|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 30, 27144-27153, July 26, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, January 29, 2002, and in revised form, May 17, 2002
E4bp4, a member of the basic
region/leucine zipper transcription factor superfamily, is
up-regulated by the interleukin-3 (IL-3) signaling pathway and plays an
important role in the anti-apoptotic response of IL-3. In this study,
we demonstrated that E4bp4 is regulated by IL-3 mainly at
the transcriptional level. Promoter analysis revealed that a GATA motif
downstream of a major transcription initiation site is essential for
E4bp4 expression in the IL-3-dependent Ba/F3
cell line. Gel shift assays demonstrated that both GATA-1 and GATA-2
proteins bind to the E4bp4 GATA site in vitro,
and the chromatin immunoprecipitation assay further confirmed the in vivo binding of GATA-1 to the E4bp4
promoter. Overexpression of GATA-1 alone transactivates the
E4bp4 reporter, whereas transactivation of the
E4bp4 reporter by GATA-2 is dependent on the stimulation of
IL-3. Last, we demonstrated that alteration of GATA-1 binding to the
GATA site by stably overexpressing GATA-1 or a GATA-1 mutant containing
only the DNA-binding domain not only modulates the expression of the
E4bp4 gene but also influences apoptosis induced by IL-3
removal. Taken together, our results suggest that the GATA factors play
an important role in transducing the survival signal of IL-3, and one
of their cellular targets is E4bp4.
GATA family proteins are a group of transcription factors
containing two related zinc fingers that mediate DNA binding (1). Among
the six known members, GATA-1, GATA-2, and GATA-3 are preferentially expressed in hematopoietic cells, whereas the other GATA factors are
expressed exclusively in nonhematopoietic tissues. GATA-1 is highly
expressed in mature erythroid cells, mast cells, and megakaryocytes
(2-4) and is expressed at a lower level in progenitor cells (5, 6) and
Sertoli cells of testis (7, 8). GATA-2 is preferentially expressed in
the stem and progenitor cell population (5, 6) and in mast cells and
megakaryocytes (9). Expression of GATA-2 is also reported in
endothelial cells (10, 11) and the nervous system (2). GATA-3 is
expressed in chicken erythroid cells, T lymphocytes, and neuronal cells
(2, 12, 13). The essential role of GATA factors in the development of
hematopoietic lineage was first established by the fact that the
functionally important GATA motifs were identified in virtually all
erythroid-specific genes (14, 15). Further characterization of the
GATA-1 and GATA-2-null embryonic stem cells through targeted gene
disruption reconfirmed their essential role in hematopoietic
differentiation phenotype (16, 17). However, these studies also
revealed that GATA-1 and GATA-2 were essential to support the viability
of red cell precursors and early progenitors, respectively, by
suppressing apoptosis (16, 17). Although several GATA-binding
motif-containing genes, including erythropoietin receptor, SCL/tal-1,
and the cytosolic glutathione peroxidase genes, possess anti-apoptotic
activity, their expression appears to be unaffected in GATA-1-deficient proerythroblasts (18). Therefore, the direct anti-apoptotic target(s) of GATA-1 in proerythroblasts remains elusive.
The E4BP4 (adenovirus E4 promoter-binding protein) protein was
initially identified by its ability to recognize and repress the
adenovirus E4 promoter (19, 20) and was subsequently identified as
NF-IL3A in T cells to be capable of binding and activating the human
IL-31 promoter (21). E4BP4 is
a protein of 462 amino acids and is a member of the basic
region/leucine zipper transcription factor superfamily (19). The
basic region/leucine zipper domain in E4BP4 is highly related to the
basic region/leucine zipper domains of Caenorhabditis
elegans cell death specification protein CES-2 (22) and acute
lymphoblastic leukemia oncoprotein E2A-HLF (23, 24). The consensus
E4BP4-binding DNA sequence, i.e. (G/A)T(G/T)A(C/T)GTAA(C/T) (19), is also similar to the consensus binding sequences for CES-2 and
E2A-HLF proteins (22). Because both CES-2 and E2A-HLF were involved in
cell death regulation, it was predicted that E4BP4 might be related to
death control in hematopoietic cells. Further experiments indeed
demonstrated that this is the case. In an IL-3-dependent
pro-B cell line, Ba/F3, E4bp4 expression was activated by
the IL-3 signaling pathway, and enforced expression of E4BP4 delayed
apoptosis caused by IL-3 deprivation without promoting cell division
(25). Furthermore, overexpression of the dominant-negative form of
E4BP4 attenuated the survival response of IL-3 (26). These findings
clearly suggest that E4bp4 is one component of the
transcription network that contributes to the survival activity of
IL-3.
In this study, we tried to determine the mechanism responsible for IL-3
induction of the survival gene E4bp4. We have demonstrated that IL-3 activation of the E4bp4 gene is regulated at the
transcriptional level. Through promoter analysis we identified that a
GATA motif downstream of a major transcription initiation site is
essential for E4bp4 expression in the
IL-3-dependent Ba/F3 cell line. We also demonstrated that
GATA-binding factors, most likely GATA-1 and GATA-2, play an important
role in the transcriptional activation of E4bp4 and are
involved in the anti-apoptotic signaling of IL-3.
Cell Cultures--
Murine IL-3-dependent pro-B cell
line Ba/F3 was maintained in medium containing murine IL-3, as
described previously (27). For restimulation experiments, 100 units/ml
of recombinant IL-3 (R & D Systems, Minneapolis, MN) was added back
to cells that had been previously cultivated in low serum medium
containing no cytokine. For culturing Ba/F3 derivatives stably
overexpressing GATA factors, the regular growth medium supplemented
with 200 µg/ml of G418 was used. Bone marrow-derived
IL-3-dependent primary cells were isolated and cultured as
described previously by Rodriguez-Tarduchy et al.
(28).
Northern Blot Analysis and Nuclear Run-on Assay--
Total
cellular RNA was isolated from Ba/F3 cells and bone marrow-derived
IL-3-dependent primary cells with the Trizol reagent kit
(Invitrogen) according to a procedure recommended by the manufacturer. Equal amounts of total RNA (30 µg) from each treatment were then subjected to the standard Northern blot analysis using probes specific
to the E4bp4 or the G3pdh
(glyceraldehyde-3-phosphate dehydrogenase) gene. Specific signals on
the Northern blot were quantified either with Instant Imager (Packard
Instrument Co., Meriden, CT) or with a densitometer (Molecular
Dynamics, Sunnyvale, CA). A nuclear run-on assay to determine the rate
of transcription of the E4bp4 gene was performed essentially
as described (29), with nuclei prepared as described by Dignam et
al. (30).
PCR Cloning of Genomic DNA--
The murine E4bp4
5'-flanking region was obtained by PCR amplification of a murine
genomic library (Genome Walker kits; CLONTECH, Palo
Alto, CA) using two sets of nested primers as recommended by the
manufacturer. One set of the nested primers contains sequences complementary to the flanking adaptors and was provided by the vendor.
The other set of the nested primers with the following sequences was
designed according to the coding sequence of E4bp4: E1
primer, 5'-GCCACCTCAGCTAAGGCAGAGTTCAGC-3', and E2 primer,
5'-AGCATCTTGTCTGAGCTGCTGGTAGGA-3'. The sequencing of this
DNA fragment confirmed that it contains 1,139 bp of the 5'-flanking
sequence (GenBankTM accession number AF512511) and the
first 77 bp of the open reading frame of the murine E4bp4 cDNA.
Primer Extension Analysis and S1 Nuclease Protection--
The
transcriptional initiation site of the E4bp4 gene was
determined by both primer extension analysis and S1 nuclease protection as described (29). The PE1 primer
(5'-AGACCGGATGGAGGAGACAAATCACTTCCCCAGTCTTC-3'; see also Fig.
2A) was used both for the primer extension as well as for
the sequencing reaction. For the S1 nuclease protection assay, a
32P-labeled single-stranded DNA probe spanning the region
between Reporter Plasmids--
The 1140-bp DNA fragment of the
E4bp4 gene spanning the region between Electroporation and Luciferase Assay--
Ba/F3 cells were
transiently transfected with plasmids by electroporation using a
Bio-Rad Gene Pulser II RF module system and assayed for luciferase
activity as described previously (31). To analyze the effect of the
dominant-negative mutant of GATA-1 in the reporter gene assays, the
electroporated cells were recovered in mIL-3-containing medium for
12 h and then deprived of mIL-3 for 8 h before restimulation
of cells with mIL-3 was initiated. Three hours after mIL-3
restimulation, the cell lysates were prepared and assayed for
luciferase activity.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared according to the method described by Dignam
et al. (30). Binding reactions were performed as described
by Wadman et al. (32). An E4bp4 GATA element
(GATA probe, 5'-TTTATTGCAGATAACCCATGAAAGGCTC-3') or a 4-bp mismatched MutGATA element (MutGATA probe,
5'-TTTATTGCAagccACCCATGAAAGGCTC-3') was used in the EMSA.
The competition and supershift experiments were performed as described
previously (31). The GATA-1-containing complex was supershifted with an
anti-GATA-1 antibody (N6; Santa Cruz Biotechnology Inc.; Santa Cruz,
CA), and the GATA-2-containing complex was supershifted with a rabbit
polyclonal anti-GATA-2 antibody (H-116; Santa Cruz Biotechnology
Inc.).
Construction of Expression Plasmids--
The murine GATA-1
cDNA encompassing the entire coding region (~1.2 kb) was
synthesized by PCR amplification of a murine cDNA library
(CLONTECH) with the following two primers: sense,
5'-GGATCCGATTTTCCTGGTCTAGGG-3', and antisense,
5'-CTTAAGTCAAGAACTGAGTGGGGC-3' (33). The PCR-amplified murine GATA-1
cDNA fragment was digested with restriction enzymes BamHI and EcoRI and cloned into the
BamHI and EcoRI sites of the pCMVflag vector to
generate the expression vector pCMVflag-mGATA-1. The expression vector
pcDNA3-HA-mGATA-2 was generated by PCR amplifying the murine GATA-2
cDNA with the following two primers: sense, 5'-GAATTCGAGGTGGCTCCTGAGCAG-3', and antisense,
5'-CTCGAGCTAGCCCATGGCAGTCAC-3'; and the resultant PCR product was
subcloned into the EcoRI and XhoI sites of the
pcDNA3 vector. The dominant-negative mutant of mGATA-1 (34)
containing only the C-terminal zinc finger (amino acids 230-336) was
generated by PCR amplification of the murine GATA-1 cDNA template
with the following two primers (N-terminal primer GATA230P5,
5'-GGATCCTTGTATCACAAGATGAAT-3'; C-terminal primer GATA336P3,
5'-TCACACCATGAAGCCACCTGC-3'). The resultant DNA fragment was
subcloned into the pcDNA3-HA vector to yield the expression vector
pcDNA3-HA-mGATA-1-dn. All constructs generated via methods involving the PCR step were confirmed by sequencing to be free of base
mutations in the amplified region.
Antibodies and Western Blot Analysis--
Antibody specific for
GATA-1 was purchased from Santa Cruz Biotechnology (N6; Santa Cruz,
CA). Antibody for FLAG tag was purchased from Sigma (M2), and antibody
for hemagglutinin (HA) tag was purchased from Roche Molecular
Biochemicals (monoclonal antibody 12CA5; Mannheim, Germany). The cell
lysates were prepared and analyzed by Western blotting as described
previously (31). The specific signals were visualized by an ECL Western
blot system (Amersham Biosciences).
Quantitation of Apoptotic Cells and Cell Viability
Analysis--
The release of nucleosome into the cytosolic fraction
was used to determine the degree of cells undergoing apoptosis and was performed as described previously (35) with an enzyme-linked immunosorbent assay kit (Roche Molecular Biochemicals). The viability of cells was determined by Trypan blue exclusion staining.
Chromatin Immunoprecipitation (ChIP) Assay--
The ChIP assay
was carried out according to a published protocol (36). Briefly,
cross-linked chromatin prepared from rapid growing cells cultured in
IL-3-containing medium was sonicated to an average size of 400 bp prior
to being immunoprecipitated with either anti-GATA-1 antibody or control
rat IgG (anti-murine CD44) at 4 °C overnight. The immunoprecipitated
chromatin, after reversal of cross-linking, was PCR-amplified with
various sets of primers that either specific to the E4bp4 or
a negative control gene (mouse mcl-1) (see below). The
amplified DNA product was then resolved by agarose gel electrophoresis
or further transferred onto membrane and subjected to Southern blot
analysis using 32P-labeled E4bp4
promoter-specific oligonucleotide containing the GATA motif
(5'-CTACTTTATTGCAGATAACCCATGAAAGGC-3'). For PCR amplification of
a specific region (F1-F5; see Fig. 6A) of the
E4bp4 genomic locus, the following sets of primers were
used: F1 (nucleotides Transcriptional Regulation of E4bp4--
E4bp4 is a
survival gene known to be activated by IL-3 in a murine pro-B cell line
Ba/F3 (25). In this study, our initial goal was to understand the
mechanism by which IL-3 regulates E4bp4 gene expression. We
first examined whether IL-3 stimulation of the E4bp4
mRNA expression could also be observed in bone marrow-derived primary IL-3-dependent cells. To address this issue, a
Northern blot analysis was carried out, and the results are shown in
Fig. 1A. As in the case of
Ba/F3 cells (lanes 1-5), IL-3 stimulation of the primary
cells resulted in an increase of the E4bp4 mRNA levels
(Fig. 1A, lanes 6-8). The induction fold
(~8-fold) and kinetics (peaks at 1 h) observed with the primary
cells were also very similar to those observed with the Ba/F3 cells. To
determine whether IL-3 stimulation of E4bp4 mRNA
expression was regulated at the transcriptional level, the nuclear
run-on assay was carried out with nascent RNA probes prepared from
Ba/F3 cells with or without IL-3 stimulation. As shown in Fig.
1B, the transcription activity of E4bp4, like
that of the positive control JunB, was markedly increased
upon IL-3 stimulation, whereas transcription of either G3pdh
or Cloning of the Mouse E4bp4 Promoter Region--
To characterize
the IL-3 signaling pathway that leads to transcriptional activation of
the E4bp4 gene, the genomic DNA spanning the
E4bp4 promoter region was PCR-amplified from the
SspI-cut, adapter-tagged murine genomic DNA using a pair of
nested primers (Fig. 2A, E1
and E2) as described under "Experimental Procedures." To further
confirm the identity of the cloned E4bp4 promoter locus, other restriction enzyme-digested adaptor-tagged murine genomic DNA was
used as a template to amplify the E4bp4 promoter region using the same set of E4bp4 nested primers. All of the
resulting DNA fragments were found to overlap with each other, and the
sequence of the longest clone is shown in Fig. 2A.
To map potential transcription start site(s) in the 5'-flanking region
of E4bp4, both primer extension and S1 nuclease protection analyses were performed. In primer extension analysis (Fig.
2B), multiple potential transcriptional initiation sites
were reproducibly observed with RNA isolated from IL-3-stimulated
cells, with one major site positioned at Functional Analysis of the Mouse E4bp4 Promoter--
To further
demarcate the critical promoter sequence in the 5'-flanking region of
E4bp4, luciferase reporters driven by various regions of the
E4bp4 promoter were transiently transfected into Ba/F3
cells, and their responses to IL-3 were analyzed. As shown in Fig.
3, the 5'-flanking DNA fragment between
GATA Site Is an Essential Promoter Element of E4bp4--
Using the
FINDPATTERNS program of the Genetics Computer Group and TFsearch
program (www.cbrc.jp/research/db/TFSEARCH.html), we identified two
putative transcriptional regulatory elements, a lymphoid
transcription factor E47 binding site-like sequence at GATA-1 and GATA-2 Bind to the E4bp4 Promoter--
To further
understand the role of the GATA element in the expression of
E4bp4 in IL-3-dependent cells, an EMSA was
performed to examine whether the Ba/F3 cells contained any DNA binding
activity for this GATA site. As shown in Fig.
5, when a 32P-labeled
oligonucleotide probe containing the E4bp4 GATA sequence (see "Experimental Procedures") was incubated with the nuclear extract prepared from Ba/F3 cells, two major binding complexes that
migrated together as a doublet were detected (complexes GATA-1 and
GATA-2 are indicated by an arrow in Fig. 5, lane
1). These bands represented a specific protein binding to the GATA
site, because complex formation was diminished by the addition of
100-fold molar excess of the unlabeled wild type probe but not by
addition of the same amount of probe containing the mutant GATA site
(Fig. 5, compare lanes 5 and 6). To confirm that
GATA family proteins bound to this site, we performed EMSA in the
presence of antibodies specifically recognizing GATA-1 or GATA-2
proteins. The selection of these two antibodies in the EMSA assay was
based on the fact that both GATA-1 and GATA-2 proteins were present in
Ba/F3 cell extracts as revealed by a Western analysis (data not shown)
and that either mouse GATA-1 or GATA-2 proteins synthesized in
vitro using the TNT-coupled reticulocyte lysate system (Promega)
also bound specifically to the E4bp4 GATA element (data not
shown). As shown in Fig. 5, among the two DNA-protein complexes,
antibody specific to GATA-1 induced a supershift of the more quickly
migrating complex of the doublet (Fig. 5, lane 2) and
generated a new complex S1, whereas antibody to GATA-2 induced a
supershift of the more slowly migrating complex of the doublet (Fig. 5,
lane 3) and generated another new complex S2. Addition of
both antibodies simultaneously supershifted the GATA-1 and GATA-2
complexes (Fig. 5, lane 4). Together, these results suggest
that both GATA-1 and GATA-2 proteins specifically recognize the
E4bp4 GATA element in vitro, but these two
proteins do not seem to be present in the same complex. Furthermore, with different salt concentrations in the binding reaction, we also
identified that the GATA-2 binding complex was more sensitive to the
ionic strength than the GATA-1 binding complex (data not shown).
We next examined whether the binding of GATA factors to the
E4bp4 promoter could be detected in vivo by a
ChIP assay (see "Experimental Procedures"). For this experiment,
the cross-linked chromatin prepared from rapid growing Ba/F3 cells
cultured in IL-3-containing medium was sonicated to an average size
of ~ 400 bp (determined after cross-linking was reversed; data
not shown) and then subjected to immunoprecipitation and subsequent PCR
amplification analyses. As shown in Fig.
6B, only the chromatin
specifically pulled down by the anti-GATA-1 antibody (lane
5) but not by an isotype-matched control antibody (lane
6) contained the DNA region that could be PCR-amplified by two
E4bp4-specific primers (mE4P5-360 and mE4P3+1; see
"Experimental Procedures") and generated the predicted 360-bp DNA
fragment (i.e. the F3 fragment illustrated in Fig.
6A). Serving as a negative control under the same
conditions, the chromatin containing the mcl-1 promoter
region (between Transactivation of the E4bp4 Promoter by GATA-1 and GATA-2--
To
demonstrate whether GATA-1 and GATA-2 can transactivate the
E4bp4 promoter, the reporter plasmid p(
To further test whether binding of GATA factors are required for IL-3
induction of the E4bp4 reporter gene, the reporter gene assay was carried out in the presence of an HA-tagged GATA-1 mutant that comprises only the C-terminal zinc finger (GATA-1-dn). This mutant
was previously shown to bind to the GATA element but failed to activate
a downstream reporter gene (34). In our hands, this mutant also
inhibited GATA-1 or GATA-2 binding to the E4bp4 GATA motif
in the EMSA experiment (data not shown), we therefore predicted that
GATA-1-dn would interfere with the binding of GATA-1 or -2 to the
E4bp4 GATA site in vivo. The results shown in
Fig. 7 indicate that when the GATA-1-dn mutant was co-expressed (an
~15 kDa protein detected with an antibody to the HA tag in Fig.
7C, lane 2) with the Regulation of Endogenous E4bp4 Expression and Cell Viability by
GATA Factors--
To further investigate the role of GATA factors in
the expression of endogenous E4bp4 gene and in the viability
response of IL-3-dependent cells, we next examined whether
constitutive overexpression of either wild type GATA-1 or GATA-1-dn
would affect the E4bp4 mRNA levels as well as the
viability response of cells. For this experiment, several Ba/F3
derivatives stably overexpressing either GATA-1 or GATA-1-dn were
established and analyzed. Fig. 8 shows the result of one representative stable line from cells transfected with each expression vector. Expression of GATA-1 or GATA-1-dn in these
stable cell lines was confirmed by Western blotting with antibodies
recognizing either GATA-1 (Fig. 8A, lane 2) or
the HA tag (Fig. 8A, lane 4). To evaluate the
effect of GATA-1 or GATA-1-dn on endogenous E4bp4 mRNA
expression, Northern blot analysis was performed with total RNA
isolated from each of these stable transfectants. Fig. 8B
shows that the E4bp4 mRNA was increased ~2-fold in
GATA-1-overexpressing cells (Fig. 8B, lane 2) but
was reduced by 60% in GATA-1-dn-expressing cells (Fig. 8B,
lane 3). These results are consistent with those observed in
the transient transfection experiments with reporter gene analysis
(Fig. 7) and therefore suggest strongly that GATA factors, most likely GATA-1 and GATA-2, indeed play an important role in transcriptional regulation of the endogenous E4bp4 gene.
We next examined whether overexpression of GATA-1 or GATA-1-dn would
affect the viability response of Ba/F3 cells in medium deprived of
IL-3. As shown in Fig. 8C, upon IL-3 removal the viability of GATA-1-overexpressing cells was sustained much longer than that of
control cells transfected with an empty vector. In contrast, under the
same conditions, the viability of GATA-1-dn-expressing cells was
significantly reduced (p < 0.001) compared with that of the control cells. Next, by using the nucleosome releasing assay, we
investigated whether overexpression of GATA-1 or GATA-1-dn would affect
the number of cells undergoing apoptosis 8 h after IL-3 removal
(apoptotic index). As shown in Fig. 8D, under such experimental conditions, the apoptotic index of the GATA-1
overexpressing cells was markedly decreased, whereas that of the
GATA-1-dn overexpressing cells was significantly increased compared
with that of the control cells (compare lanes 2-4 in Fig.
8D). Taken together, our data strongly suggest that the GATA
family transcription factor plays an important role in regulating
E4bp4 expression and is a critical component of the
viability response of IL-3 in Ba/F3 hematopoietic cells.
In this study, we present evidence that the GATA motif is
essential for the expression of E4bp4 in
IL-3-dependent Ba/F3 cells. We demonstrated that mutation
of GATA motif downstream of the major transcription start site of the
E4bp4 gene impaired not only the basal promoter activity but
also the IL-3 inducibility of this gene. In most eukaryotic genes
characterized so far, a functional enhancer element relies on the
presence of an intact basal promoter. The fact that mutation of the
GATA motif nearly completely abolished the basal promoter activity of
E4b4 made it difficult to assess whether, in this case, the
GATA motif itself plays a direct role in the IL-3 inducibility of the
E4bp4 gene. On the other hand, our preliminary data
demonstrated that the DNA binding activity of GATA factors to the
E4bp4 GATA motif is significantly increased following IL-3
stimulation (data not shown). This latter result nevertheless implies
that this GATA motif is important to the IL-3 inducibility of the
E4bp4 gene. More experiments including the employment of a
heterologous promoter system will be required to further investigate
this issue.
In addition to identifying the GATA motif to be essential for
E4bp4 expression in the IL-3-dependent cells, in
this study, we further demonstrated that the GATA transcription factors
(GATA-1 and possibly GATA-2), through binding to the GATA motif, are
required for E4bp4 gene transcription and play an important
role in the viability response of IL-3 in Ba/F3 cells. This finding is
consistent with recent observations that GATA family proteins regulate
cell viability during the development of hematopoietic cells. For
instance, analysis of mouse embryonic stem cells lacking GATA-1
revealed that GATA-1 plays an important role in supporting the
viability of red blood cell precursors by suppressing apoptosis (38). GATA-2 gene targeting experiment also suggests that the anti-apoptosis function of GATA-2 is essential for the maintenance and proliferation of the early hematopoietic progenitors and stem cells (39). Experiments
of targeted disruption of the GATA-3 gene and injection of
GATA-3-deficient, lacZ-expressing ES cells into wild type
blastocysts (40, 41) suggest that GATA-3 is required for the expansion and subsequent proliferation of T cell progenitors. Although a large
number of our studies were carried out using the
IL-3-dependent Ba/F3 cell line, IL-3 induction of
E4bp4 mRNA was readily detectable in the primary bone
marrow-derived IL-3-dependent cells, suggesting that GATA
factors are likely involved in cell death regulation in
IL-3-dependent early hematopoietic progenitors.
Although GATA transcription factors suppress apoptosis during
hematopoiesis, the underlying mechanism(s) responsible for the observed
effect remains to be determined. One likely scenario is that these GATA
factors may regulate a set of GATA element-containing genes whose gene
products in turn suppress apoptosis. Several studies aiming for
identification of the anti-apoptotic targets of GATA-1 have been
reported. For example, the Bcl-2 family member, Bcl-xL, was
shown to be induced by a conditionally activated GATA-1 fusion protein
and was essential for the survival of a GATA-1-deficient erythroid cell
line (42); Bcl-2 was demonstrated to be induced by GATA-1
transcriptional factor and to be essential for
GATA-1-dependent survival against IL-6-induced apoptosis in
the murine myeloid M1 cell line (43). Although GATA-1 activation was
closely correlated with the transcription of these two Bcl-2 family
members, evidence for a role of GATA-1 in the direct transactivation of
the promoters of Bcl-xL and Bcl-2 has not been provided.
The third example is that the expression of several erythroid-specific
GATA target genes that are linked to apoptosis regulation has been
examined in GATA-1 The involvement of GATA transcription factors in transducing survival
signals of IL-3 raises the possibility that IL-3 may modulate the
structure and thus the function of GATA factors. This possibility is
supported by a prior study demonstrating that IL-3 induced rapid serine
and threonine phosphorylation of GATA-2 in Ba/F3 cells and in the human
factor-dependent cell line TF-1 (47). Our preliminary
experiments also indicated that IL-3 stimulated the phosphorylation of
GATA-1 in Ba/F3 cells (data not shown). Although our data suggest that
phosphorylation may contribute to the increased DNA binding activity of
GATA-1 in Ba/F3 cells, two conflicting results have been reported for
the role of phosphorylation in modulating the activity of GATA-1. One
is that even though phosphorylation of GATA-1 was observed during
Me2SO-induced differentiation of murine erythroid leukemia
cells, neither DNA binding nor transcriptional activity of GATA-1 was
found to be altered in this case (48). The other is that in K562 cells
the DNA binding activity of GATA-1 increased after the induction of
differentiation, and this phenomenon was sensitive to the treatment of
phosphatase (49). It remains to be determined whether IL-3-induced
phosphorylation of GATA-1 and GATA-2 plays any role in regulating the
expression of E4bp4.
Alternatively, several studies have revealed that GATA factors,
including GATA-1, GATA-2, and GATA-3, are specifically associated with
histone acetylases and that their transcriptional activity can be
activated upon acetylation (50, 51). GATA-1 acetylation is essential
for erythroid differentiation (50), whereas GATA-3 acetylation is
required for T cell survival and homing to secondary lymphoid organs
(51). Furthermore, histone deacetylase 3 can bind to GATA-2, thereby
repressing the activity of GATA-2 in the human hematopoietic KG-1 cell
line (52). These results suggest that the transcriptional activity of
GATA family factors could be reversibly regulated through the balance
of its acetylation and deacetylation. It remains to be determined
whether the IL-3 signal can modulate the acetylation status of GATA factors.
We are grateful to Drs. Hsin-Fang Yang-Yen,
Woan-Yuh Tarn, James K. Liao, and Stuart H. Orkin for critical comments
on the manuscript.
*
This work was supported in part by an intramural fund from
Academia Sinica, Taiwan (to J. J.-Y. Y.) and Grants
NSC86-2314-B-001-026 and NSC88-2314-B-001-018 from the National Science
Council of Taiwan (to J. J.-Y. Y.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Inst. of
Biomedical Sciences, Academia Sinica. No. 128, Sec. 2, Yen-Jiou-Yuan Rd., Taipei, 115 Taiwan. Tel.: 886-2-2652-3077; Fax:
886-2-2782-9142; E-mail: bmjyen@ibms.sinica.edu.tw.
Published, JBC Papers in Press, May 21, 2002, DOI 10.1074/jbc.M200924200
The abbreviations used are:
IL, interleukin;
EMSA, electrophoretic mobility shift assay;
HA, hemagglutinin;
ChIP, chromatin immunoprecipitation.
GATA Factors Are Essential for Transcription of the Survival
Gene E4bp4 and the Viability Response of Interleukin-3
in Ba/F3 Hematopoietic Cells*
§, Graduate Institute of Life Sciences,
National Defense Medical Center and the § Institute of
Biomedical Sciences, Academia Sinica, Taipei, 115 Taiwan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
274 and 24 (see Fig. 2A) was used to form a
specific DNA-RNA hybrid prior to being digested with nuclease S1. This
probe was synthesized with the 32P-labeled PE1 primer and
the denatured plasmid DNA template p(1139/+1) e4bp4-luc (see
below) using a procedure essentially as described in Ref. 29.
1139 and +1
(relative to the translational start site) was subcloned into the
SmaI and HindIII sites of a promoter-less luciferase reporter vector pGL2-basic (Promega, Madison, WI) to generate the reporter construct p(1139/+1) e4bp4-luc. The reporter constructs of p(821/+1)e4bp4-luc, p(451/+1)e4bp4-luc,
p(205/+1)e4bp4-luc, and p(1139/-205)e4bp4-luc were generated by
isolating corresponding inserts from p(1139/+1)e4bp4-luc via
digestion with appropriate restriction enzymes and subcloning them into
the SmaI/HindIII or
SmaI/NheI sites of pGL2-basic. The reporter
plasmids of p(194/+1)e4bp4-luc, p(166/+1)e4bp4-luc,
p(135/+1)e4bp4-luc, and p(205/-138)e4bp4-luc were generated by PCR
synthesizing the corresponding fragments with an appropriate pair of
primers as described below and subsequently cloning these PCR fragments
into the multiple cloning sites of the pGL-2-basic vector. The 5'
primers for making these constructs are as follows:
mE4P5(194)NheI (5'-GCTAGCTGCCCAAGGGACTCACTG-3'), mE4P5(166)NheI (5'-GCTAGCTTTATTGCAGATAACCCA-3'),
mE4P5(135)NheI (5'-GCTAGCGACAGATTTACCCTGTGC-3'), and
mE4P5(205)NheI (5'-GCTAGCACACAGCTGCCCAAGGGA-3'). The 3'
primer for the first three constructs is mE4P3(+1)HindIII (5'-AAGCTTCAGAAAGGACCTCCTCGT-3'), and the 3' primer for the last construct is mE4P3(138) BglII
(5'-AGATCTGAGCCTTTCATGGGTTAT-3'). Plasmids p(205/+1mE) e4bp4-luc,
p(205/+1mG)e4bp4-luc, and p(205/+1mEmG)e4bp4-luc were derived from
p(205/+1)e4bp4-luc by PCR-assisted, site-directed mutagenesis of each
individual sequence element as indicated in the schemes shown in Fig.
4A. Plasmid p(1139/+1mG)e4bp4-luc was generated by
replacing the region between 205 and +1 of p(1139/+1)e4bp4-luc with
a corresponding fragment from p(205/+1mG)e4bp4-luc. All of the
constructs generated via methods involving the PCR step were confirmed
by sequencing.
1020 to 724), mE4P5-1020
(5'-ATCTTAACTTTCAAGAGAGCTGTGTTTTAA-3') and mE4P3-724
(5'-TGTTAACCTGCTGTCCCACCTCTGAGGGCC-3'); F2 (nucleotides 663 to
414), mE4P5-663 (5'-GGGCAGCACTCGTGTTCTGGTCACCATTGT-3') and
mE4P3-414 (5'-ACTTGGACCCATGCAATCCTTTCGTCTAGT-3'); F3 (nucleotides 360 to +1), mE4P5-360 (5'-TCTGCTGGACCACATAGTCCAAGGCAAAGA-3') and
mE4P3+1 (5'-CAGAAAGGACCTCCTCGTCCTACAGACCGG-3'); F4 (nucleotides +122 to
+372), mE4P5+122 (5'-CAGGTGAAGATTTGCTCCTGAACGAAGGGA-3') and mE4P3+372
(5'-TAATTTCAGGGAGAGCAGCTCAGCTTTTAA-3'); F5, (nucleotides +421 to +734),
mE4P5+421 (5'-CTCAGTAATTCCACAGCTGTCTACTTTCAG-3') and mE4P3+734
(5'-TGGTAGATGGAGGTGGAATACGTGCCCCGC-3'). The primers used to amplify the
mcl-1 gene promoter (53) are as follows: mMclP5-420
(5'-ACGAGAAAGGCTAAGGCAGGACTGC-3') and mMclP3+20
(5'-CGCCGCAGGCTGAGGGGAAGGAGCG-3').
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin was unaffected by IL-3 (Fig. 1B,
compare lanes 1 and 2). We next determined
whether the stability of E4bp4 transcripts in response to
IL-3 stimulation might also be affected. To address this issue, we
measured the half-life of the E4bp4 mRNA in cells with
or without IL-3 stimulation. As illustrated in Fig. 1C, the
levels of E4bp4 mRNA in cells with or without IL-3
stimulation both decreased dramatically within 1 h after treatment
of cells with a potent transcription inhibitor, actinomycin-D (Fig.
1C). Under the same conditions no significant decline was observed with the levels of G3pdh mRNA (data not shown).
The estimated half-life of E4bp4 mRNA was around 30 min
regardless of the presence or absence of IL-3 (Fig. 1C).
These results together with those observed with the nuclear run-on
assay suggest that IL-3 stimulation of the E4bp4 mRNA
expression is mainly regulated at the transcriptional level.
View larger version (34K):
[in a new window]
Fig. 1.
Transcriptional regulation of
E4bp4 by IL-3. A, IL-3 stimulation of
E4bp4 mRNA expression. Ba/F3 (lanes 1-5) and
bone marrow-derived primary IL-3-dependent cells
(lanes 6-8) were cultured in cytokine-free medium for
8 h and restimulated with IL-3 for various times as indicated.
Total RNA was prepared and subjected to Northern blot analysis with
32P-labeled probes specific to E4bp4 or
G3pdh. The blot was visualized by autoradiography and
quantitated by densitometry or Instant Imaging system. Expression
levels of E4bp4 mRNA were normalized to those of
G3pdh mRNA, and the fold of induction was expressed as a
relative number to the E4bp4 mRNA level at the initial
time point (lane 1). B, nuclear run-on analysis
of E4bp4, JunB, G3pdh, and
-Actin genes in Ba/F3 cells. Nuclei prepared from Ba/F3
cells deprived (; lane 1) or restimulated (+; lane
2) with IL-3 for 1 h after the initial deprivation of IL-3
for 8 h were subjected to nuclear run-on assay as described under
"Experimental Procedures." Two housekeeping genes, G3pdh
and -Actin, were used as internal hybridization controls.
The JunB gene, which is known to be induced by IL-3, was
used as a positive control. C, half-lives of
E4bp4 mRNA in cells cultured with or without IL-3. Ba/F3
cells were cultured in the absence (
) or presence (
) of IL-3 for
1 h prior to treatment of actinomycin D (50 µg/ml). At various
times after actinomycin D treatment, total RNA was isolated from each
group and subjected to Northern blot analysis with
32P-labeled probes specific to E4bp4 or
G3pdh. Quantitation and normalization of the RNA levels were
performed as described above for A. The level of
E4bp4 mRNA at each time point was converted to the
percentage of the initial E4bp4 mRNA level and plotted
to determine the half-lives. The values shown here are the averages of
two independent experiments.
View larger version (44K):
[in a new window]
Fig. 2.
Mapping of the transcriptional initiation
site of the murine E4bp4 gene. A,
nucleotide sequence of the 5'-flanking region of the murine
E4bp4 gene locus. The nucleotides are numbered relative to
the translation initiation codon ATG at +1. The positions of two 3'
primers (E1 and E2) used for the nested PCR cloning of the promoter
DNA, the PE1 primer, used for primer extension and S1 nuclease
protection analyses, and restriction sites for ScaI,
NsiI, DraI, and PvuII are
underlined. The first nucleotide (boxed T) of the
sequence is the last nucleotide of the SspI site used for
the adapter ligation step (see "Experimental Procedures"). The
major transcription initiation site mapped by primer extension at 167
is indicated by a solid arrow, and that mapped by S1
nuclease protection at 158 is indicated by a dashed arrow.
B, primer extension analysis of E4bp4 mRNA.
Total RNA (100 µg) isolated from Ba/F3 cells grown in the absence
() or presence (+) of IL-3, or control yeast RNA (200 µg) was
subjected to primer extension analysis with the PE1 primer. The
extension products were resolved by electrophoresis in a sequencing
gel. Sequencing reaction products (C, T, A, and G) of the 5'-flanking
promoter region with the same primer were run in the same gel to help
assign the transcription initiation site. The major initiation site at
167 is indicated by an arrow on the left.
C, S1 nuclease protection analysis of E4bp4
mRNA. The same RNA samples as described for B were
hybridized with a single-stranded 32P-labeled probe
synthesized with the PE1 primer (see "Experimental Procedures").
After digestion with S1 nuclease, the reaction products were resolved
in a sequencing gel. To help assign the transcription initiation site,
the sequencing reaction products as described for B were
loaded onto the same gel. An undigested free probe was also included as
a reference size marker. The mapped transcription initiation site is
indicated at 158 by an arrow.
167 (relative to the
translation start site) and a minor one at 136 (Fig. 2B,
lanes 2 and 3). The minor site at 136 was
likely to be an artifact generated because of a secondary structure
formed at the 5' end of the E4bp4 transcript, because this
band became more obscure when an identical experiment was repeated at
42 °C (data not shown). On the other hand, a single major
transcriptional initiation site at 158 was mapped when the S1
nuclease protection assay was carried out with RNA isolated from
IL-3-stimulated cells (Fig. 2C, lane 1). Under
the same conditions, neither RNA from IL-3-deprived cells nor yeast
control RNA gave any extended products (Fig. 2B, lanes
1 and 2) or protected bands (Fig. 2C,
lanes 2 and 3). An A/T-rich sequence between
167 and 158, which tends to cause breathing of double-stranded DNA
and shortening of the protected fragments in the exonuclease reaction, might have accounted for the discrepancy in mapping the transcription initiation site by these two methods. The sequence surrounding the
transcription initiation site at 167
(5'-C-T-A+1-C-T-T-T-A-3') is highly homologous to the
consensus of the initiator element of eukaryotic genes
(5'-Y-Y-A+1-N-T/A-Y-Y-Y-3') (37), suggesting that the
nucleotide at 167 is the major transcription initiation site utilized
in vivo.
1139 to +1, when placed in front of the promoter-less pGL2-basic
luciferase vector, exhibited some basal promoter activity without the
stimulation of IL-3 (Fig. 3B, compare pGL2-basic
and 1139/+1). Exposure of cells transfected with this
reporter to IL-3 resulted in an ~4-fold increase in luciferase
activity (Fig. 3), suggesting that there is a basal promoter as well as
an IL-3 response element present in this region. Reporters containing
various 5' deletions between 1139 and 205 still retained nearly
full inducibility by IL-3. However, further 5' deletions into
nucleotide 194 resulted into the decrease of induction fold to ~2.6
(Fig. 3A). Furthermore, the 166/+1 and the 205/138
constructs that lack the potential initiator consensus A at 167 or
the sequence downstream of 138, respectively, exerted a basal
promoter activity very similar to that of the other constructs
mentioned above (Fig. 3B). Both of the constructs still
responded to IL-3 induction, although their induction responses were
decreased to ~2-fold. On the other hand, the DNA fragment spanning
either between 135 and +1 or between 1139 and 205 exerted a much
weaker basal promoter activity as compared with others mentioned above.
Furthermore, neither of these latter two reporters responded to IL-3
stimulation. Taken together, these results suggest that the major
promoter element as well as the minimal IL-3 response element of
E4bp4 reside within the region between 205 and 138. Of
note, the IL-3 inducibility of any of the responsive E4bp4
reporters examined above was not as good as that of the endogenous
gene, suggesting either that the DNA element located further upstream
of 1139 in the E4bp4 promoter plays a role in this process
or that a unknown sequence element(s) present in the reporter vector
might have attenuated the full response.
View larger version (26K):
[in a new window]
Fig. 3.
IL-3 activates the E4bp4
promoter. A, schematic representation of
luciferase reporter genes driven by various regions of the
E4bp4 promoter. The E4bp4 promoter region between
1139 and +1 is shown as a solid line, and the
transcription initiation site, mapped by the primer extension
(solid arrow) or by the S1 nuclease protection (dashed
arrow) assay, is as indicated. The luciferase reporter gene is
shown as an open box with its initiation codon (ATG)
indicated as +1. B, IL-3 inducibility of the
E4bp4 promoter-luciferase constructs. Ba/F3 cells
transfected with various luciferase reporters were incubated for
12 h in the absence or presence of IL-3 (100 units/ml), after
which cell lysates were prepared and analyzed for luciferase activity.
The data shown here are from one representative assay from four
independent experiments done in duplicate with very similar results.
The IL-3 inducibility (shown as mean fold induction ± standard
deviation; n = 4) of each construct is summarized in
A as indicated.
195 and a
GATA-like site at 157, within the murine E4bp4 promoter
(Fig. 4A). A sequence
alignment between the human and mouse E4bp4 promoters
revealed that although the E47 motif was not identified in the human
sequence, the GATA motif was highly conserved between these two species
(Fig. 4A). To determine whether these sequence motifs are
essential for the basal promoter activity of E4bp4 and/or
IL-3 inducibility, we introduced clustered point mutations into either
the E47 site alone (205/+1mE), the GATA site alone (205/+1mG and
1139/+1mG), or both sites (205/+1mEmG) (Fig. 4B,
left panel) with the use of PCR and then analyzed the effect
of these mutations on promoter activity. Mutations on the GATA site not
only markedly decreased the basal promoter activity but also completely
abolished the inducibility of IL-3 (Fig. 4B, right
panel), whereas mutations on the E47 site had no effect on either
response of these reporters (Fig. 4B, right
panel). This result is consistent with the finding that the GATA
site, but not the E47 site, is conserved between the human and mouse genes and therefore suggests that the GATA site plays an essential role
in the expression of E4bp4 in IL-3-dependent
Ba/F3 cells.
View larger version (31K):
[in a new window]
Fig. 4.
Identification of promoter elements required
for E4bp4 expression in Ba/F3 cells.
A, sequence alignment of the 5'-flanking region of the human
and mouse E4bp4 genes. The top and bottom
strands represent sequences from the human and mouse genes,
respectively. Both E47 and GATA motifs are shown in bold
type, and the sequences of mutant E47 (mE) and GATA
sites (mG) are shown below each wild type
sequence. B, the GATA element is essential for
E4bp4 promoter activity in the presence or absence of IL-3.
Ba/F3 cells transfected with various reporters as schematically
indicated were processed and analyzed as described in the legend to
Fig. 3. Very similar results were obtained from four independent
experiments done in duplicate. The data shown here are from one
representative assay. The × symbol on the E47
(square) or GATA (oval) motif indicates a
mutation of that site as described in A.
View larger version (40K):
[in a new window]
Fig. 5.
Binding of GATA-1 and -2 to the
E4bp4 GATA element in vitro.
Nuclear extracts of rapidly growing Ba/F3 cells were incubated with a
32P-labeled oligonucleotide containing the E4bp4
GATA sequence. The binding reactions were performed in the absence
(lane 1) or presence of 100-fold molar excess of unlabeled
wild type (WT, lane 5) or mutant oligonucleotide
(Mut, lane 6) as a competitor (Comp),
in the presence of antibodies to GATA-1 (lane 2) or GATA-2
(lane 3), or both (lane 4). A more quickly
migrating complex is indicated by the arrow labeled GATA-1,
and a more slowly migrating one is indicated as GATA-2. S-1
and S-2 denote the GATA complexes supershifted by an
antibody (Ab) specific to GATA-1 and GATA-2,
respectively.
420 and +20) that lacks any obvious GATA-binding site
(53) was not pulled down by the GATA-1 antibody (Fig. 6B,
lane 2). The amplification of both predicted sized DNA
fragments, 360 bp for E4bp4 (Fig. 6B, lane
4) and 440 bp for mcl-1 (Fig. 6B, lane
1), from the chromatin fraction prior to the immunoprecipitation
step of the experimental procedure further supported the specificity of
this type of experiment. The 360-bp DNA fragments were also verified to
be specific to the E4bp4 gene by a Southern analysis with a
32P-labeled internal probe containing the GATA-binding
motif (Fig. 6B, bottom panel). To further confirm
that the amplification of the 360-bp DNA fragment in the experiment
shown in Fig. 6B (lane 5) was indeed due to the
in vivo binding of GATA-1 to the E4bp4 promoter
region between 360 and +1, an identical ChIP assay but with four
other sets of primers that would each amplify a specific flanking
region of the F3 fragment (Fig. 6A, F1,
F2, F4, and F5) was carried out. As
shown in Fig. 6C, whereas all five DNA fragments (F1-F5) were amplified from the input chromatin prior to
the immunoprecipitation step (lanes 1, 3,
5, 7, and 9), only the F3 fragment was
markedly amplified from the chromatin pulled down by the GATA-1
antibody (compare lane 6 with lanes 2,
4, 8, and 10). Of note, a very low level of the F2 fragment was reproducibly amplified (lane
4). Although under our experimental conditions the specific
amplification from a trace amount of a longer-sized chromatin (>500
bp) that contains the F2 region and the GATA site at position 157
could not be ruled out, this result suggests that GATA-1 may also bind to other weaker binding sites present within the F2 fragment
(e.g. the GATA-like sites at positions 607, 572, and
546). Because of the lack of a good antibody for immunoprecipitating
the GATA-2 complex in this type of assay, we were unable to address
whether GATA-2 bound to the E4bp4 GATA site in
vivo. In any event, these results suggest that the GATA family
member GATA-1 indeed efficiently associates with the endogenous
E4bp4 promoter in vivo and potentially plays an
important role in IL-3 regulation of E4bp4 expression.
View larger version (24K):
[in a new window]
Fig. 6.
Analysis of the in vivo
binding of GATA-1 to the E4bp4 promoter by the
ChIP assay. A, schematic representation of the
E4bp4 promoter region from 1020 to +734. The GATA motif at
157 is indicated as an open oval. The locations of F1-F5
PCR-amplified DNA fragments (see "Experimental Procedures") were as
indicated. B, specific precipitation of the E4bp4
promoter by the anti-GATA-1 antibody. Cross-linked chromatin was
isolated from Ba/F3 cells and immunoprecipitated (IP) with
either anti-GATA-1 (lanes 2 and 5) or control rat
IgG (anti-murine CD44, lanes 3 and 6). The input
(lanes 1 and 4) or the immunoprecipitated
chromatin (lanes 2, 3, 5, and
6), after reversal of cross-linking, was PCR-amplified with
E4bp4 primers, mE4P5-360 and mE4P3+1 (lanes
4-6) or primers (mMclP5-420 and mMclP3+20) that are specific to
the Mcl-1 promoter (lanes 1-3). The amplified
DNA products were resolved in an agarose gel (upper panel)
and subsequently subjected to Southern blot analysis using
32P-labeled E4bp4 promoter specific
oligonucleotides containing the GATA motif (lower panel).
C, specific interaction of GATA-1 with the E4bp4
GATA site. The input (lanes 1, 3, 5,
7, and 9) or the chromatin immunoprecipitated by
the GATA-1 antibody (lanes 2, 4, 6,
8, and 10) was processed and analyzed as
described for B except that various sets of primers that
would yield the F1-F5 fragments (A) as indicated were used
during the PCR amplification step. The PCR products were analyzed by
agarose gel electrophoresis.
1139/+1)e4bp4-luc
was co-transfected into Ba/F3 cells with the expression plasmids
encoding either FLAG epitope-tagged mouse GATA-1 or HA epitope-tagged
mouse GATA-2. Expression of ectopic GATA-1 and GATA-2 was confirmed by
Western blot analysis with antibodies either to the FLAG epitope or to
the HA epitope, both of which recognized a protein with predicted size
in cells transfected with the expression plasmid (Fig.
7A, lanes 2 and
4) but not in cells transfected with the empty vector (Fig.
7A, lanes 1 and 3). Without IL-3
stimulation, co-transfection with the GATA-1 expression plasmid alone
induced an ~8-10-fold increase in luciferase activity over that with
the empty vector pcDNA3 (Fig. 7B), whereas under the
same conditions, the GATA-2 protein did not seem to have any
significant transactivating activity. On the other hand, in the
presence of IL-3 signal, whereas a similar fold of transactivation was
still observed with the GATA-1 expression vector, a nearly 3-fold
activation of the 1139/+1 reporter began to be detected with the
GATA-2 construct. The lack of any transactivation activity of either
GATA-1 or GATA-2 on the same reporter with a mutant GATA site further
confirmed the importance of an intact GATA element in this experiment.
The inability of GATA-2 to transactivate the E4bp4 promoter
without the cytokine signal is not due to the presence of a HA tag in
this protein, because a similar result was obtained when a tag-less
murine GATA-2 was used to repeat this experiment (data not shown).
Thus, GATA-1 and GATA-2 appeared to transactivate the E4bp4
promoter through two slightly different mechanisms. Together, these
data suggest that GATA-1 and GATA-2 regulate the expression of
E4bp4 through a specific interaction with the GATA motif
downstream of the major transcription start site.
View larger version (31K):
[in a new window]
Fig. 7.
GATA factors regulate E4bp4
reporter activities in IL-3-dependent cells.
A, ectopic overexpression of GATA-1 and -2 in Ba/F3 cells.
The lysates from cells stably transfected with an expression plasmid
either encoding FLAG-tagged GATA-1 (lane 2) or HA-tagged
GATA-2 (lane 4) or with the corresponding empty vector
(V, lanes 1 and 3) were analyzed by
Western blot using anti-FLAG ( 
-FLAG) or anti-HA
(-HA) antibodies as indicated. The ectopically
overexpressed GATA-1 and GATA-2 proteins are indicated by
arrows. B, GATA-1 and GATA-2 transactivate the
E4bp4 reporters in a slightly different manner. Ba/F3 cells
were transfected with the reporter plasmid p(1139/+1)e4bp4-luc or
p(1139/+1mG)e4bp4-luc along with the GATA-1, GATA-2 or the control
vector as indicated. 12 h after transfection, the cells were
deprived of IL-3 for 8 h prior to restimulation with or without
IL-3 for 3 more hours. After each treatment, the cell lysates were
prepared and analyzed for luciferase activity. The data shown are the
means ± S.D. from two independent experiments done in duplicate.
C, expression of GATA-1-dn in Ba/F3 cells. The lysates
prepared from cells transfected either with an expression plasmid
encoding HA-tagged GATA-1-dn (lane 2) or with the
corresponding empty vector (lane 1) were subjected to
Western blot analysis with an antibody to the HA tag. The HA-tagged
GATA-1-dn is indicated by an arrow. D, inhibition
of IL-3-induced expression of the E4bp4 reporter gene by
GATA-1-dn. Ba/F3 cells were co-transfected with p(1139/+1) e4bp4-luc
and various combinations of the expression plasmid encoding HA-tagged
GATA-1-dn and an empty vector as indicated. The transfected cells were
then cultured either in the absence (lane 1) or the presence
of IL-3 (lanes 2-4) for 12 h before their cell lysates
were analyzed for luciferase activities. The data are the means ± S.D. from three independent experiments performed in duplicate.
1139/+1 reporter, the IL-3
inducibility of this reporter was inhibited by GATA-1-dn in a
dosage-dependent manner (Fig. 7D, lanes
3 and 4). This result again supports the notion that binding of the GATA family factors to the GATA motif is important for
E4bp4 expression in IL-3-dependent Ba/F3 cells.
View larger version (35K):
[in a new window]
Fig. 8.
Ectopic overexpression of GATA-1 or GATA-1-dn
affects the endogenous levels of E4bp4 mRNA and
the viability of Ba/F3 cells. A, establishment of Ba/F3
derivatives stably overexpressing GATA-1 or GATA-1-dn. The lysates
prepared from cells stably transfected with an expression plasmid
encoding either FLAG-tagged GATA-1 (lane 2) or HA-tagged
GATA-1-dn (lane 4) or with the corresponding empty vector
(V, lanes 1 and 3) were analyzed by
Western blot using antibodies specific to GATA-1
( -GATA-1) or the HA tag (-HA). The
arrows point to the ectopically expressed GATA-1 or
GATA-1-dn proteins. B, overexpression of GATA-1 or GATA-1-dn
influences the endogenous levels of E4bp4 mRNA. Total
RNA prepared from the control cells (lane 1) or from cells
stably overexpressing GATA-1 (lane 2) or GATA-1-dn
(lane 3) was subjected to Northern blot analysis using
cDNA probes specific to E4bp4 or G3pdh as
indicated. The relative levels of E4bp4 mRNA as compared
with that of vector control cells is indicated at the
bottom. Shown here is a representative set of data from two
independent experiments. C, ectopically overexpressed GATA-1
increases but GATA-1-dn decreases cell viability in IL-3-free medium.
Control cells (Vector, white bars) or cells
overexpressing GATA-1 (solid bars) or GATA-1-dn
(hatched bars) were deprived of IL-3 for various times as
indicated, and the number of viable cells from each treatment was
determined by a trypan blue exclusion method. The number of viable
cells of each cell line at the initial time point was set as 100%, and
those at various time points after IL-3 removal were plotted as a
relative percentage to that at the initial time point. The data are the
means ± S.D. from three independent experiments performed in
duplicate. The difference between control and GATA-1-dn overexpressing
cells is statistically significant. *, 0.001 < p < 0.01. D, overexpressed GATA-1 protects but GATA-1-dn
accelerates IL-3 deprivation-induced apoptosis in Ba/F3 cells. The
cells as indicated were deprived of IL-3 for 8 h, and the number
of cells undergoing apoptosis (apoptotic index) was determined by
nucleosome releasing assay as described under "Experimental
Procedures." The optical reading of this assay was taken as apoptotic
index in arbitrary units. The data are the means ± S.D. from two
independent experiments performed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ proerythroblasts. But none of them seems to be
directly responsible for the defect in apoptosis suppression (38). In contrast, in this study we demonstrated that expression of the newly
identified survival gene E4bp4 was very tightly regulated by
the GATA transcriptional factors through a GATA element in its
promoter. Recent studies indicated that E4bp4 mRNA was
highly expressed during IL-4-stimulated B cell differentiation (44), as
well as during differentiation of both CD4+ and CD8+ type 1 and type 2 T cells (45). These observations raised the possibility that E4bp4 may
contribute to the GATA-dependent survival effect during B
cell and T cell development (38-41). Furthermore, E4bp4 transcripts have a wide tissue distribution with a particularly high
expression level in peripheral blood leukocytes, testis, skeletal
muscle, placenta, heart, prostate, ovary, and lung (46). Interestingly,
high level expression of GATA-1 or GATA-2 has been reported in
peripheral blood leukocytes and testis (2-4, 7, 8), and other GATA
family members are preferentially expressed in tissues like heart,
prostate, ovary, and lung (15). Thus, it would be interesting to
examine whether the E4bp4 gene is also a direct target of
GATA family members (either hematopoietic or nonhematopoietic specific
or both) in the tissues mentioned above.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Martin, D. I.,
and Orkin, S. H.
(1990)
Genes Dev.
4,
1886-1898 2.
Yamamoto, M., Ko, L. J.,
Leonard, M. W.,
Beug, H.,
Orkin, S. H.,
and Engel, J. D.
(1990)
Genes Dev.
10,
1650-1662
3.
Martin, D. I.,
Zon, L. I.,
Mutter, G.,
and Orkin, S. H.
(1990)
Nature
344,
4444-4447
4.
Romeo, P. H.,
Prandini, M. H.,
Joulin, V.,
Mignotte, V.,
Prenant, M.,
Vainchenker, W.,
Marguerie, G.,
and Uzan, G.
(1990)
Nature
344,
447-449[CrossRef][Medline]
[Order article via Infotrieve]
5.
Sposi, N. M.,
Zon, L. I.,
Care, A.,
Valtieri, M.,
Testa, U.,
Gabbianelli, M.,
Mariani, G.,
Bottero, L.,
Mather, C.,
Orkin, S. H.,
and Peschle, C.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
6353-6357 6.
Leonard, M.,
Brice, M.,
Engel, J. D.,
and Papayannopoulou, T.
(1993)
Blood
82,
1071-1079 7.
Ito, E.,
Toki, T.,
Ishihara, H.,
Ohtani, H., Gu, L.,
Yokoyama, M.,
Engel, J. D.,
and Yamamoto, M.
(1993)
Nature
362,
466-468[CrossRef][Medline]
[Order article via Infotrieve]
8.
Yomogida, K.,
Ohtani, H.,
Harigae, H.,
Ito, E.,
Nishimune, Y.,
Engel, J. D.,
and Yamamoto, M.
(1994)
Development
120,
1759-1766[Abstract]
9.
Zon, L. I.,
Yamaguchi, Y.,
Yee, K.,
Albee, E. A.,
Kimura, A.,
Bennett, J. C.,
Orkin, S. H.,
and Ackerman, S. J.
(1993)
Blood
81,
3234-3241 10.
Dorfman, D. M.,
Wilson, D. B.,
Bruns, G. A.,
and Orkin, S. H.
(1992)
J. Biol. Chem.
267,
1279-1285 11.
Wilson, D. B.,
Dorfman, D. M.,
and Orkin, S. H.
(1990)
Mol. Cell. Biol.
10,
4854-4862 12.
George, K. M.,
Leonard, M. W.,
Roth, M. E.,
Lieuw, K. H.,
Kioussis, D.,
Grosveld, F.,
and Engel, J. D.
(1994)
Development
120,
2673-2686 13.
Kornhauser, J. M.,
Leonard, M. W.,
Yamamoto, M.,
LaVail, J. H.,
Mayo, K. E.,
and Engel, J. D.
(1994)
Mol. Brain Res.
23,
100-110[Medline]
[Order article via Infotrieve]
14.
Orkin, S. H.
(1992)
Blood
80,
575-581 15.
Weiss, M. J.,
and Orkin, S. H.
(1995)
Exp. Hematol.
23,
99-107[Medline]
[Order article via Infotrieve]
16.
Pevny, L.,
Simon, M. C.,
Robertson, E.,
Klein, W. H.,
Tsai, S. F.,
D'Agati, V.,
Orkin, S. H.,
and Costantini, F.
(1991)
Nature
349,
257-260[CrossRef][Medline]
[Order article via Infotrieve]
17.
Tsai, F. Y.,
Keller, G.,
Kuo, F. C.,
Weiss, M.,
Chen, J.,
Rosenblatt, M.,
Alt, F. W.,
and Orkin, S. H.
(1994)
Nature
371,
221-226[CrossRef][Medline]
[Order article via Infotrieve]
18.
Weiss, M. J.,
Keller, G.,
and Orkin, S. H.
(1994)
Genes Dev.
8,
1184-1197 19.
Cowell, I. G.,
Skinner, A.,
and Hurst, H. C.
(1992)
Mol. Cell. Biol.
12,
3070-3077 20.
Cowell, I. G.,
and Hurst, H. C.
(1994)
Nucleic Acids Res.
22,
59-65 21.
Zhang, W.,
Zhang, J.,
Kornuc, M.,
Kwan, K.,
Frank, R.,
and Nimer, S. D.
(1995)
Mol. Cell. Biol.
15,
6055-6063[Abstract]
22.
Metzstein, M. M.,
Hengartner, M. O.,
Tsung, N.,
Ellis, R. E.,
and Horvitz, H. R.
(1996)
Nature
382,
545-547[CrossRef][Medline]
[Order article via Infotrieve]
23.
Hunger, S. P.,
Ohyashiki, K.,
Toyama, K.,
and Cleary, M. L.
(1992)
Genes Dev.
6,
1608-1620 24.
Inaba, T.,
Roberts, W. M.,
Shapiro, L. H.,
Jolly, K. W.,
Raimondi, S. C.,
Smith, S. D.,
and Look, A. T.
(1992)
Science
257,
531-534 25.
Ikushima, S.,
Inukai, T.,
Inaba, T.,
Nimer, S. D.,
Cleveland, J. L.,
and Look, A. T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2609-2614 26.
Kuribara, R.,
Kinoshita, T.,
Miyajima, A.,
Shinjyo, T.,
Yoshihara, T.,
Inukai, T.,
Ozawa, K.,
Look, A. T.,
and Inaba, T.
(1999)
Mol. Cell. Biol.
19,
2754-2762 27.
Lee, S. F.,
Huang, H. M.,
Chao, J. R.,
Lin, S.,
Yang-Yen, H. F.,
and Yen, J. J.
(1999)
Mol. Cell. Biol.
19,
7399-7409 28.
Rodriguez-Tarduchy, G.,
Collins, M.,
and Lopez-Rivas, A.
(1990)
EMBO J.
9,
2997-3002[Medline]
[Order article via Infotrieve]
29.
Sambrook, J.,
and Russell, D. W.
(2001)
Molecular Cloning: A Laboratory Manual
, 3rd Ed.
, pp. 17.23-17.29, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
30.
Dignam, J. D.,
Lebovitz, R. M.,
and Roeder, R. G.
(1983)
Nucleic Acids Res.
11,
1475-1489 31.
Chen, W., Yu, Y. L.,
Lee, S. F.,
Chiang, Y. J.,
Chao, J. R.,
Huang, J. H.,
Chiong, J. H.,
Huang, C. J.,
Lai, M. Z.,
Yang-Yen, H. F.,
and Yen, J. J.
(2001)
Mol. Cell. Biol.
21,
4636-4646 32.
Wadman, I. A.,
Osada, H.,
Grutz, G. G.,
Agulnick, A. D.,
Westphal, H.,
Forster, A.,
and Rabbitts, T. H.
(1997)
EMBO J.
16,
3145-3157[CrossRef][Medline]
[Order article via Infotrieve]
33.
Tsai, S. F.,
Martin, D. I.,
Zon, L. I.,
D'Andrea, A. D.,
Wong, G. G.,
and Orkin, S. H.
(1989)
Nature
339,
446-451[CrossRef][Medline]
[Order article via Infotrieve]
34.
Visvader, J. E.,
Crossley, M.,
Hill, J.,
Orkin, S. H.,
and Adams, J. M.
(1995)
Mol. Cell. Biol.
15,
634-641[Abstract]
35.
Huang, H. M., Li, J. C.,
Hsieh, Y. C.,
Yang-Yen, H. F.,
and Yen, J. J.
(1999)
Blood
93,
2569-2577 36.
Saccani, S.,
Pantano, S.,
and Natoli, G.
(2001)
J. Exp. Med.
193,
1351-1359 37.
Smale, S. T.,
Jain, A.,
Kaufmann, J.,
Emami, K. H., Lo, K.,
and Garraway, I. P.
(1998)
Cold Spring Harb. Symp. Quant. Biol.
63,
21-31[CrossRef][Medline]
[Order article via Infotrieve]
38.
Weiss, M. J.,
and Orkin, S. H.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
9623-9627 39.
Tsai, F. Y.,
and Orkin, S. H.
(1997)
Blood
89,
3636-3643 40.
Pandolfi, P. P.,
Roth, M. E.,
Karis, A.,
Leonard, M. W.,
Dzierzak, E.,
Grosveld, F. G.,
Engel, J. D.,
and Lindenbaum, M. H.
(1995)
Nat. Genet.
11,
40-44[CrossRef][Medline]
[Order article via Infotrieve]
41.
Hendriks, R. W.,
Nawijn, M. C.,
Engel, J. D.,
van Doorninck, H.,
Grosveld, F.,
and Karis, A.
(1999)
Eur. J. Immunol.
29,
1912-1918[CrossRef][Medline]
[Order article via Infotrieve]
42.
Gregory, T., Yu, C., Ma, A.,
Orkin, S. H.,
Blobel, G. A.,
and Weiss, M. J.
(1999)
Blood
94,
87-96 43.
Tanaka, H.,
Matsumura, I.,
Nakajima, K.,
Daino, H.,
Sonoyama, J.,
Yoshida, H.,
Oritani, K.,
Machii, T.,
Yamamoto, M.,
Hirano, T.,
and Kanakura, Y.
(2000)
Blood
95,
1264-1273 44.
Chu, C. C.,
and Paul, W. E.
(1998)
Mol. Immunol.
35,
487-502[CrossRef][Medline]
[Order article via Infotrieve]
45.
Chtanova, T.,
Kemp, R. A.,
Sutherland, A. P.,
Ronchese, F.,
and Mackay, C. R.
(2001)
J. Immunol.
167,
3057-3063 46.
Lai, C. K.,
and Ting, L. P.
(1999)
J. Virol.
73,
3197-3209 47.
Towatari, M.,
May, G. E.,
Marais, R.,
Perkins, G. R.,
Marshall, C. J.,
Cowley, S.,
and Enver, T.
(1995)
J. Biol. Chem.
270,
4101-4107 48.
Crossley, M.,
and Orkin, S. H.
(1994)
J. Biol. Chem.
269,
16589-16596 49.
Partington, G. A.,
and Patient, R. K.
(1999)
Nucleic Acids Res.
27,
1168-1175 50.
Boyes, J.,
Byfield, P.,
Nakatani, Y.,
and Ogryzko, V.
(1998)
Nature
396,
594-598[CrossRef][Medline]
[Order article via Infotrieve]
51.
Yamagata, T.,
Mitani, K.,
Oda, H.,
Suzuki, T.,
Honda, H.,
Asai, T.,
Maki, K.,
Nakamoto, T.,
and Hirai, H.
(2000)
EMBO J.
19,
4676-4687[CrossRef][Medline]
[Order article via Infotrieve]
52.
Ozawa, Y.,
Towatari, M.,
Tsuzuki, S.,
Hayakawa, F.,
Maeda, T.,
Miyata, Y.,
Tanimoto, M.,
and Saito, H.
(2001)
Blood
98,
2116-2123 53.
Chao, J. R.,
Wang, J. M.,
Lee, S. F.,
Peng, H. W.,
Lin, Y. H.,
Chou, C. H., Li, J. C.,
Huang, H. M.,
Chou, C. K.,
Kuo, M. L.,
Yen, J. J. Y.,
and Yang-Yen, H. F.
(1998)
Mol. Cell. Biol.
18,
4883-4898
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. T. G. Burgess, C. Shen, L. A. Ferguson, G. T. O'Neill, K. Docherty, N. Hunter, and W. Goldmann Identification of Adjacent Binding Sites for the YY1 and E4BP4 Transcription Factors in the Ovine PrP (Prion) Gene Promoter J. Biol. Chem., March 13, 2009; 284(11): 6716 - 6724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Silvestris, P. Cafforio, M. De Matteo, N. Calvani, M. A. Frassanito, and F. Dammacco Negative Regulation of the Osteoblast Function in Multiple Myeloma through the Repressor Gene E4BP4 Activated by Malignant Plasma Cells Clin. Cancer Res., October 1, 2008; 14(19): 6081 - 6091. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-L. Yu, Y.-J. Chiang, Y.-C. Chen, M. Papetti, C.-G. Juo, A. I. Skoultchi, and J. J. Y. Yen MAPK-mediated Phosphorylation of GATA-1 Promotes Bcl-XL Expression and Cell Survival J. Biol. Chem., August 19, 2005; 280(33): 29533 - 29542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Greenbaum, A. S. Lazorchak, and Y. Zhuang Differential Functions for the Transcription Factor E2A in Positive and Negative Gene Regulation in Pre-B Lymphocytes J. Biol. Chem., October 22, 2004; 279(43): 45028 - 45035. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K.-W. Ling, K. Ottersbach, J. P. van Hamburg, A. Oziemlak, F.-Y. Tsai, S. H. Orkin, R. Ploemacher, R. W. Hendriks, and E. Dzierzak GATA-2 Plays Two Functionally Distinct Roles during the Ontogeny of Hematopoietic Stem Cells J. Exp. Med., October 4, 2004; 200(7): 871 - 882. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. C. Ozkurt, F. Q. Pirih, and S. Tetradis Parathyroid Hormone Induces E4bp4 Messenger Ribonucleic Acid Expression Primarily through Cyclic Adenosine 3',5'-Monophosphate Signaling in Osteoblasts Endocrinology, August 1, 2004; 145(8): 3696 - 3703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Chen, R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, and R. Lahesmaa Identification of Novel IL-4/Stat6-Regulated Genes in T Lymphocytes J. Immunol., October 1, 2003; 171(7): 3627 - 3635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. C. Ozkurt and S. Tetradis Parathyroid Hormone-induced E4BP4/NFIL3 Down-regulates Transcription in Osteoblasts J. Biol. Chem., July 11, 2003; 278(29): 26803 - 26809. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |