analyzed for fold induction over the level of MBP-P2 base-line activity in the absence of expression vectors. B, GATA-1 represses PU.1 transactivation of the M-CSFR promoter. CV-1 cells were transfected using the same transfection method as above with 0.5 µg of an M-CSFR promoter luciferase construct and expression vectors for GATA-1 and PU.1 as above. Promoter activity was determined 48 h after transfection and analyzed for fold induction over the M-CSFR promoter base-line activity. Means (± S.D.) are shown for four independent co-transactivation experiments. C and D, the synergy versus antagonism of GATA-1 and PU.1 is dependent upon the expression level of PU.1 relative to GATA-1, an increased amount of PU.1 reduces GATA-1/PU.1 synergistic transactivation of the MBP-P2 promoter (C). A 10-fold increase in the amount of PU.1 expression vector (0.1 µg) was compared with the amount (0.01 µg) used in transactivation experiments performed as in A. The synergy between GATA-1 and PU.1 for the MBP-P2 promoter was eliminated by the addition of an increased amount of the PU.1 expression vector (0.1 µg). D, an increased amount of PU.1 reverses GATA-1/PU.1 antagonism of the M-CSFR. A 10-fold increase in the amount of PU.1 vector (0.1 µg) was compared with the amount (0.01 µg) used in the transactivation experiments performed as described in B. The GATA-1 inhibition of PU.1-mediated transactivation of the M-CSFR promoter was overcome by the addition of an increased amount of the PU.1 expression vector. +, 0.01 µg; ++, 0.1 µg of PU.1-pECE).

These results demonstrate a significant synergy of PU.1 for the GATA-1-mediated transactivation of the MBP-P2 promoter, in contrast to the antagonism of GATA-1 for the PU.1-mediated transactivation of the M-CSFR promoter as reported previously (11) and confirmed in the current "control" experiments (Fig. 4B). The findings provide evidence for important promoter-specific and possible lineage-specific differences in the combinatorial outcome of GATA-1/PU.1 interactions in myeloid gene regulation in general and eosinophil gene regulation in particular during eosinophil development. To further evaluate the synergistic activities of GATA-1 and PU.1 for transactivation of the MBP-P2 promoter, we performed dose-response analyses using different amounts of PU.1 expression vector at an optimal concentration of the GATA-1 expression vector. The M-CSFR promoter was again evaluated in these experiments as a control for a PU.1-regulated gene that is antagonized by GATA-1. Surprisingly, we found that the synergistic transactivation of the MBP-P2 promoter by GATA-1 and PU.1 occurred in the presence of low levels of PU.1 but was completely abrogated by high levels of PU.1 (Fig. 4C). In contrast, PU.1-mediated transactivation of the M-CSFR promoter was antagonized by GATA-1 in the context of a low level of PU.1 but overcome by a high level of PU.1 (Fig. 4D). In addition, a positive interaction between PU.1 and GATA-1 was also obtained in transactivation experiments performed in the K562 erythroleukemia cell line that expresses high levels of endogenous GATA-1, such that low levels of the PU.1 expression vector doubled MBP-P2 promoter activity due to endogenous GATA-1, whereas high levels of PU.1 had no effect (Fig. 5). These findings suggest that the level of PU.1 expression is critical in determining whether GATA-1 and PU.1 functionally antagonize or synergize in the regulation of PU.1 versus GATA-1 target genes in the myeloid lineages.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   PU.1 augments MBP-P2 promoter activity in the GATA-1-expressing K562 cell line. K562 cells were transfected by electroporation with the MBP-P2 promoter construct along with a low (0.1 µg) or high (1.0 µg) dose of the PU.1-pECE expression vector and the pRL-CMV (Renilla luciferase) control vector. Mean promoter activity (± S.D.) relative to the promoterless pXP2 control luciferase vector is shown for three independent experiments. Luciferase activity was determined 6 h after transfection and normalized using the dual luciferase method.

C/EBP²⁷ Represses GATA-1 Transactivation and Blocks GATA-1/PU.1 Synergy in the Activation of the MBP-P2 Promoter-- We have shown that all four C/EBP isoforms are expressed by AML14.3D10 eosinophilic myelocytes (Fig. 2) and that C/EBP in nuclear extracts of these cells binds the C/EBP consensus site of the MBP-P2 promoter (Fig. 3C). As well, we reported previously (9) that C/EBP functionally synergizes with and physically interacts with GATA-1. We therefore sought to characterize the functional activities of the different C/EBP isoforms in terms of both direct transactivating activity and combinatorial effects with GATA-1 and PU.1. Transactivation experiments were carried out in CV-1 cells by co-transfection of the bp 117-MBP-P2 promoter construct with expression vectors for C/EBP³², C/EBP²⁷, and C/EBP¹⁴, and/or GATA-1, and/or PU.1 as shown in Fig. 6. The MBP-P2 promoter was transactivated by GATA-1 alone but not by any of the individual C/EBP isoforms (Fig. 6, A and B). In contrast, GATA-1-induced transactivation was significantly inhibited by the addition of C/EBP²⁷ but not by the C/EBP³² or C/EBP¹⁴ isoforms (Fig. 6A). Furthermore, the synergistic activity of GATA-1 and PU.1 (~75-fold induction) was completely suppressed by the addition of the C/EBP²⁷ isoform, but not by the other C/EBP isoforms (Fig. 6B). Importantly, the repressor activity of the C/EBP²⁷ isoform was not unique to the individual or combinatorial activities of GATA-1 and GATA-1/PU.1 on MBP-P2 promoter activity, as PU.1-mediated transactivation of the M-CSFR promoter was likewise blocked by C/EBP²⁷ in these experiments (Fig. 6C). In addition, the C/EBP²⁷ isoform completely blocked PU.1 synergy with endogenous GATA-1 in the K562 cell line in which the MBP-P2 promoter is active (Fig. 6D). The C/EBP²⁷ isoform contains both transactivation and putative repression domains (40, 61, 63). Its negative regulatory activity in our system may be mediated by the repression domain through either binding to the MBP promoter C/EBP site directly or by protein-protein interactions that interfere with GATA-1 DNA binding, transcriptional activity, or the physical interactions of GATA-1 with PU.1.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   C/EBP27 inhibits GATA-1 and GATA-1/PU.1 synergistic transactivation of the MBP-P2 promoter and PU.1 transactivation of the M-CSFR promoter. A, C/EBP27 inhibits GATA-1 transactivation of the MBP-P2 promoter. CV-1 cells were transfected with 0.5 µg of the MBP-P2-pXP2 luciferase reporter and also co-transfected with or without 0.2 µg of GATA-1 and 0.5 µg of C/EBP32, C/EBP27, or C/EBP14 expression vectors as indicated. Promoter activity is shown as the fold induction over the MBP-P2-pXP2 reporter alone. GATA-1 transactivation was repressed by the addition of the C/EBP27 but not by C/EBP32 or C/EBP14 isoforms. B, C/EBP27 isoform inhibits GATA-1/PU.1 synergistic transactivation of the MBP-P2 promoter. Transactivation of the MBP-P2 promoter by GATA-1, PU.1, and the C/EBP isoforms was performed as described above. Synergistic transactivation of the MBP-P2 promoter by GATA-1/PU.1 was also blocked by the addition of the C/EBP27 but not C/EBP32 or C/EBP14 isoforms. C, C/EBP27 inhibits PU.1-mediated transactivation of the M-CSFR promoter. Transactivation of a PU.1-responsive M-CSFR-pXP2 reporter construct was performed in CV-1 cells as described in Fig. 4B. 0.01 µg of PU.1 expression vector and/or 0.5 µg of C/EBP27 expression vector were used in the co-transactivation. PU.1 transactivation of the M-CSFR promoter was fully repressed by the expression of the C/EBP27 isoform. D, the C/EBP27 isoform blocks PU.1-mediated augmentation of MBP-P2 promoter activity in the K562 cell line. K562 cells were transfected by electroporation with the MBP-P2 promoter construct, a low dose of PU.1 (0.1 µg), and the C/EBP27 expression vector as indicated, along with the pRL-CMV (Renilla luciferase) control vector. Mean (± S.D.) promoter activity is expressed as the fold activity relative to the promoterless pXP2 luciferase control vector. Luciferase activity was determined 6 h after transfection and normalized using the dual luciferase method.

We showed previously that the synergistic interactions of GATA-1 and C/EBP likely occur through protein/protein interaction in that they required either a functional GATA- or C/EBP-binding site but not both (9). As well, GATA-1 and PU.1 have been shown to antagonize functionally one another's functions via protein/protein interactions, whether the target genes contain GATA-1- or PU.1-binding sites (11, 84). Because GATA-1, PU.1, and C/EBP can all bind to their respective sites in the MBP-P2 promoter (Fig. 3), we sought to determine the functional relevance of these binding sites in the combinatorial interactions of these transcription factors on this eosinophil promoter. Constructs containing mutations in the dual GATA site, the two C/EBP sites, or both PU.1 sites were generated by PCR mutagenesis using the wild type MBP-P2 promoter construct as template and tested in both CV-1 cells (Fig. 7A) and the AML14 eosinophil cell lines (Fig. 7B). Transactivation analyses were performed in CV-1 cells as described above by co-transfection of the MBP-P2 mutant and wild type constructs with GATA-1, PU.1, and C/EBP²⁷ expression vectors (Fig. 7A). Activity of the MBP-P2 promoter was essentially eliminated by the mutation of the C/EBP sites or the GATA sites, indicating a requirement for both sites in this promoter. No GATA-1-induced transactivation or GATA-1/PU.1 synergistic transactivation was seen for either the C/EBP or GATA site mutants. In contrast, mutation of the two PU.1 sites in the MBP-P2 promoter was essentially without effect, and both GATA-1-induced transactivation and GATA-1/PU.1 synergy was equivalent to that obtained for the wild type promoter. To confirm this finding for the endogenously expressed factors in eosinophils, the same MBP-P2 promoter wild type and mutant constructs were transiently transfected by electroporation into the AML14.3D10 eosinophil and AML14 parental cell lines (Fig. 7B). The results confirmed that mutation of the PU.1-binding sites was essentially without effect on MBP-P2 promoter activity compared with the wild type promoter in both cell lines, whereas mutation of either the GATA sites or C/EBP sites resulted in a significant loss of activity. We conclude from these analyses that the synergistic activity of PU.1 on GATA-1-mediated transactivation occurs independently of direct PU.1 binding to either of the two PU.1-binding sites in the MBP-P2 promoter. These results imply that the synergy of PU.1 and GATA-1 in this system is mediated via protein/protein interactions between these factors and binding of the complex through the dual GATA-binding site in the promoter. These results also confirm our prior findings that the C/EBP and GATA sites are both critical for transcriptional activation of this promoter by transactivation in either heterologous cell lines or by the endogenous factors in eosinophil progenitor lines (8, 9).

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Activity of the MBP-P2 promoter is eliminated by mutation of the GATA or C/EBP sites but not the PU.1 sites; analysis by transactivation in CV-1 cells and transfection of AML14.3D10 eosinophilic myelocytes. A, CV-1 cells were transfected with 0.5 µg of the MBP-P2 wild type or mutant constructs including a dual C/EBP site mutation, dual GATA-1 site mutation, or dual PU.1 site mutation as indicated and shown schematically in Fig. 2. Transactivation experiments were performed with or without the addition of 0.2 µg of GATA-1, 0.01 µg of PU.1, or 0.5 µg of C/EBP27 expression vectors as indicated. Promoter activity is shown as fold induction over the base-line MBP-P2-pXP2 construct alone. Transactivation of the MBP-P2 promoter by GATA-1 or GATA-1/PU.1 synergy and its repression by C/EBP27 was blocked by mutation of the C/EBP or GATA-binding sites but not the PU.1 sites. B, AML14 and AML14.3D10 cells were transiently transfected by electroporation using 10 µg of promoter constructs including the MBP-P2 wild type or dual GATA-site, C/EBP site, or PU.1 site mutation constructs as above. Luciferase activity was measured 6 h after transfection, normalized, and analyzed as fold differences compared with the promoterless pXP2 luciferase plasmid. The mean (± S.D.) for three independent transactivation or transfection experiments is shown.

Endogenous GATA-1, PU.1, and C/EBP Isoforms Expressed by Eosinophils Physically Interact; Analysis by Co-immunopre-cipitation-- To evaluate further the physical interactions that may occur among GATA-1, PU.1. and C/EBP in vivo in the eosinophil lineage, we performed co-IP experiments using whole cell lysates of the AML14.3D10 eosinophil myelocyte cell line that expresses all three transcription factors. Whole cell lysates were first immunoprecipitated with antibodies to C/EBP, GATA-1, or PU.1 and two control antibodies including non-immune rabbit and goat IgGs. Immunoprecipitates were analyzed by Western blotting using antibodies against C/EBP and GATA-1 as shown in Fig. 8. As shown on the C/EBP Western blot (Fig. 8A), antibody to C/EBP strongly immunoprecipitated both the C/EBP^32/30 and C/EBP²⁷ isoforms (lane 2). Antibodies to PU.1 and GATA-1 both co-immunoprecipitated the C/EBP^32/30 and C/EBP²⁷ isoforms (Fig. 8A, lanes 3 and 5) but not the C/EBP¹⁴ isoform (not shown). As shown on the GATA-1 Western blot (Fig. 8B), GATA-1 was efficiently immunoprecipitated by antibody to GATA-1 (lane 5), and antibodies to C/EBP and PU.1 both co-immunoprecipitated GATA-1 (lanes 2 and 3, respectively). We were unable to co-immunoprecipitate PU.1 with antibodies to either C/EBP or GATA-1 (data not shown); the low level expression of endogenous PU.1 in AML14.3D10 cells likely precludes its visualization in these co-immunoprecipitates. Alternatively, antibodies to GATA-1 and C/EBP may block their interactions with PU.1. Our co-immunoprecipitation results demonstrate for the first time that C/EBP physically interacts with both PU.1 and GATA-1 in vivo. Our results indicate that C/EBP, GATA-1. and PU.1 physically interact with one another in vivo in AML14.3D10 eosinophil myelocytes.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Co-immunoprecipitation of C/EBP, PU.1, and GATA-1 from lysates of AML14.3D10 eosinophilic myelocytes. Whole cell lysates from AML14.3D10 cells were immunoprecipitated or co-immunoprecipitated with anti-C/EBP, PU.1, and GATA-1 antibodies. Non-immune, normal rabbit IgG (lane 4), or goat IgG (lane 6) was used as the negative control. Twenty micrograms of lysate from AML14.3D10 cells was loaded on each gel to show input (lane 1). A, Western blot analysis was performed using anti-C/EBP antibody. C/EBP32/30 and C/EBP27 were immunoprecipitated by rabbit polyclonal C/EBP antibody (lane 2) but not by normal rabbit IgG (lane 4). These C/EBP isoforms were also co-immunoprecipitated with antibodies to both PU.1 (lane 3) and GATA-1 (lane 5). B, Western blot analysis was performed using GATA-1 antibody. GATA-1 was immunoprecipitated by goat polyclonal antibody to GATA-1 (lane 5) but not by non-immune, normal goat IgG (lane 6). GATA-1 was co-immunoprecipitated with antibodies to both C/EBP and PU.1 (lanes 2 and 3).

Activation Versus Repression of Eosinophil Gene Transcription by C/EBP Family Members-- Previous studies (4, 52) have shown that granulopoiesis is impaired in C/EBP- or C/EBP-deficient mice. C/EBP has been shown to play a critical role in early aspects of myeloid lineage commitment and gene transcription, whereas C/EBP has been shown to be critical for the more terminal stages of lineage differentiation and maturation (4, 40). To define further the transcriptional regulation of the MBP gene during the various stages of eosinophil development by the members of the C/EBP family, we sought to characterize their combinatorial interactions in the activation of the MBP promoter. Co-transactivation analyses were performed in CV-1 cells by co-transfection of the MBP-P2 promoter construct with expression vectors for C/EBP, C/EBP, and the three C/EBP isoforms (³², ²⁷, and ¹⁴). C/EBP or C/EBP equivalently transactivated the MBP-P2 promoter with 4-8-fold inductions, and these activities were competitively inhibited by the addition of the C/EBP¹⁴, C/EBP²⁷, and C/EBP³² isoforms as shown in Fig. 9, A and B, respectively. The C/EBP¹⁴ isoform in particular, which has no transactivating activity in this system (Fig. 6), inhibited both C/EBP- and C/EBP-induced transactivation in a dose-dependent manner, possibly by forming inactive heterodimers with these C/EBPs through their shared leucine zipper dimerization domains. The C/EBP³² and C/EBP²⁷ isoforms, which likewise have no inherent transactivating activity of their own for the MBP-P2 promoter (Fig. 6.), may also form inactive heterodimers or homodimers that compete for occupation of the functional C/EBP site in the MBP-P2 promoter, decreasing transactivation by C/EBP or -.

View larger version (19K): [in this window] [in a new window]   Fig. 9.   C/EBP- and C/EBP-mediated transactivation of the MBP-P2 promoter is inhibited by the C/EBP isoforms. Transactivation of the MBP-P2 promoter was performed in CV-1 cells by the addition of C/EBP (A) or C/EBP (B) and increasing amounts (0.5, 1.0, and 2.0 µg of plasmid DNA) of the C/EBP14, 27, and 32 isoform expression vectors. Luciferase activity was assayed 72 h after transfection. The mean (± S.D.) for three independent co-transactivation experiments is shown. All C/EBP isoforms repressed transactivation of the MBP-P2 promoter by C/EBP and C/EBP to a greater or lesser extent. Only the C/EBP14 isoform inhibited the activity of both C/EBP and - in a dose-dependent fashion.

Targeted Disruption of the C/EBP Gene Results in Only a Partial Decrease in MBP Gene Expression-- Mice with a targeted disruption of the C/EBP gene fail to produce normal mature granulocytes in their bone marrow or peripheral blood, but they instead produce neutrophils that have atypical nuclear morphology, lack expression of secondary and tertiary granule proteins, and have functional abnormalities. As well, mature eosinophils are not detectable in these mice using standard histochemical staining with Wrights'-Giemsa (4). By comparison, mice with a targeted disruption of the C/EBP gene have a profound absence of both neutrophils and eosinophils in the fetal liver (52). In C/EBP null mice, we were unable to detect any expression of MBP whatsoever, consistent with a complete block in granulocyte development (Fig. 10). In addition, there was no expression of the gene encoding murine eosinophil peroxidase and decreased expression of the gene encoding the IL-5R subunit (not shown).³ In contrast, in the C/EBP null mice, there was only a partial (~50%) decrease in MBP mRNA expression (Fig. 10), with only a partial decrease in murine eosinophil peroxidase, and no effect whatsoever on IL-5R subunit expression (not shown). Thus, although C/EBP knockout mice do not produce any terminally differentiated mature eosinophils, they must still produce eosinophil progenitors capable of expressing secondary granule protein genes such as MBP and eosinophil peroxidase, albeit at somewhat reduced levels.

View larger version (28K): [in this window] [in a new window]   Fig. 10.   Effects of targeted disruption of the C/EBP and C/EBP genes on eosinophil MBP gene expression. Semi-quantitative reverse transcriptase-PCR analysis of MBP mRNA expression in C/EBP- and C/EBP-deficient mice is shown. The GAPDH gene was used as a control for semi-quantitation of the mRNA (cDNA) sample input. The mRNA was prepared from the fetal liver (for C/EBP) or bone marrow (for C/EBP) of wild-type (+/+), heterozygote (+/), and knockout mice (/), respectively. For the wild-type and C/EBP knockout analysis (right panels), mice were first stimulated intraperitoneal with thioglycolate broth for 4.5 or 24 h prior to harvesting bone marrow cells as described previously (4) for the preparation of RNA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Basal hematopoiesis is now thought to be regulated in part by combinatorial networks of transcription factors that serve to coordinate the expression of lineage-specific genes at appropriate stages of lineage commitment, proliferation, terminal differentiation, and functional maturation (85, 86). Thus far, GATA-1, PU.1, and members of the C/EBP family (C/EBP and C/EBP) of transcriptional regulators have been found to play key roles in the commitment and terminal differentiation of myeloid progenitors to the eosinophil lineage in the avian system (1-7) and the regulation of eosinophil lineage-specific gene expression (8, 9) and development (4, 39, 52) in mammals. A physical interaction and functional antagonism between GATA-1 and PU.1 was identified recently by several groups (10-12) and was shown to play a role in the counter-regulation of myeloid versus erythroid development and transcriptional regulation of myeloid versus erythroid target genes. Moreover, the level of PU.1 expression was found to be a determinant for specifying lymphoid (B cell) versus macrophage lineage development, suggesting that graded expression of PU.1 may play a role in specifying distinct cell fates in hematopoiesis (37). However, a PU.1/GATA-1 functional antagonism appears inconsistent with the reported roles of these factors in the eosinophil granulocyte lineage. PU.1 is expressed throughout myeloid (87, 88) and eosinophil development and is induced by IL-5 in CD34+ hematopoietic progenitors.⁴ As well, PU.1 null mice lack detectable eosinophils or expression of eosinophil secondary granule protein genes such as MBP and eosinophil peroxidase,² and PU.1 may regulate eosinophil expression of two additional secondary granule ribonuclease genes, eosinophil-derived neurotoxin (EDN and RNS2) and eosinophil cationic protein (ECP and RNS3), through a PU.1-binding site in their highly conserved intronic enhancer (6). As well, GATA-1 has been shown to drive both eosinophil development (3, 7) and eosinophil gene transcription (2, 6, 8, 9).

In the present work, we have characterized a novel synergistic (as opposed to antagonistic) role for PU.1 in amplifying GATA-1-mediated gene transcription in the eosinophil lineage, an effect that is dependent on the level of PU.1 expression relative to GATA-1, and independent of PU.1 DNA-binding sites in the target promoter. Our findings support the concept that the ratio of GATA-1 to PU.1 may be one of the critical determinants in specifying the eosinophil developmental program compared with neutrophils or other myeloid lineages. Our finding of a PU.1/GATA-1 synergy, rather than antagonism, for a GATA-1-regulated target gene in the eosinophil lineage is in contrast to those reported by others (10, 84) in which PU.1 has been shown to suppress erythroid development and the expression of GATA-1-regulated erythroid genes by blocking GATA-1 binding to DNA. In the opposite direction, GATA-1 has been shown to antagonize PU.1 functions in myeloid gene expression and development when expressed ectopically in human and avian myeloid cells (11, 12). The effects of GATA-1 on PU.1 function in the myeloid lineage were found to be mediated via a direct interaction of the C-terminal zinc finger of GATA-1 with the PU.1 ets DNA-binding domain (12), in part by interfering with the binding of PU.1 co-activators such as c-Jun (11). In granulocyte development in the avian system, the expression of an "intermediate level" of GATA-1 in the context of PU.1 and C/EBP was found to be pivotal to the commitment of multipotential Myb-ets-transformed myeloid progenitors to the eosinophil lineage (3, 89). As such, antagonism of GATA-1 function by PU.1 in the eosinophil lineage would be at odds with the key role of GATA-1 function in specifying eosinophil versus neutrophil development (7, 12), and would be expected to block expression of GATA-1-regulated eosinophil genes such as MBP. In the avian system, GATA-1 was found to function as either a transcriptional activator of the EOS47 gene at low levels of expression or repressor at high levels of expression (2, 7). Thus, one possible explanation for our findings of a GATA-1/PU.1 synergy might be that PU.1 simply "titrates" the optimal level at which GATA-1 activates the MBP-P2 promoter, rather than cooperating directly with GATA-1. To eliminate this as a possibility, we first performed dose-response titrations of the GATA-1 expression vector and selected a concentration for co-transactivation experiments that provided maximum (peak) GATA-1-mediated transactivation (data not shown). In these experiments, GATA-1 functioned exclusively as a transcriptional activator in a dose-dependent fashion, with no repressor activity evident. The failure of high concentrations of PU.1 to synergize with GATA-1 in our studies is likely due to its interaction with and reduction of the available GATA-1 in the system. Although our finding that PU.1 synergizes with GATA-1 for transactivation of the eosinophil MBP gene could be a promoter-specific effect (see below), such synergy may be key to the activation and fulfillment of the hematopoietic program of the eosinophil.

The synergistic effect of PU.1 on GATA-1-mediated transactivation could potentially be mediated by a conformational change in GATA-1 when it binds to the two tandem GATA sites present in the MBP-P2 promoter. It was reported recently that the ability of GATA-1 to mediate transactivation can be modified by its DNA binding target sequences, such that high affinity tandem and/or palindromic GATA-binding sites found in several different genes including the -globin silencer (90) and testis GATA-1 promoter of rats and mice (91) may either promote or interfere with GATA-1 transactivating activity (90, 92). Only the C-terminal zinc finger of GATA-1 is capable of independently binding to a GATA consensus site and stimulating transcription, whereas the N-terminal finger shows no independent DNA binding activity but can modify binding specificity (stabilize or disrupt binding) at some naturally occurring dual GATA sites that have been shown to be critical for gene expression (93-97). The dual GATA sites in the MBP P2 promoter are canonical non-overlapping tandem GATA-binding repeats (6 nucleotides apart; Fig. 1) that could potentially interact with both the N- and C-terminal zinc fingers of GATA-1. Involvement of the N-terminal zinc finger by binding to the other GATA site in the MBP P2 promoter might trigger a conformational change leading to decreased DNA binding activity of the C-terminal zinc finger and a decrease in transactivating activity, as recently demonstrated by Trainor and colleagues (90) for the -globin silencer. In our system, a low amount of PU.1 may interact differentially or preferentially with the N-terminal zinc finger of GATA-1 and prevent this decrease in transactivating potential; this would be observed as PU.1/GATA-1 synergy for transactivation of the MBP-P2 promoter. In contrast, expression of a high amount of PU.1 could lead to interactions with both zinc fingers, thus interfering with the DNA binding activity of GATA-1 and abrogating the synergy obtained in our co-transactivation experiments. Of note, PU.1 has been shown to interact with both the C- and N-terminal zinc fingers of GATA-1 (10). Studies of the specific mechanism leading to GATA-1/PU.1 synergy for eosinophil MBP gene transcription are currently in progress.

C/EBP, an essential regulator of hepatocyte and adipocyte differentiation (49, 50), has been found to play a significant role in the early commitment of hematopoietic progenitors to myelopoiesis. C/EBP-deficient (null) mice show a selective block in the differentiation of granulocytes (both neutrophils and eosinophils) beyond the myeloblast stage (52) and fail to express G-CSF (52) or IL-6 receptors (98). In contrast, C/EBP acts downstream of C/EBP; its expression is limited principally to the myeloid lineage, and it is required for the terminal differentiation and maturation steps of granulopoiesis (40, 57, 99). Targeted disruption of the C/EBP gene results in a failure of terminal granulopoiesis, such that C/EBP null mice lack normal functionally mature neutrophils, have essentially no identifiable eosinophils by standard histochemical staining with eosin-containing dyes, and show a loss of neutrophil (4) or decreased expression of eosinophil (Fig. 10) secondary granule protein genes such as MBP. Unlike other C/EBPs, differential promoter utilization and alternative splicing of the human C/EBP gene results in the generation and expression of four distinct protein isoforms (32, 30, 27, and 14 kDa) that may play functionally distinct roles during myelopoiesis. In marked contrast, the murine gene encodes only a single full-length 34-kDa isoform translated from a single 1.3-kb mRNA (100). Although a recent study (101) reports that the murine C/EBP may function as an activator or repressor in vitro, there are no murine equivalents of the human 27- and 14-kDa C/EBP isoforms (57) we have identified as possible repressors (Fig. 11), suggesting important differences in C/EBP-mediated gene transcription in human versus murine granulopoiesis.

View larger version (41K): [in this window] [in a new window]   Fig. 11.   Model for the roles and functional interactions of myeloid transcription factors in eosinophil MBP gene expression and eosinophil development. A, C/EBP and/or C/EBP and PU.1 bind to their consensus site and initiate basal, low level transcription of the MBP gene in the earliest stages of eosinophil lineage commitment. B, GATA-1, a strong co-activator of the MBP promoter (8) and eosinophil development (7), binds to the dual GATA site in the context of C/EBP and/or -, to up-regulate activity of this eosinophil lineage-specific promoter (8). This involves a physical, synergistic interaction between GATA-1 and the C/EBPs (9). C, PU.1 and GATA-1, through a direct protein-protein interaction that alters GATA-1 conformation and/or binding to increase its activity, synergize to strongly up-regulate transcription of eosinophil lineage-specific genes such as MBP in developing eosinophil progenitors. This does not require the binding of PU.1 to its binding site in the promoter. D, C/EBP27 binds to the C/EBP site competing with C/EBP and/or - or heterodimerizes with these C/EBPs and acts as a repressor to down-regulate or turn off MBP gene transcription fully. This occurs either by blocking C/EBP and/or C/EBP function or by inhibiting GATA-1 and GATA-1/PU.1 synergistic transactivation through direct protein/protein interactions during the terminal differentiation stages of eosinophil development. E, of the C/EBP isoforms, only C/EBP14 continues to be expressed in mature blood eosinophils at high levels. This isoform binds to the critical C/EBP site and effectively blocks MBP gene transcription. As well, the lack of GATA-1 and PU.1 expression in the mature blood eosinophil helps to maintain the silence of this gene.

In the present study, we have identified novel repressor roles for both the 27- and 14-kDa human C/EBP isoforms. The C/EBP 27-kDa isoform, which contains a repressor domain with the KEEXXXPE motif identified in the RD-1 region of rat C/EBP (101) and rat, human, and chicken C/EBP (101), was found to antagonize both GATA-1 transactivation and GATA-1/PU.1 synergistic activation of the eosinophil MBP-P2 promoter, as well as PU.1-mediated transactivation of PU.1 target genes/promoters such as the M-CSFR, suggesting a potent repressor function for this isoform in myeloid development. The KEEXXXPE motif of rat C/EBP is required for both its repressor function and protein/protein interactions, as it is for the Sp3 factor (102), and this motif is also present in the RD-1 of C/EBP (103) and the inhibitory domain (ID-1) of c-Fos and FosB (104). In contrast, the 14-kDa isoform of C/EBP does not contain this repressor domain but binds DNA and was found to function effectively as a dominant negative competitor of C/EBP- and C/EBP-mediated gene transcription. Thus, two of the human C/EBP isoforms have the capacity to act as negative regulators of myeloid gene expression. Of interest, the lack of transactivating activity by any of the C/EBP isoforms for the MBP-P2 promoter is in agreement with the essential inactivity of the C/EBP³² isoform relative to other C/EBP family members for another eosinophil secondary granule protein gene, the eosinophil-derived neurotoxin (EDN, RNS2) promoter (105).

As shown by Western blotting, only the C/EBP¹⁴ isoform is expressed by mature blood eosinophils (at very high levels). Genes encoding MBP and other secondary granule proteins such as eosinophil peroxidase are down-regulated and ultimately silenced as eosinophils terminally differentiate and mature (106). These findings are consistent with a negative regulatory role for the C/EBP¹⁴ isoform in eosinophil terminal differentiation, because it lacks a transactivation or repressor domain and may act as a dominant negative regulator either by occupying the C/EBP sites in the promoters of eosinophil secondary granule protein genes, such as MBP, or by heterodimerizing with other C/EBPs (i.e. C/EBP or -), forming a functionally inactive dimer. The C/EBP²⁷ isoform, which contains both transactivation and repression domains as described above, may also act as a repressor in this process to decrease MBP gene transcription until the expression of other key regulators such as GATA-1 has ceased in the mature eosinophil (Fig. 11).

C/EBP, which has been shown to play a pivotal role during the early stages of myeloid lineage-specific gene expression and development including regulation of the G-CSF receptor (52), and eosinophil genes such as the IL-5R subunit⁵ (39), likely functions as a positive regulator for initial transcription of MBP and other eosinophil lineage-specific genes. Our current results suggest that this initial regulation is subsequently amplified through the potent functional interactions among C/EBPs ( and ), GATA-1, and PU.1 in the eosinophil lineage as modeled in Fig. 11. In the earliest stages of eosinophil lineage commitment and development, C/EBP and possibly C/EBP serve initially to activate eosinophil (e.g. MBP) gene transcription. As eosinophil differentiation progresses, GATA-1 binds and synergizes with the C/EBPs to strongly up-regulate eosinophil gene transcription, because both C/EBP (9) and C/EBP⁶ physically interact and functionally synergize with GATA-1 to activate the MBP-P2 promoter. PU.1, a central transcriptional regulator of the myeloid lineages (1, 27, 107), which is expressed continually at varying levels during myelopoiesis, may serve to either up-regulate or down-regulate eosinophil gene transcription through synergistic or antagonistic interactions with GATA-1, dependent upon its expression level relative to GATA-1 (3, 12). During the final stages of eosinophil maturation, expression of GATA-1, PU.1, and all of the C/EBP isoforms, save for C/EBP¹⁴, are significantly reduced (PU.1) or completely absent (GATA-1), resulting in the loss of eosinophil gene activation and potent repression by the dominant negative actions of the C/EBP¹⁴ isoform (Fig. 11).

This model provides a framework for in vitro and in vivo studies aimed at defining the functional versatility and roles of the various C/EBP isoforms in terms of their specific interactions with GATA-1 and PU.1, and other C/EBP family members, both in human eosinophil development and myeloid differentiation in general. Studies are in progress using chromatin immunoprecipitation and DNase I in vivo footprinting to characterize eosinophil promoter activity and occupancy in vivo for C/EBP, C/EBP, the various C/EBP isoforms, as well as GATA-1 and PU.1 during the different stages of eosinophil lineage commitment, differentiation, and terminal maturation of authentic bone marrow-derived myeloid progenitors.

	ACKNOWLEDGEMENTS

We thank Dr. Kleanthis Xanthopoulos for providing the C/EBP expression vectors; Dr. Alan Friedman for the C/EBP, -, and - expression vectors; Drs. Daniel Tenen and Pu Zhang for the M-CSFR promoter construct and mRNA from the C/EBP null mice; Dr. Richard Maki for the PU.1 expression vector; Dr. Leonard Zon for the GATA-1 expression vector; and Drs. Michael Baumann and Cassandra Paul for the AML14 parental and AML14.3D10 eosinophil cell lines. We also thank the nursing staff of the University of Illinois at Chicago General Clinical Research Center for drawing blood from normal donors; Mark Kwatia for blood donor recruitment, screening, and eosinophil purification; and Kim Hayden-Morgan for administrative secretarial support. The University of Illinois at Chicago was the recipient of General Clinical Research Center Grant M01-RR13987 from the National Institutes of Health/National Center for Research Resources.

	Note Added in Proof

Although GATA-1 null (deficient) mice have not been evaluated for defects in eosinophil development, Yu and colleagues (Yu, C., Cantor, A. B., Yang, H., Browne, C., Wells, R. A., Fujiwara, Y., and Orkin, S. H. (2002) J. Exp. Med. 195, 1387-1395) recently reported that targeted deletion of a high affinity GATA-binding site in the GATA-1 promoter leads to a selective loss of the eosinophil lineage in vivo.

	FOOTNOTES

^* This work was supported by United States Public Health Service Grant AI33043 (to S. J. A.) from NIAID, National Institutes of Health.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.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, MC536, A-312 College of Medicine West, University of Illinois, 1819 West Polk St., Chicago, IL 60612. Tel.: 312-996-6149; Fax: 312-996-5623; E-mail: sackerma@uic.edu.

Published, JBC Papers in Press, August 28, 2002, DOI 10.1074/jbc.M204777200

² J. Du, R. P. DeKoter, H. Singh, S. R. McKercher, R. A. Maki, and S. J. Ackerman, manuscript in preparation.

³ J. Du, M. J. Stankiewicz, F. Xin, J. A. Lekstrom-Himes, and S. J. Ackerman, submitted for publication.

⁴ S. J. Ackerman, unpublished observations.

⁵ J. Du, M. J. Stankiewicz, F. Xin, J. A. Lekstrom-Himes, and S. J. Ackerman, submitted for publication.

⁶ J. Du and S. J. Ackerman, unpublished results.

	ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT enhancer binding protein; MBP, eosinophil granule major basic protein; AML, acute myeloid leukemia; M-CSFR, macrophage-colony stimulating factor receptor; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation; co-IP co-immunoprecipitation, GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; G-CSF, granulocyte colony stimulating factor.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				K. Fukushima, I. Matsumura, S. Ezoe, M. Tokunaga, M. Yasumi, Y. Satoh, H. Shibayama, H. Tanaka, A. Iwama, and Y. Kanakura FIP1L1-PDGFR{alpha} Imposes Eosinophil Lineage Commitment on Hematopoietic Stem/Progenitor Cells J. Biol. Chem., March 20, 2009; 284(12): 7719 - 7732. [Abstract] [Full Text] [PDF]

				R. Bedi, J. Du, A. K. Sharma, I. Gomes, and S. J. Ackerman Human C/EBP-{epsilon} activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation Blood, January 8, 2009; 113(2): 317 - 327. [Abstract] [Full Text] [PDF]

				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]

				F. Wang and Q. Tong Transcription factor PU.1 is expressed in white adipose and inhibits adipocyte differentiation Am J Physiol Cell Physiol, July 1, 2008; 295(1): C213 - C220. [Abstract] [Full Text] [PDF]

				R. S. Viger, S. M. Guittot, M. Anttonen, D. B. Wilson, and M. Heikinheimo Role of the GATA Family of Transcription Factors in Endocrine Development, Function, and Disease Mol. Endocrinol., April 1, 2008; 22(4): 781 - 798. [Abstract] [Full Text] [PDF]

				M. E. Enos, S. A. Bancos, T. Bushnell, and I. N. Crispe E2F4 Modulates Differentiation and Gene Expression in Hematopoietic Progenitor Cells during Commitment to the Lymphoid Lineage J. Immunol., March 15, 2008; 180(6): 3699 - 3707. [Abstract] [Full Text] [PDF]

				L. A. Wagner, C. J. Christensen, D. M. Dunn, G. J. Spangrude, A. Georgelas, L. Kelley, M. S. Esplin, R. B. Weiss, and G. J. Gleich EGO, a novel, noncoding RNA gene, regulates eosinophil granule protein transcript expression Blood, June 15, 2007; 109(12): 5191 - 5198. [Abstract] [Full Text] [PDF]

				A. F. Gombart, J. Grewal, and H. P. Koeffler ATF4 differentially regulates transcriptional activation of myeloid-specific genes by C/EBP{epsilon} and C/EBP{alpha} J. Leukoc. Biol., June 1, 2007; 81(6): 1535 - 1547. [Abstract] [Full Text] [PDF]

				R. Bedi and S. J. Ackerman Differential Re-Programming of Myeloid Stem Cell Development by Activator Versus Repressor Isoforms of Human CCAAT/Enhancer Binding Protein Epsilon (C/EBP{varepsilon}). Blood (ASH Annual Meeting Abstracts), November 16, 2006; 108(11): 1177 - 1177. [Abstract] [PDF]

				A. L. Brown, C. R. Wilkinson, S. R. Waterman, C. H. Kok, D. G. Salerno, S. M. Diakiw, B. Reynolds, H. S. Scott, A. Tsykin, G. F. Glonek, et al. Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling J. Leukoc. Biol., August 1, 2006; 80(2): 433 - 447. [Abstract] [Full Text] [PDF]

				A. F. Gombart, U. Krug, J. O'Kelly, E. An, V. Vegesna, and H. P. Koeffler Aberrant expression of neutrophil and macrophage-related genes in a murine model for human neutrophil-specific granule deficiency J. Leukoc. Biol., November 1, 2005; 78(5): 1153 - 1165. [Abstract] [Full Text] [PDF]

				M. J. Stankiewicz, J. Du, and S. J. Ackerman CCAAT/Enhancer-Binding Protein {varepsilon}27 Antagonism of GATA-1 Transcriptional Activity Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation, and Does Not Require but Is Enhanced by DNA-Binding. Blood (ASH Annual Meeting Abstracts), November 16, 2004; 104(11): 1468 - 1468. [Abstract]

				J.-N. Bastie, N. Balitrand, F. Guidez, I. Guillemot, J. Larghero, C. Calabresse, C. Chomienne, and L. Delva 1{alpha},25-Dihydroxyvitamin D3 Transrepresses Retinoic Acid Transcriptional Activity via Vitamin D Receptor in Myeloid Cells Mol. Endocrinol., November 1, 2004; 18(11): 2685 - 2699. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/45/43481    most recent M204777200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Du, J. Articles by Ackerman, S. J. Search for Related Content PubMed PubMed Citation Articles by Du, J. Articles by Ackerman, S. J. Social Bookmarking                      What's this?