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of June 15, 2017.
The Crucial Role of GATA-1 in CCR3 Gene
Transcription: Modulated Balance by
Multiple GATA Elements in the CCR3
Regulatory Region
Byung Soo Kim, Tae Gi Uhm, Seol Kyoung Lee, Sin-Hwa
Lee, Jin Hyun Kang, Choon-Sik Park and Il Yup Chung
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2010; 185:6866-6875; Prepublished online 1
November 2010;
doi: 10.4049/jimmunol.1001037
http://www.jimmunol.org/content/185/11/6866
The Journal of Immunology
The Crucial Role of GATA-1 in CCR3 Gene Transcription:
Modulated Balance by Multiple GATA Elements in the CCR3
Regulatory Region
Byung Soo Kim,* Tae Gi Uhm,* Seol Kyoung Lee,* Sin-Hwa Lee,† Jin Hyun Kang,*
Choon-Sik Park,† and Il Yup Chung*
E
osinophils are granulocytes that are implicated in a wide
variety of allergic diseases, including bronchial asthma.
Eosinophils appear to be key effector cells in allergic
diseases such as asthma; there is a positive correlation between
increased numbers and activation of eosinophils and the severity
of allergic symptoms (1, 2). Eosinophils develop in bone marrow,
exit to the bloodstream, and enter inflamed tissues in response to
proinflammatory mediators. Their transfer to the blood and tissues
is attributed largely to CCR3 activity (3, 4). CCR3, an essential
surface marker for eosinophils, is a major chemokine receptor that
is strongly, if not exclusively, expressed on eosinophils and that, in
concert with IL-5R, mediates eosinophil trafficking in the body
(5, 6). Targeted disruption of the genes that encode CCR3 and/or
its ligand eotaxin has been shown to reduce the recruitment of
*Division of Molecular and Life Sciences, College of Science and Technology,
Hanyang University; and †Division of Allergy and Respiratory Medicine, Genome
Research Center for Allergy and Respiratory Diseases, Soonchunhyang University
Hospital, Ansan, South Korea
Received for publication March 30, 2010. Accepted for publication September 22,
2010.
This work was supported by Korea Science and Technology Foundation Grant 20090072520. B.S.K. and J.H.K. were supported partially by Brain Korea 21, Korea
Research Foundation.
Address correspondence and reprint requests to Prof. Il Yup Chung, Division of
Molecular and Life Sciences, College of Science and Technology, Hanyang University, 1271 Sa-3-dong, Ansan, Gyeonggi-do 426-791, South Korea. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: AML, acute myeloid leukemia; BA, butyric acid;
C-finger, C-terminal finger; ChIP, chromatin immunoprecipitation; DN-GATA-1,
dominant-negative GATA-1; EDN, eosinophil-derived neurotoxin; m, mutant probe;
MBP, major basic protein; siRNA, small interfering RNA; w, wild-type probe.
Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001037
eosinophils to the blood and airways after Ag challenge in animal
models of asthma (7–10). Likewise, administration of anti-CCR3
or anti-eotaxin Abs significantly reduces the numbers of eosinophils in the airways and circulation (11, 12). Several small molecule antagonists of CCR3 have been developed to inhibit eosinophil accumulation and airway hyperresponsiveness (13, 14).
A recent study revealed that CCR3 is expressed, along with
IL-5Ra, in eosinophil progenitors at a very early stage in human
eosinophil development (15). Hence, analysis of the transcription
factors that control CCR3 expression may offer insights into the
mechanisms behind the commitment of common myeloid progenitors to the eosinophil lineage. The CCR3 gene (Mendelian
Inheritance in Man No. 601268) is located on chromosome 3p21.3
and consists of at least four exons (16). The critical sequence that
accounts for transcriptional regulation of CCR3 has been mapped
to exon 1 and its flanking sequences (16–19). Several lines of
evidence have demonstrated unequivocally that GATA-1 plays an
integral role in eosinophil development and the regulation of
eosinophil-specific genes. Disruption of a high-affinity doublepalindromic GATA site in the murine GATA-1 promoter results
in the selective loss of the eosinophil lineage in vivo (20). GATA1–transduced human CD34+ hematopoietic progenitor cells exclusively give rise to eosinophil lineage cells in vitro, whereas
GATA-1–deficient mice fail to develop eosinophil progenitors in
the fetal liver (21). GATA-1 reprograms avian myeloblastic cell
lines to eosinophils, and its expression level fine-tunes development of the eosinophil lineage (22, 23). GATA-1 is expressed
in human peripheral eosinophils and eosinophilic cell lines (24),
and eosinophil progenitors are found exclusively within cells activating GATA-1 transcription (25). The promoters of the genes
that encode the eosinophil-specific major basic proteins (MBPs),
Charcot-Leyden crystal protein and eosinophil-derived neurotoxin
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GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of
many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell
types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring
exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of
endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease
in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1
bound to virtually all seven putative GATA elements present in exon 1–intron 1, the first GATA site in exon 1 exhibited the highest
binding affinity for GATA-1 and was solely responsible for GATA-1–mediated transactivation. The fourth and fifth GATA sites in
exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1–mediated transcription of eosinophilspecific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore,
mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest
that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the
CCR3 gene. The Journal of Immunology, 2010, 185: 6866–6875.
The Journal of Immunology
Materials and Methods
Cell culture
Human lung epithelial A549 and NCI-H292 cells and K562 cells were
maintained in RPMI 1640 medium (Welgene, Seoul, Korea), and human
embryo kidney 293T cells were maintained in DMEM (Welgene). All of
the growth media were supplemented with 10% FBS, penicillin (100 U/
ml), and streptomycin (100 mg/ml). K562 cells differentiated in the presence of 0.5 mM butyric acid (BA; Sigma-Aldrich, St. Louis, MO). IL-5 (10
ng/ml) then was added on day 1. Thereafter, K562 cells were incubated for
up to 7 d. Peripheral blood eosinophils were isolated from atopic individuals. After RBCs had been sedimented in 6% dextran–dextrose in 0.1 M
EDTA (pH 7.4), the leukocyte-rich cell suspension was layered on Percoll
solution (1.070 g/ml) and centrifuged at 400 3 g for 20 min at 4˚C.
Enriched eosinophil fractions were incubated with anti-CD16 mAbconjugated microbeads (Miltenyi Biotec, Auburn, CA), and contaminating neutrophils were removed through negative selection using a magneticactivated cell sorter (BD Pharmingen, San Diego, CA). Mononuclear cells
were isolated from umbilical cord blood as described previously (34)
and plated at a density of 1 3 106 cells per milliliter in IMDM (Welgene)
supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100
mg/ml), and HEPES (25 mM). Cells were cultured initially with GM-CSF
(10 ng/ml), IL-3 (10 ng/ml), and IL-5 (10 ng/ml). GM-CSF and IL-3 were
omitted from culture from days 7 and 14 onwards, respectively. Half of the
culture medium was replaced weekly with fresh medium enriched with the
appropriate cytokines. By culture day 28, eosinophilic cells comprised 40–
80% of the total cell population, as determined morphologically on the
basis of granule formation, and the shapes of the nuclei were visualized
through staining with Diff-Quick solution (Sysmex, Kobe, Japan). This
study was approved by the Soonchunhyang University Hospital Institutional Review Board (Protocol SCHBC-IRB-06-04).
CCR3 and GATA-1 mRNA expression
Total mRNA was extracted from four different cell lines, peripheral blood
eosinophils, and umbilical cord blood-derived eosinophils using TRI reagent
(Molecular Research Center, Cincinnati, OH). First-strand cDNA was
synthesized from 4 mg total RNA using SuperScript II RNase Reverse
Transcriptase (Invitrogen Life Technologies) in a 20 ml reaction including
the random primers, deoxynucleotide triphosphates (0.5 mM), MgCl2
(2.5 mM), and DTT (5 mM). Reverse transcription was performed at 42˚C
for 1 h and followed by heat inactivation at 70˚C for 15 min. Thussynthesized cDNA was amplified for 30 cycles with Ex Taq DNA polymerase (TAKARA, Shiga, Japan). The following primers were used in the
amplification: CCR3 forward primer 59-ATGGCATGTGTAAGCTCCTCTCAG-39 and CCR3 reverse primer 59-TTGCTCCGCTCACAGTCATTTCC-39; GATA-1 forward primer 59-GCCCTGACTTTTCCAGTACC-39 and
GATA-1 reverse primer 59-CGAGTCTGAATACCATCCTTCC-39; GAPDH
forward primer 59-CGTCTTCACCACCATGGAGA-39 and GAPDH reverse primer 59-CGGCCATCACGCCACAGTTT-39.
Flow cytometry
Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15 min at
4˚C, washed with 2% BSA in PBS, and then stained with a PE-conjugated
anti-human CCR3 Ab (R&D Systems, Minneapolis, MN) or isotypematched Ab (BD Pharmingen). Fluorescence staining was analyzed using a FACSCalibur flow cytometer in conjunction with CellQuest software
(Becton Dickinson, San Jose, CA).
Western blot analysis
Cells were lysed in RIPA buffer (50 mM Tris-Cl [pH 7.4], 0.1% NaN3, 1%
Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM Na3VO4,
1 mM NaF, and protease inhibitor mixture [Calbiochem, San Diego, CA])
supplemented with 0.4 M NaCl. Lysates were centrifuged, and the resulting supernatants were subjected to Western blot analysis. Thirty micrograms of the cell lysates were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were
blocked with 5% nonfat dry milk. Blots were probed with anti-GATA (H200; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GAPDH Abs
(Santa Cruz Biotechnology) and then with an anti-rabbit HRP-conjugated
Ab (Cell Signaling Technology, Beverly, MA). Immunostained proteins
were detected using an ECL detection system (Amersham Biosciences).
ChIP analysis
Physical associations between GATA-1 and the CCR3 regulatory region in
A549 and K562 cells were analyzed using a ChIP assay kit (Millipore,
Bedford, MA), according to the manufacturer’s instructions. Cells were
treated with formaldehyde for 10 min at 37˚C, incubated in lysis buffer,
and sonicated to shear the chromatin. The cross-linked protein–DNA complexes were immunoprecipitated with specific Abs raised against GATA-1
(N6; Santa Cruz Biotechnology), histone 3 dimethyl K9, histone 3 trimethyl K9, and histone 3 acetyl K9 (Abcam, Cambridge, U.K.). Rat IgG
(Sigma-Aldrich) was used as a negative control for GATA-1, and an antihistone 3 Ab (Abcam) was used as a positive control. After the Ab-bound
complexes were pulled down with protein A-agarose/salmon sperm DNA
(Millipore), the cross-links were reversed. DNA was recovered through
phenol/chloroform extraction and ethanol precipitation and used as a
template for PCR amplification. The sequences of the regulatory region of
the CCR3 gene analyzed in the ChIP assay are shown in Fig. 1A, and the
primers used in the amplification are listed in Supplemental Table I.
Reporter gene plasmids, mutagenesis, and luciferase assay
Regions of exon 1 and/or proximal intron 1 of the CCR3 gene shown in
Fig. 1 were PCR-amplified and cloned into the pGL3-Basic luciferase reporter vector (Promega, Madison, WI). The PCR primers that were used are
listed in Supplemental Table II. Mutation of GATA sequences was performed through conventional overlap extension PCR. All 59-GATA-39 sequences in exon 1 and proximal intron 1 of the CCR3 gene were mutated to
59-TCGC-39. Amplification was performed using four primers (including
two overlapping primers for each mutation) and Pyrobest Taq (TAKARA)
in a 50 ml reaction. The resulting PCR products were cloned into a pGL3
vector, and their sequences were confirmed by sequence analysis. The
primers used in the overlap extension PCR are listed in Supplemental
Table III. Cell lines were transfected with the reporter plasmids using
Lipofectamine 2000 reagent (Invitrogen Life Technologies). pSV-b-galac-
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(EDN), contain functional GATA elements at which GATA-1
interacts with other transcription factors to activate transcription
(26–28). Zimmermann et al. (19) showed that GATA-1 binds to a
double-GATA element within exon 1 of the CCR3 gene. However,
the functional relevance and precise role of these GATA elements
in the transcriptional regulation of the CCR3 gene remain to be
determined.
GATA-1, one of six members of the GATA family, binds to
consensus (A/T)GATA(A/G) sequences through a DNA-binding
domain comprising two characteristic zinc finger motifs (29–31).
The C-terminal finger (C-finger) is essential for GATA-1 function,
because it is both necessary and sufficient for high-affinity binding
to consensus sequences (32). The N-terminal finger, which does
not bind DNA independently, influences C-finger binding. It may
stabilize or disrupt binding or alter the binding specificity of the Cfinger (33). More importantly, even high-affinity binding of GATA1 does not necessarily lead to transactivation (33). In general,
GATA-1 does not bind to its DNA recognition sequences with
equal affinity, even those that include an identical consensus GATA
binding sequence. The basis for this differential binding affinity
might lie in differences in the sequences flanking the consensus
GATA binding sequence and the orientation and spacing of the
GATA elements to which GATA-1’s two zinc fingers bind.
Despite the fact that numerous studies have focused on CCR3
gene expression, CCR3 ligand specificity, the development of
CCR3 antagonists, and the role of CCR3 in eosinophil trafficking
in vitro and in vivo, there is a surprising paucity of information on
CCR3 gene transcription at the molecular level. In the current study,
we explored the molecular basis of CCR3 gene expression and
focused on GATA-1’s specific mode of action. By analyzing constructs containing a few dozen point mutants and a deletion mutant
of CCR3 regulatory sequences with chromatin immunoprecipitation (ChIP) and EMSA assays, we identified a functional GATA
element. This GATA element, which was not recognized previously as being important, does not appear to meet the criteria that
define the proposed double-GATA element. Moreover, additional
GATA elements within the regulatory regions of the CCR3 gene
may participate in the fine-tuning of CCR3 gene transcription.
6867
6868
MAPPING A FUNCTIONAL GATA-1 BINDING SITE IN THE CCR3 GENE
tosidase (Promega) was cotransfected as an internal control. Cells were lysed
36 h posttransfection. Lysate luciferase activity was measured using the
Luciferase Assay System (Promega), whereas b-galactosidase activity was
assessed, through ortho-nitrophenyl-D-galactopyranoside hydrolysis, using
a b-Galactosidase Enzyme Assay System Kit (Promega). Variations in
transfection efficiency were corrected through normalization to b-galactosidase activity. K562 cells were cotransfected with dominant-negative
GATA-1 (DN-GATA-1), which was described previously (21), to probe the
involvement of GATA-1 in reporter transactivation.
RNA interference
Small interfering RNA (siRNA)-dependent knockdown experiments were
conducted in K562 cells using ON-TARGETplus SMARTpool siRNA
GATA-1 and nontargeting (scrambled) control pools (Dharmacon, Chicago,
IL). Briefly, K562 cells were cotransfected with siRNA and the reporter
plasmid (Ex1-In1) using DharmaFECT Duo Transfection Reagent (Dharmacon). Thirty-six hours after transfection, luciferase activities were measured. An aliquot of the transfected cells was taken for later GATA-1 protein level analysis by immunoblot.
Nuclear extract preparation and EMSA
Statistical analysis
Statistical analyses were performed using Microsoft Excel and SPSS,
version 13.0 (SPSS, Chicago, IL). For in vitro analyses, differences between
independent groups and samples were evaluated using a nonparametric
Kruskal-Wallis H test for continuous data. When significant differences
were found, a Mann-Whitney U test was performed to detect differences
between two independent samples. A p value ,0.05 was considered to be
statistically significant.
Results
Expression of CCR3 and GATA-1 mRNA and protein in cell
lines, developing eosinophils, and peripheral blood eosinophils
GATA-1 is a key player in both eosinophil development (20, 21, 35)
and the regulation of CCR3 expression (3, 19). We tested whether
there was any correlation between the levels of expression of the
genes encoding these two proteins. It has been shown that human
lung epithelial A549 and NCI-H292 cells express CCR3 mRNA
and protein (36, 37), whereas A549 and K562 erythroleukemia
cells express GATA-1 mRNA and protein (38, 39). Notably, K562
cells express high levels of GATA-1. We found CCR3 mRNA and
protein to be modestly produced in human epithelial cells, including A549, NCI-H292, and 293T cells (Fig. 2A). In contrast,
CCR3 expression was barely detectable in K562 cells but became
evident after induction of differentiation Peripheral blood eosinophils, as expected, constitutively expressed CCR3 mRNA and
protein. All four cell lines analyzed expressed GATA-1 mRNA
and protein with the following rank order: K562 . 293T . A549
. NCI-H292 (Fig. 2B). GATA-1 protein was not detectable in
peripheral blood eosinophils, whereas its mRNA was barely detectable, the former being consistent with a previous finding (39).
Next, we examined CCR3 and GATA-1 expression during the
differentiation of umbilical cord blood-derived eosinophils. As we
reported previously (34), CCR3 mRNA and protein levels gradually increased during the 28 d of culture. The GATA-1 mRNA level,
GATA-1 binding to sequences in exon 1 and proximal intron 1
of the CCR3 gene
It was demonstrated previously that the key regulatory sequences
for CCR3 gene transcription reside not only in exon 1 (17, 18) but
also in proximal intron 1 (16). A subsequent study identified
a palindromic double-GATA site in exon 1 of the CCR3 gene as
a GATA-1 binding site, as judged by EMSA, but did not analyze
its functional effects (19). To investigate the involvement of these
regions of the CCR3 gene in GATA-1 binding, four different
regions were selected for ChIP analysis (Fig. 1A): a promoter sequence (2280 to 298 bp); exon 1 (+1 to +161 bp); proximal
intron 1 (+162 to +405 bp); and a protein-coding sequence located
∼22 kbp downstream of exon 1. To avoid confusion, it should be
mentioned that the numbering was according to the longest exon 1
(161 bp in length), as described previously (16). Thus, the length
of the exon 1 is 67 bp longer than that of exon 1 identified by
Zimmermann and his colleagues (17) (the first nucleotide of the
exon 1 that those authors described corresponds to the 68th nucleotide of the exon 1 that we defined in this study; Fig. 1C). The
ChIP assay was performed on cross-linked genomic DNA from
A549 cells. An anti–GATA-1 Ab was able to detect exon 1- and
intron 1-bound GATA-1 but failed to pull down GATA-1–bound
promoter and the protein-coding CCR3 DNA sequences (Fig. 3).
No signal was detected with control Ab. An anti-histone 3 Ab
efficiently pulled down histone–DNA complexes in the same region, allowing us to compare ChIP efficiency and/or specificity.
These results suggest that GATA-1 binds to sequences in exon 1
and proximal intron 1 of the CCR3 gene in cells that modestly and
constitutively express GATA-1 and CCR3. Thus, the intron 1 sequence also may contribute to transactivation of the CCR3 gene by
GATA-1. In addition, the failure of GATA-1 to bind to promoter
sequences upstream of exon 1 suggests a minimal role for these
sequences in CCR3 transactivation (16, 17).
Both exon 1 and intron 1 contain sequences that are important
for transactivation by GATA-1. On the basis of our ChIP data and
the findings of previous studies (16, 18, 19), we investigated the
contribution of exon 1 and the intron 1 sequences to transcription
activation of the CCR3 gene. To this end, we generated reporter
plasmids bearing sequences from exon 1 (named Ex1), its 39
proximal intron 1 sequence (In1), or both exon 1 and intron 1
(Ex1-In1) (Fig. 1B). Four different cell lines were transfected with
these three constructs, and luciferase activities were measured.
The three reporters produced almost identical patterns of transactivation in all four cell lines studied. The Ex1-In1 construct
yielded the highest transactivation in all cells tested, whereas Ex1
and even In1 retained substantial transactivation, especially in
293T and K562 cells, which expressed higher levels of GATA-1
than A549 and NCI-H292 cells. Levels of reporter transactivation
were essentially proportional to the endogenous levels of GATA-1
(Fig. 4A). In support of the results, the transactivation of the three
constructs was reduced by half by DN-GATA-1 (Fig. 4B). The
inhibitory response by DN-GATA-1 was dose-dependent, even in
the case of the In1 construct (Fig. 4C). Furthermore, introduction
of GATA-1 siRNA led to a decrease in transcription activity of
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Nuclei obtained by centrifugation after lysis with RIPA buffer containing
0.15 M NaCl were resuspended in RIPA buffer containing 0.4 M NaCl.
Lysates were centrifuged, and the resulting supernatants (nuclear extracts)
were analyzed by EMSA. Four micrograms of the nuclear extracts were
incubated with 32P-labeled probe for 20 min in binding buffer (50 mM
Tris-Cl [pH 7.5], 20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM
DTT, 250 mM NaCl, and 0.25 mg/ml poly[dI-dC]•poly[dI-dC]). For
supershift analysis, extracts were incubated with anti–GATA-1 (M-20;
Santa Cruz Biotechnology), anti–GATA-2 (Santa Cruz Biotechnology),
anti-PU.1 (Santa Cruz Biotechnology), acute myeloid leukemia (AML)-1a
(Santa Cruz Biotechnology), or control Abs (Sigma-Aldrich), followed by
radiolabeled probe. Reaction mixtures were analyzed through separation on
4% polyacrylamide gels. The wild-type and mutant probe sequences that
were used are listed in Supplemental Table IV.
which was monitored along with GAPDH used as a loading control
by a multiplex PCR, peaked on culture day 21, declining slightly
thereafter. GATA-1 protein was not detectable in fresh mononuclear cells obtained from umbilical cord blood but was induced steadily as differentiation progressed (Fig. 2C). Therefore,
it may be concluded that CCR3 expression generally parallels
the expression of GATA-1, although these two genes are not
always expressed coincidentally, as shown by the data from the
K562 cells.
The Journal of Immunology
6869
the CCR3 reporter with a concomitant reduction of GATA-1 protein levels in K562 cells in a dose-dependent manner, whereas
scrambled siRNA had no effect (Fig. 4D). These data suggest that
GATA-1 is a key transactivator of the CCR3 gene and that the
sequences involved in regulation by GATA-1 may reside not only
in exon 1 but also in its proximal intron 1.
FIGURE 2. Correlation between the levels of
expression of CCR3 and GATA-1 in various cell
types. A and B, Expression of CCR3 and GATA-1
mRNA and protein was analyzed in cell lines
and peripheral blood eosinophils. Protein expression was analyzed by FACS (CCR3) and
Western blotting (GATA-1), and mRNA expression was analyzed by RT-PCR. C, CCR3 and
GATA-1 expression in developing eosinophils.
Unfractionated umbilical cord blood mononuclear
cells were cultured as described previously (34).
Expression of GATA-1 and GAPDH mRNA was
analyzed by multiplex RT-PCR. GAPDH mRNA
and protein were used as loading controls. The
results shown are representative of three to five
independent experiments.
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FIGURE 1. CCR3 gene structure, reporter plasmid
constructs, and the nucleotide sequence of exon 1 and
proximal intron 1. A, CCR3 gene structure and the
fragments amplified in the ChIP assay are shown.
Open and filled boxes indicate untranslated and protein-coding CCR3 gene sequences, respectively. Open
ovals represent GATA or GATC sites. Exons and
introns are not drawn to scale. B, On the basis of the
results shown in Fig. 3, the relative importance of
three constructs to CCR3 gene transcription was
evaluated. C, Nucleotide sequence of the Ex1-In1
construct containing exon 1 and proximal intron 1.
Numbering is relative to the transcription start site
(asterisk) identified by Vijh et al. (16). The open box
identifies exon 1. All putative GATA sites and the
GATC site located at +244 to +247 bp are highlighted
by shading. The arrow indicates the first nucleotide of
the partial exon 1 sequence identified by Zimmermann et al. (19). Dashed lines indicate the probe
sequences used to generate the EMSA data shown in
Fig. 6. The sequences of probes 2 and 3 and probes 4
and 5 partially overlap.
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MAPPING A FUNCTIONAL GATA-1 BINDING SITE IN THE CCR3 GENE
FIGURE 3. GATA-1 binds to regulatory sequences in exon 1 and proximal intron 1 of the CCR3 gene. A549 cells were fixed in formaldehyde and
sonicated. A rat anti-human GATA-1 Ab (N6; Santa Cruz Biotechnology)
was used to pull down DNA fragments bound to GATA-1. An anti-histone 3
Ab and a rat IgG were used as positive and negative controls, respectively.
D.W indicates no DNA template. The pulled-down DNA fragments were
amplified by conventional PCR using the primers listed in Supplemental
Table I. Different fragments were named as shown in Fig. 1A: Fragment 1,
the proximal promoter; Fragment 2, exon 1; Fragment 3, proximal intron 1;
Fragment 4, the protein-coding region of the CCR3 gene. The results shown
are representative of three independent experiments.
Inhibitory roles of the fourth, fifth, and eighth GATA sites in
CCR3 transcription
Interestingly, mut4, mut5, and mut8 displayed significantly augmented transcription (compared with that of the wild-type Ex1-In1
construct), suggesting that the fourth, fifth, and eighth GATA sites
inhibit transcriptional activation. To confirm their inhibitory roles,
we created further point mutants, mut9 and mut10, which carried
mutations in the fourth and fifth GATA sites in Ex1 (rather than in
Ex1-In1), respectively. Both significantly increased transcription
(Fig. 5B). Similarly, mutation of the eighth GATA site in In1 led to
a profound increase in transcriptional activity. Moreover, mutations in these three GATA elements partially reversed the reduction in transcriptional activity resulting from mutation of the
transcriptionally functional GATA site (mut1): mut1–4, mut1–5,
and mut1–8 all displayed higher transcription than mut1 (Fig. 5C).
FIGURE 4. Transactivation of CCR3 is related to levels of endogenous
GATA-1 and is inhibited by DN-GATA-1 and GATA-1 siRNA. A, Four
different cell lines were transfected with Ex1-In1, Ex1, and In1 reporter
constructs, whose transcription activities then were measured. B, K562
cells were cotransfected with DN-GATA-1 and each of the three constructs. C, The Ex1-In1 and In1 constructs were introduced to K562 cells
together with increasing concentrations (0.01, 0.05, and 0.1 mg) of DNGATA-1. Data represent the mean 6 SEM of at least three independent
experiments. D, K562 cells were cotransfected with the Ex1-In1 construct
and GATA-1 siRNA or scrambled siRNA (1, 5, 10, and 25 nM). Results are
the average of two independent experiments that are set in triplicate. The
aliquot of the transfected cells was tested for GATA-1 protein levels by
Western blot analysis.
These data suggest that 1) the first GATA site is solely responsible
for transactivation by GATA factors; 2) the fourth and fifth GATA
sites, previously reported to be key elements mediating transactivation, in fact negatively modulate GATA-1 activity; and 3) a
GATA site located in intron 1 also participates in the negative
regulation of CCR3 gene transcription.
GATA-1 binding is not correlated with transactivation. Using
EMSA, we examined whether the binding of GATA-1 to these
GATA sites paralleled CCR3 transactivation. Nuclear extracts from
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The first GATA site within exon 1 is involved in GATA-1–mediated transcription. Because the results from the transient transfection experiments suggested that sequences from both exon 1
and intron 1 mediate transactivation by GATA-1, we evaluated the
functional relevance of individual GATA sites to transactivation
of the CCR3 gene. There are seven putative GATA sites in the
Ex1-In1 sequence (Fig. 1C). GATA-1 binding occurs in two adjacent GATA sites within exon 1 (19), which correspond to the
fourth and fifth GATA sites depicted in Fig. 1C. It also should be
mentioned that the first exon initially identified by Zimmermann
et al. (17) was found subsequently to be longer (16). The sequence
identified by Zimmermann et al. (17) spanned +68 to +161 bp of
exon 1 and thus lacked the first three GATA sites (Fig. 1C). To
map the sequences responsible for functional GATA-1 binding,
we created a series of point mutants in which the 59-GATA-39 of
each GATA-containing sequence was replaced with 59-TCGC-39.
K562 cells then were transfected with these constructs. Only the
point mutant (mut1) in which the first GATA site was disrupted
exhibited diminished transactivational activity. In agreement with
that result, a mutant (del1) lacking the first GATA site exhibited
similarly reduced activity. A decrease in transactivation was not
observed in any of the other point mutants. A mutation in the
nonconsensus GATC recognition sequence (mut6) also had no
effect (Fig. 5A). These patterns of transactivation were not confined to K562 cells: similar results were obtained with A549 cells,
which modestly express both CCR3 and GATA-1 (data not
shown).
The Journal of Immunology
6871
K562 cells yielded the highest level of binding of GATA-1 to the
first GATA site and a similar level of binding to the fourth GATA
(Fig. 6A). The fifth and eight GATA sites exhibited intermediate
levels of GATA-1 binding, and the second, third, and seventh
GATA sites exhibited much less binding. As expected, GATA-1
did not bind to the sixth site, whose sequence was GATC instead
of GATA. Thus, binding affinity did not correlate with the extent
of activation. Binding complexes formed at the first (Fig. 6B),
fourth, fifth, and eighth GATA sites (Supplemental Fig. 1) included GATA-1, as revealed by a supershift assay involving an
anti–GATA-1 Ab. Abs raised to transcription factors, including
GATA-2, PU.1, and AML-1a (whose encoding mRNAs were
expressed in K562 cells, as analyzed by RT-PCR; data not shown),
did not affect the binding of GATA-1 to the first (Fig. 6B) and
other GATA elements (Supplemental Fig. 1). Our results suggest
that the DNA-bound complex does not contain any the aforementioned transcription factors (apart from GATA-1). Furthermore, the binding of GATA-1 to a hot probe containing the first
GATA site was efficiently competed out by cold probes carrying
the fourth and eighth GATA sites and less efficiently so by a cold
probe carrying the fifth GATA site (Fig. 6C). Other combinations
of hot and cold probes yielded similar patterns of inhibition
(Supplemental Fig. 2), indicating that GATA-1 in isolation efficiently binds to any of these GATA elements. When nuclear ex-
tracts from different cell lines expressing varying amounts of
endogenous GATA-1 were allowed to bind to a radiolabeled probe
carrying the first GATA site, a DNA–protein complex was formed
(Fig. 6D). No binding complexes were detected when a probe
carrying the first GATA site was incubated with nuclear extracts
from peripheral blood eosinophils (data not shown).
Treatment with BA induces CCR3 mRNA expression without
enhancing GATA-1 binding and transactivation of the CCR3 gene.
When K562 cells were treated with BA followed by IL-5, expression of CCR3 mRNA was induced, reaching a peak at day
5, and then declined to basal levels by day 7. Surface CCR3 expression was preceded by CCR3 gene transcription (Fig. 7A). A
similar response was observed in HL-60 cells (data not shown).
When the Ex1-In1 reporter plasmid was introduced to K562 cells
that had been induced to differentiate through exposure to BA and
IL-5 for 3 d, transactivation increased 3.6-fold compared with that
of nontreated cells (Table I). However, this increase appeared to
result from increased transfection efficiency in BA-treated cells,
as demonstrated by a comparable increase (4.6-fold) in transactivation activity upon transfection with the pGL-Basic vector.
Accordingly, levels of GATA-1 mRNA and protein were not increased by BA treatment (data not shown). To evaluate whether
the increase in transactivational activity was caused by increased
GATA-1 binding in vivo, we performed ChIP assays using ge-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. Influence of individual
GATA sites in the regulatory regions of
the CCR3 gene on transactivation. A, An
array of single-point mutant constructs
for GATA sites and a deletion construct
lacking the first GATA. Asterisks indicate
statistical significance (compared with
Ex1-In1). B, Transactivation of singlepoint mutants. Asterisks indicate statistical significance compared with Ex1 for
mut9 and mut10 and In1 for mut11. C,
Transactivation of double-point mutants.
Asterisks (mut1–4, mut1–5, and mut1–8)
indicate statistical significance compared
with mut1. Open and filled symbols indicate wild-type and mutant GATA sites,
respectively. Data represent the mean 6
SEM of six or more independent experiments. *p , 0.05; **p , 0.01.
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MAPPING A FUNCTIONAL GATA-1 BINDING SITE IN THE CCR3 GENE
nomic DNA isolated from undifferentiated cells and differentiated
K562 cells. In untreated cells (as in A549 cells), GATA-1 weakly
occupied the GATA elements in exon 1 and intron 1. BA treatment
had little effect on GATA-1 binding (Fig. 7B), suggesting that
butyric treatment does not stimulate the binding of GATA-1 to the
GATA elements. Moreover, we examined whether there would be
differential binding of GATA-1 to the positively and negatively
acting GATA elements within exon 1 in uninduced and maximally
induced CCR3 mRNA states. ChIP assays were performed with
the two additional primer sets shown in Fig. 7C and K562 cells
treated with or without BA and IL-5. We found that both positively and negatively acting GATA sites were bound to GATA-1
regardless of CCR3 expression levels, lowering the likelihood of
differential binding of GATA-1 to these GATA sites in this cell
FIGURE 7. Expression of CCR3, GATA-1 binding, and the modification
of histone 3 at K9 in the regulatory regions of the CCR3 gene during BAinduced differentiation of K562 cells. A, K562 cells were cultured with 0.5
mM BA for 1 d and then with IL-5 for up to 7 d. CCR3 mRNA levels and
cell surface expression of CCR3 protein were measured. The results shown
are representative of three experiments. B–D, K562 cells were cultured
with BA for 1 d and then with IL-5 for an additional 2 d. The cultures were
subjected to ChIP analysis using Abs raised against GATA-1 (B, C) and
modified forms of histone 3 (D). In B, different fragments were named as
shown in Fig. 1A. In C, two additional primer sets were used for the ChIP
assay, and the open box and ovals represent exon 1 and GATA sites within
exon 1, respectively. In D, the results shown are representative of three
independent experiments (the results of the other two experiments revealed
similar patterns of histone modification).
type. Instead, acetylation of histone 3 at K9 increased in the
studied regions of the CCR3 gene, whereas methylation of histone
3 at K9 remained unchanged (Fig. 7D), suggesting that BA influences chromatin remodeling.
Discussion
We mapped a GATA site necessary for GATA-1–mediated transactivation to exon 1 of the CCR3 gene. ChIP analysis initially
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. Binding of GATA-1 to the putative GATA sites. A, Eight
different radioactive probes (indicated in Fig. 1C) were incubated with nuclear extracts from K562 cells. Competitors were used to test the specificity
of binding: w and m represent wild-type and mutant sequence-harboring cold
probes, respectively. The sequence 59-GATA-39 in the w probes was replaced
with 59-TCGC-39 in the m probes, except for probe 6, in which the 59-GATC39 was replaced with 59-AGCT-39. B, Nuclear extracts from K562 cells were
incubated with Abs raised against different transcription factors, followed by
32
P-labeled probe 1. S and C indicate specific and control Abs, respectively.
C, Probe 1 was incubated with varying concentrations (10 and 50 molar
excess) of unlabeled probes containing the first, fourth, fifth, and eighth
GATA sites. D, Probe 1 was incubated with nuclear extracts from the cell
lines tested in the transfection experiments. Arrows and the arrowhead indicate specific binding and a supershift, respectively.
The Journal of Immunology
6873
Table I. Transactivation of CCR3 reporter plasmids in K562 cells
treated and untreated with BA
2BA
+BA
pGL3-Basica
Ex1-In1a
Fold Increase
(Ex1-In1/pGL3-Basic)b
4.0 6 0.3
18.4 6 0.2
177.9 6 7.4
641.5 6 7.1
44.3 6 1.8
34.8 6 0.4
a
Empty vector (pGL3-Basic) and the Ex1-In1 reporter construct were introduced
by transfection to K562 cells that had been treated with BA for 1 d and cultured with
IL-5 for an additional 2 d. Values represent luciferase activities.
b
To compare reporter transactivation in untreated and treated K562 cells, the
luciferase activity for Ex1-In1 construct was divided by that for the empty vector.
Transfections were performed in triplicate. Data represent the mean 6 SEM of two
independent experiments.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
enabled us to narrow our search for GATA-1 binding sites to
sequences in exon 1 and its proximal intron 1 rather than sequences
upstream of exon 1 (Fig. 3). Subsequently, transient transfection
experiments involving reporter genes linked to sequences containing exon 1, intron 1, or both showed that both exon 1 and intron 1 contain sequences that regulate GATA-1–mediated transactivation. Moreover, transactivation was essentially proportional
to levels of endogenous GATA-1 (Fig. 4). More conclusively, only
two mutants, one in which the first GATA site was disrupted and
a second in which it was missing, exhibited severely impaired
transcription (Fig. 5). In addition, the first GATA site exhibited
high-affinity binding for GATA-1 (Fig. 6). This functional GATA
element is different from the one that was proposed previously to
be critical for GATA-1–mediated transactivation of the CCR3 gene
(19): the authors of a previous report claimed, based on the results
of EMSA analysis, that the fourth and fifth GATA sites constituted
a canonical double-GATA site responsible for the regulation of
CCR3 expression by GATA-1. Moreover, the functional GATA site
does not meet the criteria that define the proposed double-GATA
element, which is postulated to be a key sequence for the regulation
of eosinophil-specific genes by GATA-1 (3). Such double-GATA
sites have been identified in several genes. Notably, a doubleGATA sequence in the mouse GATA1 gene is essential for the activation of its promoter. Furthermore, ablation of this palindromic
GATA site results in loss of the eosinophil lineage (20). Similar
double-GATA sequences are present in the promoter or enhancer
regions of the eosinophil-specific genes encoding CCR3, MBP, and
human IL-5Ra (3). However, it is not clear whether the doubleGATA site is essential for the transcriptional activation of eosinophil-specific genes. Charcot-Leyden crystal protein (27) and
gp91phox (40) are encoded by eosinophil-specific genes that
contain a single-GATA site in their promoters, and mutation of
these GATA sites significantly suppresses transcriptional activity.
In addition, although the MBP P2 promoter contains a double-GATA
site, mutation of one of the two GATA subsites is sufficient to inhibit transcriptional activity (26). Elsewhere, the two GATA elements in the EDN promoter are ∼600 bp apart. A mutation in either
site sharply reduces promoter activity (28). Although these studies do
not completely rule out the possible requirement for a double-GATA
site for transactivation, neither do they support the double-GATA
hypothesis.
A second important observation in the current study was that the
level of transactivation of the CCR3 gene by GATA-1 may depend
on the net effect of its interaction with GATA elements that act
as both enhancers and repressors of transactivation. Indeed, the
fourth GATA site binds to GATA-1 with high affinity (comparable
to that of the first GATA site; Fig. 6A) without increasing transactivation (Fig. 5A, 5B). Similarly, the fifth and eighth GATA sites,
which displayed intermediate levels of GATA-1 binding, made no
positive contribution to transactivation. Instead, point mutations of
these three sites resulted in a significant increase in transcriptional
activity (Fig. 5A, 5B), indicating that these GATA sites may act as
negative cis elements controlling transactivation of the CCR3 gene
by GATA-1. We then hypothesized that the reduction in transcriptional activity resulting from mutation of the first GATA site
might be, at least in part, reversed by mutation of the GATA sites
that negatively regulate transcription. Indeed, this was the case
(Fig. 5C). Consequently, the level of transcriptional activation
does not necessarily correlate with DNA binding affinity. This
finding is in agreement with a previous report (33), which demonstrated that GATA-1’s two zinc fingers interact to influence
DNA binding and transactivation: the N-terminal finger (and adjacent linker region) alters the binding specificity of the C-finger
and even inhibits the ability of the C-finger to recognize consensus
GATA sequences. Moreover, because high-affinity binding does
not always lead to transactivation, some GATA sites may have
other roles. Indeed, the fourth, fifth, and eighth GATA sites in the
Ex1-In1 sequence of the CCR3 gene may act to inhibit transactivation. We therefore reason that transactivation of the CCR3
gene by GATA-1 may be influenced by the nature of the two
GATA sites that the two zinc fingers of GATA-1 engage during
binding. Even if these GATA sites are not located in tandem in the
regulatory region, they may be brought into proximity through
looping, because it was shown that DNA bending may be induced
by the binding of GATA-1 (41) and possibly other transcription
factors. In this sense, the more favorable scenario for transactivation would involve simultaneous binding of GATA-1 to the
first and second or sixth GATA sites and not the fourth, fifth, or
eighth GATA sites, mutations in which increased transcription.
These three GATA sites may act as sinks for GATA-1, reducing
the amount of protein available for binding to the positive regulatory elements. Furthermore, although eosinophil-specific genes
commonly have GATA elements in their regulatory regions,
GATA-1 binding may affect GATA-containing promoters or enhancers in individual eosinophil-specific genes differently, because
these genes differ in the number and spacing of GATA sites in their
regulatory regions. The number and spacing of these GATA elements may collectively influence transactivation, in a sequencespecific manner, by altering the conformation of GATA-1.
Our EMSA and point mutation analyses allowed us to determine
the GATA-1 binding affinities and roles of individual GATA elements in exon 1 and intron 1 of the CCR3 gene. However, it
remains to be shown whether these individual GATA elements
have similar GATA-1 binding affinities and roles in the context of
an intact CCR3 gene with a particular chromatin structure and
pattern of DNA methylation. Examination of the exon 1 and intron
1 sequences reveals that the fourth and fifth GATA elements immediately follow CpG dinucleotide sequences (fourth, 59-tatccg39, and fifth, 59-tatcg-39, where underlined and bold text denotes
the GATA elements and CpG sites, respectively). A recent study
showed that CpG-poor promoters are hypermethylated in
somatic cells, which does not preclude their activity (42). Given
that DNA methylation interferes with the binding of transcription
factors (43), methylation of these GATA sites would be expected
to greatly influence binding affinity for GATA-1 and to have
functional consequences.
Although GATA-1 acts as a central regulator of CCR3 gene
transcription, it is not likely to be sufficient for the induction of
CCR3 mRNA expression. First, K562 cells do not constitutively
express CCR3 mRNA despite abundant expression of GATA-1
(Fig. 2). Second, BA induces CCR3 mRNA without increasing
cellular GATA-1 levels, binding of GATA-1 to sequences in the
CCR3 regulatory regions (Fig. 7), and reporter gene transactivation (Table I). Moreover, transcription factors such as C/EBP
6874
MAPPING A FUNCTIONAL GATA-1 BINDING SITE IN THE CCR3 GENE
Acknowledgments
We thank Dr. Atsushi Iwama (Chiba University, Chiba, Japan) for providing
DN-GATA-1 and Dr. Sung Hee Choi (Hanvit Clinic, Ansan, Korea) for providing experimental materials.
Disclosures
The authors have no financial conflicts of interest.
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