Cooperative Interaction of EWS with CREB-binding Protein
Selectively Activates Hepatocyte Nuclear Factor 4-mediated
Transcription*
Natsumi
Araya
§,
Keiko
Hirota
§,
Yoko
Shimamoto
§,
Makoto
Miyagishi
§,
Eisaku
Yoshida§,
Junji
Ishida
§,
Setsuko
Kaneko¶,
Michio
Kaneko¶,
Toshihiro
Nakajima
§, and
Akiyoshi
Fukamizu
§**
From the
Center for Tsukuba Advanced Research
Alliance, Aspect of Functional Genomic Biology, the
§ Institute of Applied Biochemistry, Tsukuba, Ibaraki
305-8577, and the ¶ Department of Pediatric Surgery, Institute of
Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
Received for publication, October 7, 2002, and in revised form, November 22, 2002
 |
ABSTRACT |
The EWS gene when
fused to transcription factors such as the ETS family
ATF-1, Wilms' tumor-1, and nuclear
orphan receptors upon chromosomal translocation is thought to
contribute the development of Ewing sarcoma and several malignant
tumors. Although EWS is predicted to be an RNA-binding protein, an
inherent EWS nuclear function has not yet been elucidated. In this
study, we found that EWS associates with a transcriptional co-activator
CREB-binding protein (CBP) and the hypophosphorylated RNA polymerase
II, which are included preferentially in the transcription
preinitiation complex. These interactions suggest the potential
involvement of EWS in gene transcription, leading to the hypothesis
that EWS may function as a co-activator of CBP-dependent
transcription factors. Based on this hypothesis, we investigated the
effect of EWS on the activation of nuclear receptors that are activated by CBP. Of nuclear receptors examined, hepatocyte nuclear factor 4-dependent transcription was selectively enhanced
by EWS but not by an EWS mutant defective for CBP binding. These
results suggest that EWS as a co-activator requires CBP for hepatocyte nuclear factor 4-mediated transcriptional activation.
 |
INTRODUCTION |
The EWS gene was originally identified in Ewing's
sarcoma with the t(11,22) chromosomal translocation where it is fused
to the ETS transcription factor Fli-1 gene (1). Subsequent
studies indicated that other ETS transcription factor genes fuse to the EWS gene and produce fusion proteins in Ewing's sarcoma. In
addition to the fusion with ETS transcription factors in Ewing's
sarcoma, the EWS gene has been shown to form fusion proteins
with ATF-1 in malignant melanoma of soft parts, WT-1 in desmoplastic
small round cell tumors, and nuclear orphan receptors in myxoid
chondrosarcomas (2). In EWS fusion proteins, the N-terminal domain
(NTD)1 of EWS is retained,
whereas the C-terminal domain of EWS is replaced by corresponding
fusion partners. However, the roles of EWS in normal cellular functions
and the mechanisms whereby EWS fusion proteins lead to these malignant
tumors remain unclear.
EWS contains the transcriptional activation domain in its NTD and the
RNA-recognition motif and arginine-glycine-glycine repeats (RGG), both
of which are found in RNA-binding proteins (3) in its C-terminal
domain. Transcriptional and post-transcriptional processing are closely
coupled events in vivo (4), and based on its structural
features, it is likely that EWS participates in RNA transcription and
mRNA synthesis. Furthermore, the NTD of EWS associates with the
basal transcription factor TFIID (5) and with certain subunits of the
RNA polymerase II complex (5, 6). On the other hand, EWS interacts with
the splicing factors SF1 (7), U1C (8), TASR-1/TRSR-2 translocation
liposarcoma protein-associated serine-arginine protein (9), and Y-box
protein-1 (10). A current expectation is that EWS may act as an
adaptor molecule linking between gene transcription and mRNA
processing by interacting with RNA polymerase II and the splicing
factors (2).
Interestingly, in this study, we indicated the interaction of EWS with
the transcriptional co-activator CBP and the hypophosphorylated RNA
polymerase II. CBP enhances many DNA-binding transcriptional activator
proteins including nuclear receptors and other signal-regulated activators by its histone acetyltransferase activity and recruitment of
RNA polymerase II-dependent basal transcription complex or other cofactors to target gene promoters (11, 12). Therefore, these
identified interactions suggest the potential involvement of EWS in
gene transcriptional activation. However, the fact that EWS does not
have any significant DNA-binding motifs or binding activity to specific
gene promoters prompted us to propose the hypothesis that EWS may
function as a co-activator of CBP-dependent DNA-binding
transcription factors. Based on this hypothesis, we investigated the
possible effects of EWS on transactivation mediated by nuclear
receptors. We showed that EWS selectively enhances the nuclear receptor
hepatocyte nuclear factor 4 (HNF4)-mediated transcription cooperatively
with CBP. These results suggest that EWS not only functions as an
adaptor molecule for splicing events but also as a co-activator with
CBP for transcriptional initiation.
 |
EXPERIMENTAL PROCEDURES |
Plasmids--
Human EWS deletion mutants were generated by
PCR-based subcloning into pGEX5X-1 (Amersham Biosciences) or
FLAG-tagged pcDNA3 (Invitrogen). pHNF4x8-tk-Luc and p
REx2-tk-Luc
have been described previously (13, 14). pPPREx3-sv-Luc was constructed
by ligating the three copies of the peroxisome proliferator-activated
receptor (PPAR
) binding site encompassing the nucleotide region
(
104 to
92) of the 3-hydroxy-3-methyl-glutaryl-CoA synthase to the pGL3-SV40 promoter/luciferase fusion vector (Promega). Apolipoprotein CIII (ApoCIII)-Luc and glucose 6-phosphatase (G6P)-Luc were constructed by PCR-based methods. Human ApoCIII promoter (
890 to +44)
or human G6P promoter (
826 to +3) fragment was
inserted into pPicaGene-Basic vector II. Angiotensinogen 13-Luc
was constructed by digesting a 1266-bp (
1222 to +44) fragment from
human angiotensinogen promoter 13cat, which has been described
previously (15), into pPicaGene-Basic vector II (Toyo-ink).
pcDNA3-mCBP-HA and pcDNA3-mCBP-FLAG were generated by
inserting full-length mouse CBP into HA or FLAG-tagged pcDNA3. pcDNA3-HA-HNF4
was generated by reverse
transcription-PCR-based cloning into HA-tagged pcDNA3.
Antibodies--
Anti-EWS rabbit polyclonal antibody was
generated against a GST-EWS-(246-504). Anti-HNF4 rabbit
polyclonal antibody was generated against a GST-HNF4-(133-366).
Anti-CBP rabbit polyclonal antiserum-(5614) was described previously
(16). Anti-HA monoclonal antibody (12CA5) was purchased from Roche
Molecular Biochemicals; anti-FLAG monoclonal antibody (M2) was from
Sigma; anti-HNF4
(C-19) and anti-RNA polymerase II (N-20) polyclonal
antibodies were from Santa Cruz Biotechnology; and anti-RNA pol II
monoclonal antibodies (8WG16 and H14) were from BAbCo.
Cells Culture and Transfections and Reporter Gene
Assays--
HEK293T cells and HepG2 cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum, and
HEK293 cells were maintained in minimum Eagle's medium containing 10%
fetal bovine serum. For the reporter gene assay, transfections were
performed by the calcium phosphate method. A Rous sarcoma virus-
-galactosidase plasmid was included in each transfection experiment to control for transfection efficiency. The luciferase activity was measured with an ARVOTMSX (Wallac Berthold).
The values were normalized to
-galactosidase activity as an internal control.
Immunoprecipitation--
HEK293T cells were transfected using
FuGENE 6 reagents (Roche Molecular Biochemicals), and nuclear extracts
were prepared as described previously (13). Nuclear extracts were
incubated with anti-FLAG coupled with protein G-agarose in buffer A (20 mM HEPES, pH 7.9, 100 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, 5%
glycerol, 1 mM Na3VO4, 5 mM NaF, and protease inhibitors) for 4 h at 4 °C.
The binding complexes were washed with the same buffer. For
co-immunoprecipitation of endogenous EWS, CBP, and HNF4, nuclear
extracts of HepG2 cells were subjected to immunoprecipitation with
anti-EWS, anti-HNF4, or anti-CBP-(5614) in Co-immunoprecipitation
buffer (10 mM HEPES, pH 7.5, 100 mM KCl, 0.1%
Nonidet P-40, and protease inhibitors).
GST Pull-down Assay--
The GST fusion protein of each EWS
mutant was expressed in Escherichia coli strain TopXF'
(Invitrogen) and purified using glutathione-Sepharose beads (Amersham
Biosciences). Protein extracts of HEK293T cells were incubated with
each GST fusion protein bound to resin in 1 ml of buffer A for 8 h
at 4 °C. After washing the beads with buffer A, bound proteins were
fractionated by SDS-PAGE and analyzed Western blotting.
Immunofluorescence--
HEK293T or HEK293 cells were plated onto
glass coverslips. HEK293T cells were transfected using FuGENE 6 reagents. After 24 h, cells were fixed with 6% paraformaldehyde,
permeabilized with 0.1% Triton X-100, and blocked with 1% bovine
serum albumin. Cells then were incubated with the primary antibody,
mouse anti-FLAG (1:500 dilution), rabbit anti-EWS (1:250 dilution),
mouse anti-Pol IIa (1:200 dilution), or mouse anti-Pol IIo (1:100
dilution) antibody followed by staining with Alexa Fluor 488 anti-mouse
IgG, Alexa Fluor 488 anti-rabbit IgG, or Alexa Fluor 568 anti-mouse IgG
second antibody (1:2000 dilution each; Molecular Probes).
Immunofluorescence was analyzed under a confocal microscope (TCS 4D, Leica).
 |
RESULTS |
Physical Association of EWS with CBP and RNA Polymerase II--
To
isolate CBP-interacting proteins, we used a GAL4 DNA-binding domain
fused to CBP as a bait in a yeast two-hybrid screening assay (13) in
which we identified EWS as a CBP-binding protein. To biochemically
characterize the interaction of EWS with CBP, we constructed a series
of EWS deletion mutants fused to GST and bacterially expressed products
of them for the following assays (Fig.
1A). GST pull-down assays were
performed on this series of EWS deletions with HEK293T lysates
expressing HA-tagged CBP. Western blotting of the bound proteins using
anti-HA antibody showed that CBP was specifically retained on beads
coupled with GST-EWS-(1-656), GST-EWS-(1-333), and GST-EWS-(83-227)
(Fig. 1B). These results show that EWS can specifically
interact with CBP through its amino acids 83-227 located on the NTD of
EWS.

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Fig. 1.
Map of the CBP interaction domain in EWS.
A, schematic representation of EWS and its deletion
mutants in this study. These elements in the C-terminal domain of EWS
were commonly found in RNA-binding proteins (3). B, in
vitro binding assay using cell extracts from HEK293T cells
transfected with HA-tagged CBP expression plasmid and GST or EWS
deletion mutants fused to GST. Western blot analysis was performed with
anti-HA antibody (12CA5).
|
|
Two isoforms of RNA polymerase II (Pol II) exist in vivo,
the hypophosphorylated (Pol IIa) and the hyperphosphorylated (Pol IIo)
forms required for transcriptional initiation and elongation, respectively. Pol IIa is shown to possess the hypophosphorylated C-terminal domain of the largest subunit and interacts with a range of
general transcription factors at the promoter (17, 18). We investigated
the relationship of EWS with different forms of Pol II using three
anti-Pol II antibodies: N-20, which recognizes the common NTD of both
Pol IIa and Pol IIo large subunits; H14, which only recognizes Pol IIo;
and 8WG16, which only recognizes Pol IIa in immunofluorescence staining
and Western blotting. To test the possible interaction of EWS with
distinct forms of Pol II, we first examined their cellular
localization. The staining patterns of Pol IIa and Pol IIo were
compared with nucleoplasmic EWS distribution in double-labeling
experiments followed by a confocal microscopy. The overlay images
showed that the nucleoplasmic structures of EWS, and both Pol II forms
partially overlapped (Fig.
2A). To further confirm the
biochemical interaction of EWS with both Pol IIa and Pol IIo, we
performed GST pull-down assays using HEK293 nuclear extracts. GST-EWS
combined with both Pol IIa and Pol IIo in vitro, whereas GST
alone did not (Fig. 2B). As shown in Fig. 2C,
both EWS and CBP associated with the preinitiation form of Pol II (pol
IIa), whereas EWS but not CBP co-immunoprecipitated with Pol IIo
in vivo. These results suggest that EWS is spatially and
physically included in a part of both of the preinitiation and
elongation Pol II complexes.

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Fig. 2.
In vitro and in vivo
association of EWS with Pol II. A, co-localization of
endogenous EWS and Pol IIa or Pol IIo by immunofluorescence staining.
Fixed HEK293 cells were double-labeled with anti-EWS antibody
(panels a and d) and 8WG16 against Pol IIa
(panel b) or H14 against Pol IIo (panel e),
whereas coincident signals are seen in yellow in the overlay
pictures obtained by a confocal microscopy (panel c and
f). B, interaction of EWS with Pol II in
vitro. Nuclear extracts from HEK293 cells were incubated with GST
or GST-EWS-(1-656). Bound Pol II was visualized by Western blot
analysis using anti-Pol II antibodies N-20, 8WG16, or H-14.
C, co-immunoprecipitation of Pol II using FLAG-tagged EWS or
CBP. Nuclear extracts from HEK293T cells transfected with FLAG-tagged
EWS, or CBP expression plasmids were immunoprecipitated with anti-FLAG
antibody (M2) and subjected to immunoblotting with anti-Pol II (N-20,
8WG16, or H14) or anti-FLAG (M2) antibodies.
|
|
Selective Enhancement of HNF4-mediated Transactivation by
EWS--
To examine the potential role of EWS in nuclear
receptors-mediated transactivation, we evaluated the effects of EWS on
the activation of three nuclear receptors including HNF4, retinoic acid
receptor (RAR), and PPAR
by transfection experiments. A role for EWS
as a co-activator in HNF4-mediated transactivation was first tested
using HNF4-specific reporter, pHNF4x8-tk-Luc (13). As shown in Fig.
3A, transfection of HEK293
cells with the reporter alone or with HNF4 resulted in activation (Fig.
3A1), which was stimulated 5-fold by co-transfection of EWS
(Fig. 3A, 1 and 2). We also examined
the effect of EWS on RAR-mediated transactivation using RAR-specific
reporter, p
REx2-tk-Luc (14). HEK293 cells express the endogenous RAR
as the reporter activity was sufficiently activated by its cognate
ligand, even without co-transfection of RAR expression plasmids (Fig.
3B1). Co-transfection of EWS repressed this activity by an
RAR-specific ligand (Fig. 3B, 1 and
2). Similar experiments were carried out using PPAR
with pPPREx3-sv-Luc. As shown Fig. 3C, no effect of EWS was found
on the activity of this receptor. Taken together, these results suggest that EWS is a selective co-activator for HNF4-mediated
transactivation.

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Fig. 3.
Selective enhancement of HNF4-mediated
transactivation by EWS. A, HEK293 cells were transiently
transfected with 50 ng of pHNF4x8-tk-Luc reporter plasmid and absence
(open bars) or presence (filled bars) of 25 ng of
HNF4 and 100 ng of EWS expression plasmid. B, HEK293
cells were transfected with 100 ng of p REx2-tk-Luc for retinoic acid
receptor and 100 ng of EWS expression plasmid. After transfection,
cells were treated with Phenol Red-free minimum Eagle's medium with
10% fetal bovine serum either alone (open bars) or with 1 µM all-trans-retinoic acid (filled
bars). C, HEK293 cells were transfected with 10 ng of
pPPREx3-sv-Luc for peroxisome proliferator-activated receptor and
100 ng of EWS expression plasmid. After transfection, cells were
incubated in the absence (open bars) or presence
(filled bars) of 500 µM Bezafibrate. The
results are the means ± S.E. of at least three independent
experiments performed in duplicate. DMSO,
Me2SO.
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Formation of EWS, CBP, and HNF4 complex and Cooperative Enhancement
of HNF4-dependent Transactivation--
HNF4, which belongs
to nuclear receptor superfamily, is a liver-enriched DNA-binding
transcription factor. Binding sites for HNF4 have often been found in
the regulatory regions of a large number of genes involved in fatty
acid metabolism (19), gluconeogenesis (20), and blood pressure control
(15) and in determining the hepatic phenotype (21). To test for
transcriptional co-activations by EWS on the endogenous promoter
sequences, we carried out transfection experiments using luciferase
reporter plasmids containing each HNF4-targeted gene promoter. EWS
enhanced ApoCIII (Fig. 4A),
G6P (Fig. 4B), or angiotensinogen (Fig. 4C)
reporters, which were activated by HNF4 in a dose-dependent
manner. To confirm the interaction of HNF4, CBP, and EWS under more
physiological conditions, endogenous EWS, HNF4, and CBP in HepG2
nuclear extracts were co-immunoprecipitated with anti-EWS, anti-HNF4,
and anti-CBP antibodies, respectively. As shown in Fig. 4D,
endogenous EWS, HNF4, and CBP could be efficiently co-immunoprecipitated with endogenous CBP/HNF4, CBP/EWS,
and EWS/HNF4, respectively. To assess the regulatory effects of EWS and
CBP on HNF4-mediated transactivation, we performed the reporter assay. Transfected HNF4 activity evaluated by co-transfection with an HNF4-tk-Luc reporter was further increased 3-4-fold by overexpression of EWS or CBP. When co-transfected together, both vectors induced reporter activity 13-fold in HNF4-transfected but not in
non-transfected cells, suggesting that these transcription factors
function cooperatively in HNF4-dependent transactivation
(Fig. 4E).

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Fig. 4.
Formation of EWS, CBP, and HNF4 complex and
cooperative enhancement of HNF4-dependent
transactivation. Enhancement of endogenous promoter sequences by
EWS. HEK293 cells were transfected with 10 ng of ApoCIII-Luc
(A), G6P-Luc (B), or 50 ng of angiotensinogen
13-Luc reporter plasmid (C), 25 ng of HNF4 expression
plasmid, and the indicated amounts of EWS expression plasmid. These
results represent the mean ± S.E. of at least three experiments.
D, co-immunoprecipitation of endogenous CBP/HNF4, CBP/EWS,
and EWS/HNF4 using anti-EWS, anti-HNF4, or anti-CBP antibodies. Nuclear
extracts from HepG2 cells were immunoprecipitated with anti-EWS
( EWS; left), anti-HNF4 ( HNF4;
center), and anti-CBP ( CBP; right) antibodies
or normal rabbit IgG as a control and then were subjected to
immunoblotting with anti-EWS, anti-CBP-(5614) or anti-HNF4 (C-19)
antibodies. E, EWS and CBP cooperatively enhance
HNF4-mediated transactivation. Cells were transfected with 50 ng of
pHNF4x8-tk-Luc reporter plasmid, 25 ng of HNF4 expression plasmid,
100 ng of EWS expression plasmid, and 250 ng of CBP expression plasmid.
This result is the means ± S.E. of at least three independent
experiments performed in duplicate.
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|
Requirement of CBP for EWS Function--
To determine whether the
interaction with CBP is required for EWS-mediated activation, we
constructed EWS
CBP, which lacks the CBP-binding region (amino acids
83-227) (Fig. 5A). We first compared expression patterns of FLAG-tagged EWS or EWS
CBP in transfected HEK293 cells by immunofluorescence and Western blot analyses with an anti-FLAG antibody. As shown Fig. 5, B and
C, FLAG-tagged EWS
CBP as well as EWS was localized in the
nucleus, and these expression levels were nearly equal. We next tested the binding activity of EWS
CBP with CBP or HNF4 by GST pull-down assays. As shown in Fig. 5D, EWS
CBP lost the interaction
with transfected HA-tagged CBP (upper panel) but still bound
to HA-tagged HNF4
(lower panel). We then assessed the
effect of EWS
CBP on HNF4-mediated transactivation by transient
transfection assay. In HEK293 cells, an HNF4-tk-Luc reporter was
inactive because of little expression of endogenous HNF4 protein (Fig.
5E, lanes 1-3). The luciferase reporter was
activated up to 3-fold by co-transfection with HNF4 (Fig.
5E, lane 4). The addition of EWS resulted in
additional 2.5-fold activation (Fig. 5E, lane 5).
However, in the presence of HNF4, co-transfection with EWS
CBP did
not activate the HNF4-dependent transactivation (Fig.
5E, lane 6). These results suggest that interaction with CBP is required for the activity of EWS on
HNF4-dependent transactivation through the CBP-binding
region.

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Fig. 5.
Requirement of CBP for EWS function.
A, schematic representation of EWS CBP, which lacks the
amino acids 83-227 in EWS. B, subcellular localization of
EWS and EWS CBP. HEK293T cells were transfected with FLAG-tagged EWS
(panels a and d) and EWS CBP (panels
b and e) expression plasmids or an empty plasmid
(panels c and f) and were stained with anti-FLAG
antibody (M2) (panels a, b, and
c) or Hoechst (panels d, e, and
f). C, Western blot analysis of FLAG-tagged EWS
and EWS CBP. Cell extracts from HEK293 cells transfected with
FLAG-tagged EWS or EWS CBP expression plasmid were immunoblotted with
anti-FLAG antibody (M2). D, pull-down assays with EWS or
EWS CBP fused to GST and extracts from HEK293T cells overexpressing
HA-tagged CBP (upper panel) or HNF4 (lower
panel). Western blot analyses were performed with an anti-HA
antibody (12CA5). E, EWS but not EWS CBP enhances the
HNF4-dependent transactivation. HEK293 cells were
transfected with 50 ng of pHNF4x8-tk-Luc reporter plasmid, 25 ng of
HNF4 , and 100 ng of EWS or EWS CBP expression plasmid followed by
luciferase assay. This result is the means ± S.E. of at least
three independent experiments performed in duplicate.
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|
 |
DISCUSSION |
EWS was originally identified as a fusion protein with Fli-1 in
Ewing's sarcoma and was later found in several malignancies in which
the NTD of EWS was fused to the DNA-binding domain of the ETS family
such as ATF-1, WT-1, CHN, and C/EBP homologous protein (2). For
all of the above malignancies, the EWS fusion proteins are thought to
act as potent transcriptional activators in a manner that is dependent
on the transcriptional activation domain in the NTD of EWS (22).
However, the transcriptional function of native EWS is not very well
understood. It has been shown that transcriptional events are closely
coupled with processing events in vivo (17, 18). The
C-terminal domain of the largest subunit of Pol II plays a central role
in this coupling by reversible phosphorylation during the transcription
cycle. Once initiation of transcription begins, the hypophosphorylated
form of C-terminal domain (Pol IIa) becomes hyperphosphorylated (Pol
IIo) during the transition from the preinitiation complex to the
elongation-processing complex, and this form of pol II is able to
recruit mRNA processing factors (23). EWS was previously reported
to interact with only Pol IIo as an adaptor molecule to recruit
splicing factors in elongation stages (2).
In this study, we identified the interaction of EWS with CBP and with
both of the preinitiation (Pol IIa) and the elongation (Pol IIo) forms
of Pol II. CBP associates only with Pol IIa (24) (Fig. 2) and activates
transcriptional initiation as a
bridging/co-activating/chromatin-remodeling factor. These findings
suggest that EWS may activate transcriptions in collaboration with CBP
and basal transcription machinery including the preinitiation form (Pol
IIa) of Pol II complex.
A previous study (25) showed the interaction of EWS with CBP/p300 and
the activation of c-fos, Xvent-2, and Erb2
promoters by EWS. Because EWS does not have any obvious
DNA-binding domain, it has been described that EWS may be recruited to
target promoters by protein-protein interaction with DNA-binding
transcription factors and act as a co-activator. However, a target
DNA-binding transcription factor has not yet been identified. We
demonstrated that EWS selectively potentiates the DNA-binding HNF4
transcription factor-mediated transactivation (Fig. 3). As transient
transfection analyses using the ApoCIII, G6P, and
angiotensinogen promoters illustrated that these were
activated by HNF4 in conjunction with EWS but not in the absence of
HNF4, HNF4 is a high affinity site for the entry of EWS to the
target promoters. On the other hand, EWS repressed RAR-mediated
transcription (Fig. 3B) and did not associate with RAR under
the conditions used in this assay (data not shown). This finding
suggested that RAR is not a direct target molecule of EWS and that the
repression by EWS artificially arises from squelching CBP, RNA
polymerase II, TFIID, or other factors in the transcriptional complex
for RAR.
We found that EWS interacted with CBP and HNF4 and played cooperatively
with CBP to further reinforce the transactivation of HNF4 (Fig. 4). In
addition, the co-activational activity of EWS was abolished by the
deletion of the CBP-binding region of EWS (amino acids 83-227) (Fig.
5). In previous studies, Ohno et al. (26) and Lessnick
et al. (27) demonstrate that the transcriptional activity of
EWS is included in amino acids 83-265 when fused to the DNA-binding
domain of GAL4 or Fli-1. These findings reveal that the
co-activator function of CBP mediates the transactivation ability of
EWS or EWS fusion proteins through the NTD.
Although we demonstrated that EWS interacts with CBP and the
preinitiation form of Pol II and enhances HNF4-mediated transcription in a manner dependent on the co-activator function of CBP, additional EWS functions were suggested by other studies. For example, EWS contains motifs like RNA-binding proteins and actually binds to RNA
in vitro (28), the elongation-mRNA processing form of
pol II (Pol IIo) (Fig. 2) (9, 10), and the splicing factors (7-9). Furthermore, it has been shown that EWS-Fli-1 can antagonize the splicing of model mRNA constructs in cells (9, 10). In this point,
PPAR
co-activator-1, which has also been reported as an HNF4
co-activator (29), can function as both transcriptional co-activator
and splicing regulator (30). Thus, our present findings provide the
possibility that a variety of HNF4-dependent physiological
functions including fatty acid metabolism, gluconeogenesis, and blood
pressure control may be exerted efficiently as results of coupling of
between transcription and mRNA processing by EWS and/or PPAR
co-activator-1.
Because EWS is a ubiquitously expressed factor (31) as compared with
the limited expression profile of PPAR
co-activator-1 in the brown
fat, kidney, heart, and brain under the normal conditions (32), it is
likely that EWS participates in various biological events through other
nuclear receptors or CBP-directed DNA-binding transcription factors. In
contrast, we confirmed that a EWS fusion gene, EWS-Fli-1,
lost the activity as a co-activator by a minimal reporter gene system
using HNF4 recognition site (data not shown). It is expected that
impaired (lost or acquired) biological systems of native EWS caused by
a gene fusion event may be related to the tumorigenesis. Therefore,
further analysis regarding the native EWS function will be helpful to
understand the physiological significance of EWS in addition to the
mechanisms of transformation by EWS fusion proteins.
 |
ACKNOWLEDGEMENT |
We thank the Fukamizu laboratory members for
helpful discussion and encouragement.
 |
FOOTNOTES |
*
This work was supported in part by Grant JSPS-RFTF
97L00804 from the "Research for the Future" Program (The Japan
Society for the Promotion of Science), a grant-in-aid for Scientific
Research on Priority Areas and a grant-in-aid for Scientific Research
(A) from the Ministry of Education, Science, Sports, and Culture of Japan, and the Research Grant 11C-1 for Cardiovascular Diseases from
the Ministry of Health, Labor, and Welfare.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: Center for Tsukuba
Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. Tel./Fax: 81-298-53-6070; E-mail: akif@tara.tsukuba.ac.jp.
Published, JBC Papers in Press, November 28, 2002, DOI 10.1074/jbc.M210234200
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ABBREVIATIONS |
The abbreviations used are:
NTD, N-terminal
domain;
Luc, luciferase;
tk, thymidine kinase;
CREB, cAMP-response
element-binding protein;
CBP, CREB-binding protein;
HNF4, hepatocyte
nuclear factor 4;
GST, glutathione S-transferase;
PPAR, peroxisome proliferator-activated receptor;
HA, hemagglutinin;
GST, glutathione S-transferase;
G6P, glucose 6-phosphatase;
Apo, apolipoprotein;
RAR, retinoic acid receptor;
Pol, polymerase.
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