|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3447-3458
Mechanisms of Transcription in Eosinophils: GATA-1, but not GATA-2,
Transactivates the Promoter of the Eosinophil Granule Major Basic
Protein Gene
By
Yuji Yamaguchi,
Steven J. Ackerman,
Naoko Minegishi,
Masaki Takiguchi,
Masayuki Yamamoto, and
Toshio Suda
From the Department of Cell Differentiation, Institute of Molecular
Embryology and Genetics and the Department of Molecular Genetics,
Kumamoto University School of Medicine, Kumamoto; the Departments of
Biochemistry and Molecular Biology, College of Medicine, University of
Illinois at Chicago; the Department of Biochemistry, Tohoku University
School of Medicine, Sendai; and the Center of Tsukuba Advanced Research
Alliance, University of Tsukuba, Tsukuba, Japan.
 |
ABSTRACT |
Granule major basic protein (MBP) is expressed exclusively in
eosinophils, basophils, and placental trophoblasts. To identify the
cis-elements and transcription factors involved in regulating MBP expression, we subcloned 3.2 kb of sequence upstream of the exon 9 transcriptional start site (P2 promoter) and serial 5 deletions into
the pXP2 luciferase reporter vector. An 80% decrement in promoter
activity was obtained when MBP sequences between bp  117 to 67
were deleted. To identify transcription factors that bind to and
transactivate through the bp 117 to 67 region, we first compared
the upstream genomic sequences of human and murine MBP; a potential
GATA binding consensus site was conserved in the 50-bp region between
the two genes. To determine which GATA proteins bind this consensus
site, we performed electrophoretic mobility shift assays (EMSAs), which
showed that both GATA-1 and GATA-2 can bind to this consensus site. To
determine the functionality of this site, we tested whether GATA-1 and
GATA-2, either individually or in combination, can transactivate the
MBP promoter in the Jurkat T cell line. Cotransfection with a GATA-1
expression vector produced 20-fold augmentation of MBP promoter
activity, whereas GATA-2 had no activity. In contrast, combined
cotransfection of GATA-1 and GATA-2 decreased the ability of GATA-1 to
transactivate the MBP promoter by approximately 50%. Our results
provide the first evidence for a GATA-1 target gene in eosinophils, a
negative regulatory role for GATA-2 in MBP expression, and possibly
eosinophil gene transcription in general during myelopoiesis.
| |
INTRODUCTION |
EOSINOPHILS, primarily tissue-dwelling
granulocytes that constitute 1% to 5% of normal circulating
leukocytes, play an important cytotoxic effector role in host immune
defenses against multicellular helminth parasites and in immune
responses to certain malignancies. Eosinophils are recognized to be
potent inflammatory effector cells that contribute to the pathogenesis
of a wide variety of allergic diseases and idiopathic syndromes,
especially asthma and the hypereosinophilic syndrome. The recognition
of eosinophil involvement in the inflammatory pathogenesis of allergic,
parasitic, and certain neoplastic diseases has generated considerable
interest in characterizing the molecular basis for the commitment and
differentiation of myeloid progenitors to the eosinophil lineage in the
greater context of myeloid and hematopoietic development.
Eosinophil differentiation from hematopoietic stem cells and myeloid
progenitors is regulated primarily by cytokines interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
IL-5.1-3 Although IL-3 and GM-CSF each allow proliferation of eosinophil progenitors, IL-5 is specifically involved in their terminal differentiation, functional activation, and prolonged survival
in tissues.4 IL-5 has been shown to support the
proliferation and terminal differentiation of eosinophilic precursors
in vitro in clonal cell culture assays and to activate mature
eosinophils for enhanced effector functions.3 Furthermore,
IL-5 has been shown to play a major role in eosinophilopoiesis in
vivo.5-7 Although IL-3 and GM-CSF participate in the
proliferation and differentiation of stem cells to the multipotential
myeloid and eosinophil progenitor pools, and homeostatic levels of
eosinophil differentiation in mice, IL-5 is an eosinophil-specific
cytokine that is required for both the terminal differentiation
and amplification of eosinophil development as well as eosinophilic
inflammatory responses to proceed.8
The molecular basis for the commitment of myeloid progenitors to the
eosinophil lineage has not been determined. It is generally believed
that specific combinations of transcription factors determine the
lineage fate of hematopoietic progenitors.9 Promoter
regions of genes expressed specifically in immature myeloid cells have been shown to be regulated by a number of factors including PEBP2/CBFs, C/EBPs, PU.1, and c-Myb.10-12 Furthermore, the
synergy in transcription of promoters activated by GATA-1, Sp1, EKLF
(erythroid Krüppel-like factor), SCL/tal-1, and NF-E2 plays an
important role in regulating activity of erythroid promoters.13-16 These insights and understanding have been
obtained through detailed analyses of the promoters for genes expressed selectively in the myeloid or erythroid lineages.
Eosinophils contain a number of enzymatic and nonenzymatic inflammatory
and cytotoxic mediators in their secondary (specific) granules. These
highly cationic proteins include the ribonucleases eosinophil-derived
neurotoxin (EDN, RNS2), eosinophil cationic protein (ECP, RNS3),
eosinophil peroxidase (EPO, the eosinophil counterpart of neutrophil
myeloperoxidase), and the granule major basic protein (MBP). In
addition, the Charcot-Leyden crystal (CLC) protein (eosinophil
lysophospholipase/galectin-10) is a major cytosolic, granule, and
nuclear protein. We and others have examined the 5 promoter regions of
these granule protein genes for consensus transcription factor binding
sites that could be relevant to their coordinate expression during
eosinophil differentiation and granulogenesis. We have previously shown
increased expression of mRNA for the GATA-binding proteins GATA-1, -2, and -3 during eosinophil differentiation of myeloid leukemic cell lines
and constitutive expression in mature blood eosinophils and
basophils.17 Based on these findings, we hypothesized that
GATA-binding proteins play an important role in eosinophil gene
regulation, differentiation, and maturation. However, functional
characterization of the promoters for the genes encoding
EPO,18 CLC,19,20 and EDN21,22 has
thus far failed to provide direct evidence for transcriptional regulation by GATA-binding proteins.
To identify transcription factors involved in regulating the commitment
and differentiation of hematopoietic progenitors toward the
eosinophilic lineage, and the coordinate expression of genes encoding
specific granule proteins in this process, we have analyzed the
cis-acting element and DNA-binding proteins that may regulate the expression of gene encoding MBP. Our findings indicate a major positive regulatory role for GATA-1 and negative regulatory role for
GATA-2 in MBP gene transcription, providing the first evidence for a
GATA-regulated target gene in the eosinophil lineage.
| |
MATERIALS AND METHODS |
Cell cultures.
The eosinophil-committed subline of the HL-60 promyelocytic leukemic
cell line, HL-60-C15 (a gift of Dr Steven Fishkoff, Lederle Laboratory,
New York), was maintained in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS; JRH Biosciences, Lenexa, KS), 2 mmol/L L-glutamine,
and passaged twice weekly.23 HL-60-C15 cells
(2 × 105 cells/mL) were induced with 0.5 mmol/L
n-butyrate (Sigma) for 48 hours as previously described.17
Another eosinophil-committed cell line (YJ-11), established by Yuji
Yamaguchi (manuscript submitted), was also maintained in RPMI 1640 supplemented with 8% FBS. Other myeloid and nonmyeloid cell lines used
in these studies including the basophilic KU812, myelomonocytic U937,
T-lymphocytic Jurkat, and the trophoblast cell lines, BeWo,
NUC-1, Jeg-3, and Jar (a gift of Dr Shigeru Saito,
Department of Obstetrics and Gynecology, Nara Medical
University), were maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% FBS.
Nuclear run-on transcription assay.
Nuclear run-on assays were performed as previously
described.24 The following DNAs were used to prepare slot
blots: a 0.85-kb fragment containing the complete coding region of MBP
in pUC12; a full-length cDNA of c-myc in pcDNAIneo; a 1.6-kb
fragment of human CD11b cDNA; a full-length cDNA of human
myeloperoxidase (MPO) in pcDNAIneo; a full-length cDNA of human
 -actin in pBluescriptII KS(); a 1.9-kb EcoRI/Sal
I fragment containing the human 18S rRNA cDNA in pBR322 (kindly
provided by Dr Howard A. Young, Laboratory of Molecular
Immunoregulation, NCI, Bethesda, MD); and pBR322 vector alone.
Autoradiograms were exposed at 80°C with an intensifying screen.
Northern blot analysis.
Total RNA was prepared from uninduced and butyrate-induced HL-60-C15
cells by the guanidium isothiocyanate method.25 The RNA (15 to 20 µg/lane) was denatured in formamide-formaldehyde and
electrophoresed in 1% agarose-formaldehyde gels. The RNA was then
transferred to Hybond-N nylon membranes (Amersham International plc,
Buckinghamshire, UK) and the blots probed sequentially with full-length
cDNA of MBP26 and 18S rRNA27 cDNA for internal
control of RNA loading. Hybridization with the random-primed DNA probes
was performed at 42°C in 50% formamide, 6× standard saline citrate
(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). The
filter was then stripped and rehybridized to an 18S rRNA probe.
Primer extension.
Total RNA was extracted from HL-60-C15 cells by homogenizing the cells
in 4 mol/L guanidine isothiocyanate followed by sedimentation through
5.35 mol/L CsCl. Poly(A)+ RNA was separated by
Oligotex-dT30 (Takara Shuzo, Kyoto, Japan) and digested with RNAse-free
DNAse I for 30 minutes at 37°C. The primer extension
followed the method of Kong et al28 and Li et
al.29 Briefly, DNAse-free RNA samples were annealed with the primer 5-CCACCCAGAGACCTTCCTGG-3 by heating to 70°C and
cooling slowly to 50°C. Primer extension was performed at 50°C for
15 minutes in a final volume of 40 µL of 50 mmol/L Tris (pH 8.0), 50 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol (DTT), 52.5 ng/mL bovine serum albumin (BSA), 7 U/mL RNAsin, 0.03 mmol/L each
of dATP/dGTP/dTTP, 0.03 mmol/L [ -32P] dCTP, and 10 U
of AMV reverse transcriptase. A further 10 U of reverse transcriptase
was added and the reaction was continued for another 15 minutes. After
phenol/chloroform extraction and ethanol precipitation, the primer
extension product was redissolved in water plus sequencing stop
solution and electrophoresed onto a 6% polyacrylamide/8 mol/L urea
gel. The gel was fixed, dried, and exposed to x-ray film.
Plasmids for transient transfections.
The promoterless luciferase plasmid pXP2 was used for all promoter
studies. A 3.2-kb BamHI/BglII human MBP genomic
fragment was filled in with Klenow enzyme and subcloned into the
Sma I site of the pXP2 promoterless luciferase plasmid. The
resulting construct contained approximately 3.2 kb of 5-flanking DNA
and extended 3 to bp +47. Unidirectional deletions of the
3.2-kb/MBP-luciferase construct were prepared using Exonuclease
III as described.30 Dideoxysequencing using
Sequenase (Amersham International plc) was performed to identify the
positions of the Exonuclease III (Promega, Madison, WI) end points.
Expression vectors for human GATA-1 and human GATA-2 under control of
the elongation factor-1 promoter, pEF-GATA1 and pEF-GATA2, were
prepared by cloning the human GATA-1 or GATA-2 cDNAs inserted into
pEF-BSSHII, a modified version of the expression vector
pEF-BOS.31 Plasmids expressing murine C/EBP, human
C/EBP, and murine C/EBP under control of the human elongation
factor-1 promoter, pEF-mC/EBP, pEF-hC/EBP, and pEF-mC/EBP,
respectively, were described previously.32
Transient transfections.
DNA was prepared and cells transfected by electroporation as previously
described using 1.5 × 107 cells in 500 µL with minor
modification.18 Briefly, the HL-60-C15 cells were
electroporated at 280 V, 960 µF, and the YJ, KU812, Jurkat, HeLa,
Raji, and U937 cell lines at 300, 280, 250, 150, 280, and 300 V, 960 µF, respectively, conditions previously optimized for these lines.
Luciferase activity in cell lysates prepared 4 to 6 hours
posttransfection 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, 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-human growth
hormone (pCMV-hGH) plasmid or CMV-Gal plasmid was used for
normalization of transfection efficiency among the different cell
lines, different plasmid DNA preparations, and individual transfection
experiments. Growth hormone production or -galactosidase activity
was measured by a commercially available radioimmunoassay kit (Nichols
Institute Diagnostics, San Juan Capistrano, CA) or -galactosidase
enzyme assay kit (Promega Co, Madison, WI), respectively. Individual transfection experiments were repeated at least three times, and the
results are shown as mean RLU/ng of hGH/mL or RLU/mU of
-galactosidase/mL (±SEM).
Electrophoretic mobility shift assay (EMSA).
COS cells transfected with the GATA-1 or GATA-2 expression vectors,
pEF-GATA1 and pEF-GATA2, were obtained 24 hours after electroporation
with these plasmids. Nuclear extracts were prepared as
described33 and protein concentrations were determined
using the Bradford assay (BioRad, Richmond, CA). Nuclear extracts (8 to
12 µg) were incubated with radiolabeled oligonucleotides (0.1 to 1 ng) and then subjected to electrophoresis as described
previously.18 An anti-GATA-1 monoclonal antibody (MoAb)
for supershift analysis was obtained commercially (Santa Cruz
Biotechnology, Santa Cruz, CA). 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
(a mutated GATA consensus site is underlined): 93/58 bp
(WT) (5-AAGTGATGAAATGGTCCTTATCAGCCTTGCTATCTC-3), 93/58 bp GATA
(5-AAGTGATGAAATGGTCCGGCGACGCCTTGCTATCTC-3).
Reverse transcription-polymerase chain reaction (RT-PCR).
We used two oligonucleotide primers for GATA-1 (A;
5-GGAGCCCTCTCAGCTCAGC-3, B; 5-GCCACCAGCTGGTCCTTCAG-3)17
and human -actin oligonucleotides (C; 5-GTCGTCGACAACGGCTCCGG-3, D;
5-AAGGTGTGGTGCCAGATTTTCTCCA-3)34 as the internal control.
Primers were synthesized using a DNA synthesizer (Model 381A; Applied
Biosystems Inc, Foster, CA). The synthesized length of cDNA fragment
for human GATA-1 and human -actin was 470 bp and 220 bp,
respectively. The isolated RNA was reverse-transcribed in a total
volume of 20 µL in buffer containing 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L DTT, a 10 mol/L dNTP
mixture, 100 pmol random hexamer oligonucleotides (Promega, Madison,
WI), and 7.5 U of AMV reverse transcriptase (Promega). Double-stranded
DNA was then synthesized from the cDNA with 1 U of Thermus
aquaticus (Taq) polymerase (Promega) and two pairs of
oligonucleotides (human GATA-1 and human -actin), using 25 PCR
cycles on a Perkin-Elmer Cetus PCR 1000 Thermocycler
(Perkin-Elmer-Cetus, Norwalk, CT). Each cycle included denaturation at
94°C for 1 minute, reannealing of the primers and fragment at 55°C
for 2 minutes, and polymerization at 72°C for 2 minutes.
Southern blot analysis of PCR amplified human GATA-1.
A portion of each sample was separated by electrophoresis on a 5%
polyacrylamide gel, then transferred to a Hybond-N membrane (Amersham).
Samples immobilized on the membrane were hybridized to a
32P-labeled probe of human GATA-1 or human -actin cDNA
at 42°C in 50% formamide, 6× SSC, 0.2% Ficoll-PVP, 0.1% SDS. The
filter was washed twice in 2× SSC containing 0.2% SDS at 55°C for
30 minutes and 0.2× SSC with 0.2% SDS at 57°C for 60 minutes, then autoradiographed for 12 hours using an intensifying screen.
| |
RESULTS |
Expression of mRNA encoding MBP is transcriptionally upregulated during
eosinophilic differentiation of HL-60-C15 cells.
It has previously been shown that treatment of HL-60-C15 cells with
n-butyrate induces eosinophilic differentiation and granulation of the
cell line,17 with a concomitant increase in steady-state levels of mRNAs encoding all the major eosinophil granule cationic proteins (ECP, EDN, EPO, and MBP) and the CLC protein.19
For EPO and CLC protein we have shown by nuclear run-on analysis that the increased levels of steady-state mRNA are in part transcriptionally mediated during n-butyrate induction of HL-60-C15 eosinophilic differentiation.18,19 To determine whether MBP expression
is likewise transcriptionally regulated during eosinophilic
differentiation, nuclear run-on analysis was performed on both
uninduced and 48-hour n-butyrate-induced HL-60-C15 cells
(Fig 1). The transcription rate for the MBP
gene increased fivefold with n-butyrate-induced eosinophilic
differentiation over the constitutive rate in uninduced cells. The
transcriptional rate of the CD11b gene increased marginally whereas
myeloperoxidase (MPO) and myc transcription decreased during
eosinophilic differentiation. These results indicate that MBP gene
expression is transcriptionally upregulated during eosinophilic differentiation of the HL-60-C15 cell line, in contrast to the downregulation of genes (eg, MPO) specifically expressed during neutrophil and monocyte development.
View larger version (70K):
[in this window]
[in a new window]
| Fig 1.
Nuclear run-on assay of HL-60-C15 cells induced with
n-butyrate. Nuclear run-on transcription analysis was performed with nuclei isolated from uninduced and 48-hour n-butyrate (0.5 mmol/L) induced HL-60-C15 cells. Equivalent numbers of counts were hybridized to duplicated filters containing equimolar amounts of the indicated probes.
|
|
The P2 promoter in the 5 region of MBP gene is specific for the
eosinophil lineage.
The MBP gene contains a total of 14 exons, including nine upstream
exons (exons 1-9) comprising the 5 untranslated region and five coding
exons (exons 10-14) (see Fig 3A).29,35 Two transcripts,
major 1.0-kb and minor 1.6-kb messages, are observed in developing and
mature eosinophils. Although the 1.6-kb mRNA transcript is derived from
exons 1-8 and exons 10-14, the 1.0-kb mRNA transcript is derived from
exon 9 and the coding exons, consistent with a previous report (see Fig
3A).26 Exons 8 and 10 are separated by a 9-kb, noncoding
region containing exon 9, the only upstream exon of the 1.0-kb
transcript. Li et al29 have reported that the 1.6 and 1.0 kb mRNAs arise by differential splicing of alternate MBP transcripts
from the distal and proximal promoters, respectively, located 32 kb
apart in the genomic DNA. The distal promoter that drives expression of
the 1.6-kb transcript and the proximal promoter that drives expression
of the 1.0-kb transcript have been designated the P1 and P2 promoters,
respectively (see Fig 3A).29 Human MBP has been shown to be
expressed in both basophils36 and placental cells37 in addition to eosinophils. Therefore, we have
determined which promoter, P1 or P2, is specific for the eosinophil and
basophil lineages by analyzing expression of the 1.6-kb and 1.0-kb MBP transcripts in eosinophilic, basophilic, and trophoblastic cell lines
by Northern blot analysis (Fig 2). In
eosinophilic and basophilic cell lines, YJ-11 and KU812, respectively,
the major MBP mRNA transcript was 1.0 kb, with only minor expression of
the 1.6-kb transcript. In contrast, only the 1.6-kb transcript was
observed in all trophoblastic cell lines examined. These results
suggest that the P2 promoter driving the 1.0-kb mRNA transcript is
specific for the eosinophil and basophil lineages whereas the P1
promoter is specifically used for placental trophoblast lineage
expression.
View larger version (19K):
[in this window]
[in a new window]
View larger version (32K):
[in this window]
[in a new window]
| Fig 3.
(A) Map of the human MBP gene and the
positively acting cis-elements of MBP P2 promoter. The
structure of the MBP gene was based on the study of Li et
al.29 (B) Identification of the transcriptional start site of the MBP gene (P2). Ten micrograms of
poly(A)+ RNA from YJ-11 cells was subjected to primer
extension and the resulting radiolabeled DNA product was
electrophoresed on a 6% denaturing polyacrylamide gel next to M13mp18
DNA-sequence size markers.
|
|
View larger version (54K):
[in this window]
[in a new window]
| Fig 2.
Northern blot analysis for mRNA encoding eosinophil
granule MBP in various leukemic cell lines. Lane 1, human basophilic
leukemia cell line, KU812; lanes 2 through 5, human choriocarcinoma
cells, BeWo (lane 2), Jeg-3 (lane 3), Nuc-1 (lane 4), and Jar (lane 5); lane 6, human eosinophil-committed leukemic cell line, YJ-11. The blot
was sequentially probed with an MBP cDNA and an 18S rRNA cDNA probes to
control for RNA loading.
|
|
Identification of the transcriptional start site in the P2 promoter
region of MBP gene.
The above results suggested that the 5 region upstream of exon 9, ie,
the putative P2 promoter region, is specific for the eosinophil
lineage. Therefore, we have defined the transcriptional start site in
the MBP P2 promoter region using primer extension analysis. As shown in
Fig 3B, one minor and two
major bands (start sites) were detected. We have designated the adenine
residue located 78 bp upstream of the end of exon 9, observed as the
first major band in the primer extension analysis (Fig 3B), as the
major transcriptional start site for the P2 promoter of MBP gene.
Deletion analysis of the MBP P2 promoter demonstrates the presence of
positive and negative regulatory elements.
Our Northern analysis of the MBP transcripts expressed in eosinophil
and basophil versus trophoblast cell lines indicated that the putative
P2 promoter (region immediately upstream of exon 9) should be specific
for the eosinophil and basophil lineages. We analyzed the ability of
this region to drive reporter gene (luciferase) activity in HL-60-C15
cells transiently transfected with various MBP P2 promoter constructs
in the promoterless pXP2 expression vector to determine whether this
region was functionally active and to locate the cis-element(s)
required for MBP promoter activity. An MBP genomic fragment containing
3.2 kb of sequence upstream of the exon 9 transcriptional start site
was subcloned into the pXP2 luciferase vector. When transiently
transfected into HL-60-C15 cells, the 3.2-kb/MBP-luciferase (luc)
promoter construct reproducibly expressed greater than 50-fold more
luciferase activity than the promoterless pXP2 control. To localize
regulatory elements in the MBP P2 promoter, a series of eight deletion
mutants were produced from the 3.2-kb/MBP construct using
exonuclease III and the PCR. No significant decrease in promoter
activity was observed when sequences between 3.2 kb and 177 bp
were deleted (Fig 4). Further deletion of
the region between bp 177 and 117 resulted in a threefold
increase in activity. In contrast, deletion of additional sequence to
bp 67 produced a significant, 75% decrease in promoter activity
when compared with the bp 117 construct. These results suggest that
basal MBP promoter activity is controlled by both negative and positive
elements in the bp 177 to 117 and bp 117 to 67 segments of
the gene, respectively.
View larger version (23K):
[in this window]
[in a new window]
| Fig 4.
Functional activity of pXP2-MBP (P2) luciferase
constructs in the eosinophilic HL-60-C15 cell line. Promoter activities
of additional 5 deletion mutants of the MBP-pXP2 construct in
HL-60-C15 cells. Fifteen micrograms of each MBP-pXP2 construct was
transfected along with 10 µg of pBluescript II KS() as carrier
DNA. Luciferase activities have been normalized for the amount of hGH
produced by cotransfection with a control CMV-hGH expression vector.
Corrected RLU for each construct are shown as the percent activity
relative to the mean activity of the longest 3.2-kb MBP (P2) promoter
construct. The mean ± SEM for three replicate experiments is shown.
|
|
Lineage-specificity of the MBP P2 versus P1 promoters.
The lineage specificity of the MBP P2 promoter was assessed by
transfecting the 117 bp/MBP-pXP2 construct into eosinophilic (YJ-11
and HL-60-C15), nonhematopoietic (HeLa), lymphoid (Raji), and
myelomonocytic (U937) cell lines (Fig 5A). As shown by Northern blot
analysis in Fig 5B, MBP mRNA expression was
observed in YJ-11 and HL-60-C15 cells, but not in HeLa, Raji, and U937
cells. The MBP promoter was approximately 4 to 7 times less active in
HeLa, Raji, and U937 cells than in HL-60-C15 cells (Fig 5A). To compare the specificity and activity of the P2 versus P1 MBP promoters in the
eosinophil lineage, we also prepared a pXP2 construct containing the
MBP P1 promoter (MBP-P1-pXP2). An MBP genomic fragment containing 285 bp of sequence upstream of the transcriptional start site for exon 1 was subcloned into the promoterless pXP2 vector. The MBP-P1-pXP2
construct was essentially inactive compared with the pXP2 control in
transient transfections of the YJ-11 and HL-60-C15 eosinophil cell
lines (data not shown). These results suggest that the MBP P1 promoter
is inactive in the eosinophil lineage and that usage of the P2 promoter
predominates for MBP expression during eosinophil development.
View larger version (22K):
[in this window]
[in a new window]
| Fig 5.
(A) Comparative activity of the MBP P2 promoter in
eosinophil (YJ-11, HL-60-C15), myelomonocytic (U937), lymphoid (Raji), and nonhematopoietic (HeLa) cell lines. Fifteen micrograms of the
117/MBP-luc promoter construct was transfected, along with 10 µg
of pBluescript II KS() as carrier DNA, into each cell line. Promoter
(luciferase) activities of the 117/MBP-luc construct were normalized
based on the measurement of growth hormone expression from a control
cotransfected CMV-hGH expression vector in the various cell lines. (B)
Northern blot analysis for mRNA encoding eosinophil granule MBP in the
various cell lines. Fifteen micrograms of total RNA isolated from the
indicated human cell lines was hybridized to the MBP cDNA. Lane 1, YJ-11; lane 2, HL-60-C15; lane 3, HeLa; lane 4, Raji; and lane 5, U937.
Hybridization of the same filter to a 18S rRNA probe is shown as
control for amounts of RNA.
|
|
GATA-1 and GATA-2 bind to a GATA consensus site in the MBP P2
promoter.
To identify transcription factors that bind to and transactivate the
50-bp positively acting region from bp 117 to 67, we first
compared the upstream genomic sequences for human35 and murine MBP.38 A potential GATA binding consensus site was
conserved in this region in both genes (Fig 3A). It has been reported
that both GATA-1 and GATA-2 play crucial roles in the differentiation of myeloid cells.9 To address the question of whether
GATA-1 or GATA-2 binds to the potential GATA-binding site in the MBP P2
promoter, EMSAs were performed. An EMSA with nuclear extracts from
COS-7 cells which were transiently transfected with either GATA-1 or
GATA-2 expression vectors and those from a human eosinophil-committed leukemia cell line, YJ-11, and an oligonucleotide spanning the MBP P2
promoter from position bp 93 to 58, showed specific complex formation for GATA-1 or GATA-2 (Fig
6A and B, lanes 2 and 6). The
DNA-protein complex formed by the nuclear extracts YJ
cells with the oligonucleotides containing GATA-binding site of the MBP
P2 promoter represents GATA-1 or GATA-2 proteins (Fig 6A, lane 5).
Formation of the GATA-1 or GATA-2 complexes could be blocked by an
excess of the unlabeled "self" oligonucleotides (Fig 6A, lanes 2, 4, and 6; and Fig 6B, lanes 3 and 7), but was not blocked by a mutated
GATA consensus site oligonucleotide (Fig 6B, lanes 4 and 8).
Furthermore, a small but definite supershift of the GATA-1 complex was
obtained by the addition of an anti-GATA-1 antibody to the EMSA
binding reaction (Fig 6B, lane 5). To determine whether or not there
was a difference in the binding affinities of GATA-1 and GATA-2 for the
MBP GATA consensus site, competition experiments were performed using a
labeled oligonucleotide from bp 93 to 58 and nuclear extracts
from COS-7 cells transfected with the GATA-1 or GATA-2 expression
vectors. Unlabeled oligonucleotide competitors were added in increasing
molar amounts to the binding reactions. These competitive DNA binding
studies showed that the binding of the oligonucleotide with GATA-1 was
more efficiently competed than that of GATA-2 (Fig
7A). Quantitation of the competition efficiency indicated that the binding affinity of GATA-1 for the GATA
consensus site in the MBP P2 promoter was approximately twofold greater
than that of GATA-2 (Fig 7B).
View larger version (44K):
[in this window]
[in a new window]
View larger version (21K):
[in this window]
[in a new window]
| Fig 6.
Identification of GATA-1 or GATA-2 binding to
the MBP promoter by gel-shift assay. (A) A double-stranded MBP promoter
oligonucleotide extending from position 93 to 58 bp was
end-labeled with [ -32P] ATP and incubated with 1 µg
of double-stranded poly(dI-dC) in the presence of 12 µg nuclear
proteins from COS 7 cells which were transiently transfected with
GATA-1 (lanes 1 and 2) or GATA-2 (lanes 3 and 4) expression vectors,
and in the presence of 12 µg nuclear extracts from YJ-11 cells (lanes
5 and 6). A 50-fold molar excess of each unlabeled oligonucleotide
(Competitors), identical to the probe in the binding reaction, was
added (lanes 2, 4, and 6). The arrow represents GATA-1- or
GATA-2-specific bands, which are inhibited by unlabeled
oligonucleotides spanning bp 93 to 58 of MBP P2 promoter region.
(B) A double-stranded MBP promoter oligonucleotide extending from
position 93 to 58 bp was end labeled with [-32P]
ATP and incubated with 1 µg of double-stranded poly(dI-dC) in the
presence of 8 µg nuclear proteins from COS 7 cells (lane 1), those
from COS 7 cells which were transiently transfected with GATA-1 (lanes
2 through 5), or GATA-2 (lanes 6 through 8) expression vectors. The
following unlabeled double-stranded competitor oligonucleotides were
added at a 100-fold molar excess over the probe oligonucleotide: MBP bp
93 to 58 (lanes 3 and 7) and MBP mutated GATA-consensus site
oligonucleotide (lanes 4 and 8). In lane 5, anti-GATA-1 antibody was
added to the reaction mixture.
|
|
View larger version (83K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
| Fig 7.
Analysis for the binding affinity of GATA-1 and GATA-2 to
the GATA consensus site in the MBP promoter by EMSA. (A) Competitor with the unlabeled oligonucleotides was added using increasing amounts
of 0 to 160-fold molar excess as indicated. A double-stranded MBP
promoter oligonucleotide extending from bp 93 to 58 was end-labeled and incubated with 1 µg of poly (dI-dC) in the absence of
nuclear extract (lane 1) or in the presence of 8 µg of nuclear extracts from COS 7 cells which was transfected with GATA-1 (lanes 2 through 6) or GATA-2 (lanes 7 through 11) expression vectors. (B)
Quantitation of the competition efficiency of the MBP promoter oligonucleotide for GATA-1 or GATA-2 binding. The radioactivity of each
band was quantitated with a BAS-2000II radioanalysis imaging system
(Fujix, Tokyo, Japan).
|
|
GATA-1, but not GATA-2, transactivates the MBP P2 promoter.
The results described above indicated that both GATA-1 and GATA-2 are
capable of binding to the GATA consensus site (bp 76 to 71),
albeit with somewhat different affinities. To determine whether this
GATA binding site is functional, we analyzed the ability of GATA-1 and
GATA-2, either individually or in combination, to transactivate the MBP
P2 promoter in the GATA-1 and GATA-2 negative Jurkat cell line.
Cotransfection with the GATA-1 expression vector alone produced a
20-fold increase in MBP P2 promoter activity, whereas GATA-2 alone had
no effect, but did decrease GATA-1 transactivation by 50% in the
combined cotransfection (Fig 8A). In
addition, the combined cotransfection of GATA-2 along with GATA-1
decreased the ability of GATA-1 to transactivate the MBP promoter in a
dose-dependent fashion (Fig 8B). These findings indicate that GATA-2
acts as a competitive inhibitor for GATA-1 in the transactivation of
the MBP promoter. Constructs containing the intact MBP P2 promoter with
the GATA motif (bp 76 to 71), and a mutated promoter with a 6-bp
linker substitution for the GATA-binding site (bp 76 to 71), were
cotransfected into the GATA-1 negative Jurkat cell line along with the
human GATA-1 expression vector pEF-GATA-1 (Fig
9). Mutation of the GATA-binding site
in the context of the most active bp 117/pXP2 MBP P2 promoter
construct resulted in a 90% loss of promoter activity in response to
GATA-1 transactivation (Fig 9). These results show the functional
importance of GATA-1 in regulating MBP promoter activity and likely MBP
gene expression.
View larger version (29K):
[in this window]
[in a new window]
| Fig 8.
Transactivation of the MBP P2 promoter by GATA-1 and
GATA-2 (A) and competitive inhibition by GATA-2 (B). Transactivation of
the MBP P2 promoter (bp 117/MBP-pXP2) in Jurkat cells, in which
neither GATA-1 nor GATA-2 transcription factors are expressed. The
T-lymphocytic Jurkat cell line was transfected by the electroporation method with 15 µg of the MBP P2 promoter construct 117 MBP-luc along with one of the following expression constructs: pXP2-MBP (control), 20 µg of pEF-BOS (the control plasmid containing
elongation factor promoter without cDNAs); pXP2-MBP + GATA-1, 10 µg
of pEF-hGATA-1 and 10 µg of EF-BOS; pXP2-MBP + GATA-2, 10 µg of
pEF-hGATA-2 and 10 µg of pEF-BOS; pXP2-MBP + GATA-1 + GATA-2, 10 µg of pEF-hGATA-1 and 10 µg of pEF-hGATA-2. Luciferase activity was
measured 24 hours after transfection and normalized for transfection
efficiency based on the activity of a cotransfected -galactosidase
expression vector (CMV-GAL). Data are shown as the mean of three
independent experiments (±SEM).
|
|
View larger version (8K):
[in this window]
[in a new window]
| Fig 9.
Mutations of the GATA-binding sites diminish the MBP P2
promoter activity. Jurkat cells were cotransfected with either pXP2-MBP containing a wild-type GATA-binding site, or pXP2-MBP containing a
mutated GATA-binding site, bp 76 to 71, along with 10 µg of pEF-hGATA-1. The error bar represents the SEM for three independent experiments. Luciferase activity was normalized to -galactosidase activity produced from a cotransfected CMV-Gal plasmid.
|
|
Transcriptional activation of the MBP P2 promoter by C/EBP family
members.
We have identified a C/EBP consensus binding site only 7 bp upstream of
the functional GATA-binding site in the MBP P2 promoter. Therefore, we
addressed whether C/EBP families (C/EBP, C/EBP, and C/EBP) can
transactivate the MBP P2 promoter. As shown in Fig
10, luciferase activity was increased
10-fold by cotransfecting the plasmids expressing C/EBP or C/EBP,
while cotransfection with the C/EBP expression vector produced a
20-fold increase. These data indicate that C/EBP family members and
GATA-1 may be able to cooperatively activate MBP gene expression.
View larger version (16K):
[in this window]
[in a new window]
| Fig 10.
Transactivation of the MBP P2 promoter by C/EBP,
C/EBP, and C/EBP. Ten micrograms of pXP2 or pXP2-MBP was
transfected into Jurkat cells along with 10 µg of pEF-mC/EBP,
pEF-hC/EBP, or pEF-mC/EBP. pCMV-Gal was included as an
internal control. Luciferase and -galactosidase activities were
assayed 24 hours later. Data are shown as the mean of three independent
experiments (±SEM).
|
|
Induction of GATA-1 mRNA expression during IL-5-induced eosinophil
differentiation of normal human bone marrow progenitors.
We previously showed that GATA-1 mRNA expression was upregulated in
both the HL-60-C15 and HL-60(3 + C5) cell lines when n-butyrate or
BCGF-II (as a source of IL-5), respectively, was used to induce eosinophilic differentiation.17 Results from our
experiments demonstrating GATA-binding protein transactivation of the
MBP P2 promoter suggest that GATA-1 plays an important role in
regulating aspects of eosinophil-lineage commitment, differentiation,
and gene regulation. To characterize the expression of the GATA-binding proteins during eosinophil development from normal bone marrow (BM)-derived myeloid progenitors, we assessed the steady-state levels
of GATA-1 and GATA-2 mRNAs in normal human BM mononuclear cells
stimulated to differentiate into eosinophils with IL-5. As shown by
Northern blot analysis in Fig 11A,expression of mRNAs for all the major eosinophil granule-associated
proteins, including MBP, increased significantly in BM progenitors
cultured with IL-5 for 3 days, with a continued increase throughout the
culture period. We used this ex vivo system for inducing eosinophil
development of myeloid progenitors to analyze the expression of mRNAs
encoding the GATA-binding proteins. A semiquantitative RT-PCR method
was used for mRNA expression of GATA-1 and a Northern blot analysis was
used for that of GATA-2. As shown in Fig 11B, GATA-1 expression decreased markedly from day 0 to day 3 in the absence of IL-5. In
contrast, GATA-1 mRNA levels persisted in the presence of IL-5 for the
duration of the culture period. These findings suggest that GATA-1
expression is maintained during IL-5-stimulated terminal differentiation of eosinophils from BM myeloid progenitors. On the
other hand, GATA-2 mRNA was expressed constitutively in normal human BM
mononuclear cells both in the presence and absence of exogenous IL-5
(Fig 11C).
View larger version (67K):
[in this window]
[in a new window]
View larger version (41K):
[in this window]
[in a new window]
View larger version (42K):
[in this window]
[in a new window]
| Fig 11.
(A) Northern blot analyses of mRNA for eosinophil
granule proteins in normal human bone marrow (hBM) cells cultured with
IL-5. hBM cells were cultured in the presence or absence of the
supernatant (10% vol/vol) from X63Ag8-653-mIL-5 myeloma cells as a
source of IL-5. Total RNA was obtained at days 0, 3, 6, 9, and 12 of culture. Each lane contains 8 µg of total RNA. Exposure to x-ray film
was for 18 hours. (B) mRNA expression for hGATA-1 in normal hBM cells
cultured with IL-5. Southern blot analysis of RT-PCR products amplified
from 1 µg of total RNA extracted from hBM cells cultured in the
presence or absence of the supernatant from X63Ag8-653-mIL-5 myeloma
cells as a source of IL-5. Total RNA was obtained at days 0 (lane 1), 3 (lanes 2 and 6), 6 (lanes 3 and 7), 9 (lanes 4 and 8), and 12 (lanes 5 and 9) of culture. The blot was probed simultaneously with hGATA-1 cDNA
and -actin as an internal control. Each lane contains 1/10th of the
amplification products from each time point. (C) Northern blot analysis
of mRNA for hGATA-2 in hBM cells stimulated with (IL-5) or without (FCS
alone) the supernatant from X63Ag8-653-mIL-5 myeloma cells as a source
of IL-5. Total RNA was obtained at days 0, 3, 6, and 9 of culture. Each
lane contains 3.5 µg of total RNA.
|
|
| |
DISCUSSION |
We have functionally characterized the P2 promoter for the human
eosinophil granule MBP gene, which is used exclusively during the
differentiation of myeloid progenitors to the eosinophil granulocyte lineage. We have previously reported that the expression of two other
eosinophil genes encoding eosinophil peroxidase (EPO) and Charcot-Leyden crystal protein (lysophospholipase/galectin-10) is
transcriptionally regulated during butyrate-induced eosinophilic differentiation of HL-60-C15 cells.18,19 As shown in this
study, expression of the MBP gene is also transcriptionally regulated during eosinophil differentiation. In addition, expression of the mRNA
encoding the eosinophil granule-associated proteins, MBP, EPO, ECP, and
EDN, increases along with eosinophil development and then decreases
with maturation of the cells.39 These results suggest that
transcription factors involved in the coordinate regulation of the
eosinophil granule protein genes may be associated with the process of
commitment and differentiation of hematopoietic progenitors toward the
eosinophilic lineage.
We have demonstrated the capacity of GATA-1 to bind to and
transactivate the MBP P2 promoter. These results provide the first evidence for a GATA-1 target gene in the eosinophil lineage. We have
previously suggested that gene transcription during eosinophil and
basophil hematopoietic development is regulated in part by members of
the GATA-binding family of transcription factors.17 In
particular, eosinophils, basophils, and eosinophil-differentiated myeloid cell lines were shown to express GATA-1 and GATA-3 in an
inducible fashion.17 In this study we have shown that
GATA-1 expression is sustained in human BM cells stimulated with IL-5. Although GATA-1 expression induced by the addition of IL-5 in ex vivo
system of human BM cells may be a phenomenon in an artificial system,
the increased expression of GATA-1 has been observed not only in the BM
cells stimulated with IL-5 but also in the cell line capable of
differentiating toward the eosinophil lineage (data not shown). Kulessa
et al40 have shown that stable clones containing
peroxidase-positive granules characteristic of eosinophils develop from
Myb-Ets-transformed myeloblasts after transfection with a GATA-1
expression vector. They speculate that lineage determination by GATA-1
depends in part on its intracellular levels. The multipotential cell
line 416B with the highest level of GATA-1 expression exhibited megakaryocytic features, whereas cell lines with approximately fourfold
lower levels displayed properties consistent with eosinophil differentiation.40 These results suggest that GATA-1 is one of the transcription factors involved in regulating the commitment and
differentiation of myeloid progenitors to the eosinophil lineage. Based
on previously published reports, the GATA-binding proteins are
speculated to be expressed in the various hematopoietic lineages as
follows: GATA-1 in erythrocytes, megakaryocytes, mast cells, eosinophils, and basophils; GATA-2 in mast cells, megakaryocytes, erythrocytes, monocytes, neutrophils, eosinophils, and basophils; GATA-3 in T cells, mast cells, and eosinophils; and GATA-4 in an adult
T-cell leukemia cell line, ATL-16T.41 As noted above, we
have shown that mRNAs for GATA-1, GATA-2, and GATA-3 are expressed in
mature eosinophils.17 Our current results indicate 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 by approximately 50%. We have also shown that
eosinophilic cell lines and human BM-derived progenitors express mRNAs
for GATA-1 and GATA-2, with constitutive expression of GATA-2 and
inducible expression of GATA-1 during eosinophil differentiation.17 The hematopoietic expression of GATA-2
in these systems overlaps with that of GATA-1, although GATA-2 is also
expressed more widely. During erythroid differentiation, it has been
shown that the expression of GATA-2 decreases as GATA-1 expression
increases.42 Furthermore, the forced expression of GATA-2
arrests erythroid differentiation.43 These findings suggest that GATA-2 functions as a transdominant negative regulator for hematopoietic myeloid progenitors capable of differentiating toward the
eosinophil lineage.
It has been reported that NF-M, the chicken homolog of C/EBP,
induces eosinophil differentiation in a multipotent progenitor cell
line transformed by the Myb-Ets oncoprotein.44 We have identified a C/EBP consensus binding site only 7 bp upstream of the
functional GATA-binding site in the MBP P2 promoter, and shown that
cotransfection with C/EBP, C/EBP, and C/EBP expression vectors
effectively transactivates the MBP P2 promoter (Fig 10). These findings
suggest that the family of C/EBPs, in particular C/EBP and , may
also be associated with eosinophil differentiation. Of interest,
C/EBP null (knock-out) mice have recently been shown to have
impaired eosinophil development.45 Müller et
al44 speculate that the balance between three transcription
factors, c-Myb, GATA-1, and C/EBP, plays a decisive role in
hematopoietic lineage determination. They suggest that in the
multipotent precursor cell line (MEP), the expression of GATA-1 in the
absence of C/EBP results in differentiation toward the thrombocytic
and erythroid lineages, the expression of C/EBP in the absence of
GATA-1 drives differentiation toward the myelomonocytic
lineage, whereas the expression of both factors allows differentiation
toward the eosinophilic lineage.44 We have obtained results
demonstrating expression of both GATA-1 and C/EBP concomitant with
eosinophilic differentiation of the HT93A cell line, which can
differentiate from uncommitted blastic cells toward eosinophil and
neutrophil lineages after treatment with retinoic acid (data not
shown). From our results to date, we conclude that transcription
factors involved in fulfillment of the eosinophilic differentiation
program likely include GATA-1, C/EBP( and ), and c-Myb. Recent
studies of mice lacking functional GATA-1, C/EBP, and c-Myb genes
have shown impaired hematopoietic development of erythroid, mast cell,
and certain myeloid lineages.45-47 However, the eosinophil
lineage has yet to be evaluated in any of these knockout mice. However,
recent analysis of the eosinophil lineage in C/EBP null mice has
demonstrated impaired eosinophil development (complete absence of
eosinophils) along with impaired G-CSF signaling and neutrophil
development.45 Based on our current findings, we would
predict that eosinophil development is impaired wholly or in part in
mice also lacking expression of the GATA-1. In the absence of more
profound effects on eosinophil development, we would expect that
expression of certain key eosinophil genes such as MBP should be
lacking in GATA-1 null mice. The regulation of hematopoietic
differentiation from lineage commitment through terminal maturation is
likely determined by combinations of transcription factors, their
cofactors, as well as interactions among the factors. Continued
elucidation of the transcriptional mechanisms that regulate eosinophil
development and expression of eosinophil granule-derived mediators of
inflammation and tissue damage such as MBP may provide new targets for
drug development and therapeutic intervention in eosinophil-induced
inflammation in allergic diseases.
| |
FOOTNOTES |
Submitted August 14, 1997;
accepted December 22, 1997.
Supported by Grants-in-Aid from the Ministry of Education, Science and
Culture of Japan (to Y.Y.) and from the National Institutes of Health,
USA (AI33043) and the W.M. Keck Foundation (to S.J.A.).
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.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Gerald J. Gleich for providing the MBP genomic gene; Kenji
Kishi for a gift of KU812 cells; Shigeru Saito for a gift of
trophoblast cell lines; Daniel G. Tenen for providing myc, CD11b, and
MPO cDNAs; and Shizuo Akira for providing the human C/EBP cDNA.
| |
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 supports the terminal differentiation and proliferation of murine eosinophilic precursors.
J Exp Med
167:43,
1988[Abstract/Free Full Text]
4.
Yamaguchi Y,
Hayashi Y,
Sugama Y,
Miura Y,
Kasahara T,
Kitamura S,
Torisu M,
Mita S,
Tominaga A,
Takatsu K,
Suda T:
Highly purified murine interleukin-5 (IL-5) stimulates eosinophil function and prolongs in vitro survival: IL-5 as an eosinophil chemotactic factor.
J Exp Med
167:1737,
1988[Abstract/Free Full Text]
5.
Coffman RL,
Seymour BWP,
Hudak S,
Jackson J,
Rennick D:
Antibody to interleukin-5 inhibits helminth-induced eosinophilia in mice.
Science
245:308,
1989[Abstract/Free Full Text]
6.
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]
7.
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]
8.
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]
9.
Shivdasani RA,
Orkin SH:
The transcriptional control of hematopoiesis.
Blood
87:4025,
1996[Free Full Text]
10.
Takahashi A,
Satake M,
Yamaguchi-Iwai Y,
Bae S-C,
Lu J,
Maruyama M,
Zhang YW,
Oka H,
Arai N,
Arai K,
Ito Y:
Positive and negative regulation of granulocyte-macrophage colony-stimulating factor promoter activity by AML1-related transcription factor, PEBP2.
Blood
86:607,
1995[Abstract/Free Full Text]
11.
Smith LT,
Hohaus S,
Gonzalez DA,
Dziennis SE,
Tenen DG:
PU.1 and C/EBP regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells.
Blood
88:1234,
1996[Abstract/Free Full Text]
12.
Mucenski ML,
McLain K,
Kier AB,
Swerdlow SH,
Schreiner CM,
Miller TA,
Pietryga DW,
Scott JW Jr,
Potter SS:
A functional c-myb gene is required for normal fetal hepatic hematopoiesis.
Cell
65:677,
1991[Medline]
[Order article via Infotrieve]
13.
Miller IJ,
Bieker JJ:
Identification of a novel, erythroid-specific murine transcription factor structually related to the Kruppel family of nuclear proteins.
Mol Cell Biol
13:2776,
1993[Abstract/Free Full Text]
14.
Shivdasani RA,
Mayer E,
Orkin SH:
Absence of blood formation in mice lacking the T-cell leukemia oncoprotein tal-1/SCL.
Nature
373:432,
1995[Medline]
[Order article via Infotrieve]
15.
Shivdasani RA,
Orkin SH:
Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.
Proc Natl Acad Sci USA
92:8690,
1995[Abstract/Free Full Text]
16.
Tsai S-F,
Martin DIK,
Zon LI,
D'Andrea AD,
Wong GW,
Orkin SH:
Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells.
Nature
339:446,
1989[Medline]
[Order article via Infotrieve]
17.
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: Potential role in gene transcription.
Blood
81:3234,
1993[Abstract/Free Full Text]
18.
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]
19.
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]
20. (abstr, suppl 1)
Sun Z,
Wong IC,
Yergeau DA,
Paul CC,
Baumann MA,
Tenen DG,
Ackerman SJ:
An enhancer-like element, CTTCCT (a PEA3, ets-type consensus sequence), regulates transcription of the gene encoding human Charcot-Leyden crystal (CLC) protein.
Blood
86:31a,
1995
21.
Tiffany HL,
Handen JS,
Rosenberg HF:
Enhanced expression of the eosinophil-derived neurotoxin ribonuclease (RNS2) gene requires interaction between the promoter and intron.
J Biol Chem
271:12387,
1996[Abstract/Free Full Text]
22.
Handen JS,
Rosenberg HF:
Intronic enhancer activity of the eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3) genes is mediated by an NFAT-1 consensus binding sequence.
J Biol Chem
272:1665,
1997[Abstract/Free Full Text]
23.
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]
24.
Satterthwaite AB,
Borson R,
Tenen DG:
Regulation of the gene for CD34, a human hematopoietic stem cell antigen, in KG-1 cells.
Blood
75:2299,
1990[Abstract/Free Full Text]
25.
Chirgwin JM,
Przybyla AE,
MacDonald RJ,
Rutter WJ:
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:5294,
1979[Medline]
[Order article via Infotrieve]
26.
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]
27.
Radzioh D,
Clayton M,
Varesio L:
Interferon-alpha, -beta, and -gamma augment the levels of rRNA precursors in peritoneal macrophages but not in macrophage cell lines and fibroblasts.
J Immunol
139:805,
1987[Abstract]
28.
Kong TH,
Coates ARM,
Butcher PD,
Hickman CJ,
Shinnick TM:
Mycobacterium tuberculosis expresses two chaperonin-60 homologs.
Proc Natl Acad Sci USA
90:2608,
1993[Abstract/Free Full Text]
29.
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 two promoters.
Biochem J
305:921,
1995
30.
Henikoff S:
Unidirectional digestion with exonuclease III targeted breakpoints for DNA sequencing.
Gene
28:351,
1984[Medline]
[Order article via Infotrieve]
31.
Mizushima S,
Nagata S:
pEF-BOS, a powerful mammalian expression vector.
Nucleic Acids Res
18:5322,
1990[Free Full Text]
32.
Chowdhury S,
Gotoh T,
Mori M,
Takiguci M:
CCAAT/enhancer-binding protein (C/EBP) binds and activates 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]
33.
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]
34.
Nakajima-Iijima S,
Hamada H,
Reddy P,
Kakunaga T:
Molecular structure of the human cytoplasmic beta-actin gene: Interspecies homology of sequences in the intron.
Proc Natl Acad Sci USA
82:6133,
1985[Abstract/Free Full Text]
35.
Barker RL,
Loegering KC,
Arakawa KC,
Pease LR,
Gleich GJ:
Cloning and sequence analysis of the human gene encoding eosinophil major basic protein.
Gene
86:285,
1990[Medline]
[Order article via Infotrieve]
36.
Ackerman SJ,
Kephart GM,
Habermann TM,
Greipp PR,
Gleich GJ:
Localization of eosinophil granule major basic protein in human basophils.
J Exp Med
158:946,
1983[Abstract/Free Full Text]
37.
Wasmoen TL,
Coulam CB,
Benirschke K,
Gleich GJ:
Association of immunoreactive eosinophil major basic protein with placental septa and cysts.
Am J Obstet Gynecol
165:416,
1991[Medline]
[Order article via Infotrieve]
38.
Larson KA,
Horton MA,
Madden BJ,
Gleich GJ,
Lee NA,
Lee J:
The identification and cloning of a murine major basic protein gene expressed in eosinophils.
J Immunol
155:3002,
1995[Abstract]
39.
Gruart V,
Truong MJ,
Plumas J,
Zandecki M,
Kusnierz JP,
Prin L,
Vinatier D,
Capron A,
Capron M:
Decreased expression of eosinophil peroxidase and major basic protein messenger RNAs during eosinophil maturation.
Blood
79:2592,
1992[Abstract/Free Full Text]
40.
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]
41.
Yamagata T,
Nishida J,
Sakai R,
Tanaka T,
Honda H,
Hirano N,
Mano H,
Yazaki Y,
Hirai H:
Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.
Mol Cell Biol
15:3830,
1995[Abstract]
42.
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]
43.
Griegel K,
Lim K-C,
Plank C,
Beug H,
Engel J,
Zenke M:
Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrest erythroid differentiation in a hormone-dependent manner.
Genes Dev
7:1097,
1993[Abstract/Free Full Text]
44.
Müller C,
Kowenz-Leutz E,
Grieser-Ade S,
Graf T,
Leutz A:
NF-M (chicken C/EBP) induces eosinophilic differentiation and apoptosis in a hematopoietic progenitor cell line.
EMBO J
14:6127,
1995[Medline]
[Order article via Infotrieve]
45.
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]
46.
Pevny L,
Simon MC,
Robetson E,
Klein WH,
Tasi S-H,
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]
47.
Screpanti I,
Romani L,
Musiani P,
Modesti A,
Fattori E,
Lazzaro D,
Sellitto C,
Scarpa S,
Bellavia D,
Lattanzio G,
Bisoni F,
Frati L,
Cortese R,
Gulino A,
Ciliberto G,
Costantini F,
Poli V:
Lymphoproliferative disorder and imbalanced T-helper response in C/EBP-deficient mice.
EMBO J
14:1932,
1995[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
D. Nozawa, N. Suzuki, M. Kobayashi-Osaki, X. Pan, J. D. Engel, and M. Yamamoto
GATA2-dependent and region-specific regulation of Gata2 transcription in the mouse midbrain
Genes Cells,
May 1, 2009;
14(5):
569 - 582.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. D. Dyer, M. Czapiga, B. Foster, P. S. Foster, E. M. Kang, C. M. Lappas, J. M. Moser, N. Naumann, C. M. Percopo, S. J. Siegel, et al.
Eosinophils from Lineage-Ablated {Delta}dblGATA Bone Marrow Progenitors: The dblGATA Enhancer in the Promoter of GATA-1 Is Not Essential for Differentiation Ex Vivo
J. Immunol.,
August 1, 2007;
179(3):
1693 - 1699.
[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]
|
|
|
|
|
|
|
H. Iwasaki, S.-i. Mizuno, R. Mayfield, H. Shigematsu, Y. Arinobu, B. Seed, M. F. Gurish, K. Takatsu, and K. Akashi
Identification of eosinophil lineage-committed progenitors in the murine bone marrow
J. Exp. Med.,
June 20, 2005;
201(12):
1891 - 1897.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Monticelli, D. C. Solymar, and A. Rao
Role of NFAT Proteins in IL13 Gene Transcription in Mast Cells
J. Biol. Chem.,
August 27, 2004;
279(35):
36210 - 36218.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Grass, M. E. Boyer, S. Pal, J. Wu, M. J. Weiss, and E. H. Bresnick
GATA-1-dependent transcriptional repression of GATA-2 via disruption of positive autoregulation and domain-wide chromatin remodeling
PNAS,
July 22, 2003;
100(15):
8811 - 8816.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
J. P. Justice, M. T. Borchers, J. J. Lee, W. H. Rowan, Y. Shibata, and M. R. Van Scott
Ragweed-induced expression of GATA-3, IL-4, and IL-5 by eosinophils in the lungs of allergic C57BL/6J mice
Am J Physiol Lung Cell Mol Physiol,
February 1, 2002;
282(2):
L302 - L309.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Umetani, C. Mataki, N. Minegishi, M. Yamamoto, T. Hamakubo, and T. Kodama
Function of GATA Transcription Factors in Induction of Endothelial Vascular Cell Adhesion Molecule-1 by Tumor Necrosis Factor-{{alpha}}
Arterioscler Thromb Vasc Biol,
June 1, 2001;
21(6):
917 - 922.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Zimmermann, B. L. Daugherty, J. L. Kavanaugh, F. Y. El-Awar, E. A. Moulton, and M. E. Rothenberg
Analysis of the CC chemokine receptor 3 gene reveals a complex 5' exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements
Blood,
October 1, 2000;
96(7):
2346 - 2354.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. T. Magness, A. Tugores, and D. A. Brenner
Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells
Blood,
June 1, 2000;
95(11):
3568 - 3577.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Yang, S. Suzuki, L. J. Hao, Y. Fujii, A. Yamauchi, M. Yamamoto, M. Nakamura, and A. Kumatori
Eosinophil-specific Regulation of gp91phox Gene Expression by Transcription Factors GATA-1 and GATA-2
J. Biol. Chem.,
March 24, 2000;
275(13):
9425 - 9432.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Yamaguchi, H. Nishio, K. Kishi, S. J. Ackerman, and T. Suda
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
Blood,
August 15, 1999;
94(4):
1429 - 1439.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Iwama, J. Pan, P. Zhang, W. Reith, B. Mach, D. G. Tenen, and Z. Sun
Dimeric RFX Proteins Contribute to the Activity and Lineage Specificity of the Interleukin-5 Receptor alpha Promoter through Activation and Repression Domains
Mol. Cell. Biol.,
June 1, 1999;
19(6):
3940 - 3950.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Giembycz and M. A. Lindsay
Pharmacology of the Eosinophil
Pharmacol. Rev.,
June 1, 1999;
51(2):
213 - 340.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. A. Plager, D. A. Loegering, D. A. Weiler, J. L. Checkel, J. M. Wagner, N. J. Clarke, S. Naylor, S. M. Page, L. L. Thomas, I. Akerblom, et al.
A Novel and Highly Divergent Homolog of Human Eosinophil Granule Major Basic Protein
J. Biol. Chem.,
May 14, 1999;
274(20):
14464 - 14473.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Moriuchi, H. Moriuchi, and A. S. Fauci
GATA-1 Transcription Factor Transactivates the Promoter for CCR5, a Coreceptor for Human Immunodeficiency Virus Type 1 Entry
Blood,
February 15, 1999;
93(4):
1433 - 1435.
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract