From the Institut de Génétique Moléculaire de Montpellier, IFR24, CNRS, 1919 Rte. de Mende, Montpellier 34293, France
Received for publication, August 28, 2000, and in revised form, November 7, 2000
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ABSTRACT |
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Proliferative signals lead to the rapid and
transient induction of the c-fos proto-oncogene by
targeting the ternary complex assembled on the serum response element
(SRE). Transactivation by both components of this complex, serum
response factor (SRF) and the ternary complex factor Elk-1, can be
potentiated by the coactivator CREB-binding protein (CBP). We report a
novel interaction between the bromodomain of CBP, amino acids
1100-1286, and Elk-1. DNA binding and glutathione
S-transferase pull-down assays demonstrate that binding
requires Elk-11-212 but not the C-terminal transactivation
domain. Competition and antibody controls show that the bromocomplex
involves both SRF and Elk-1 on the c-fos SRE and uniquely
Elk-1 on the E74 Ets binding site. Interestingly, methylation
interference and DNA footprinting analyses show almost indistinguishable patterns between ternary and bromocomplexes, suggesting that CBP-(1100-1286) interacts via Elk-1 and does
not require specific DNA contacts. Functionally, the bromocomplex blocks activation, because cotransfection of CBP-(1100-1286) reduces RasV12-driven activation of SRE and E74 luciferase reporters. Repression is relieved moderately or strongly by linking the
bromodomain to the N- or C-terminal transactivation domains of CBP,
respectively. These results are consistent with a model in which CBP is
constitutively bound to the SRE in a higher order complex that would
facilitate the rapid transcriptional activation of c-fos by
signaling-driven phosphorylation.
One response of cells to extracellular stimuli is the activation
of signaling pathways that lead to changes in gene expression mediating
proliferation, differentiation, and apoptosis. A major messenger system to these downstream
events is the various MAPK1 signaling pathways that
play a key role in this response by the activation of immediate early
genes like the proto-oncogene c-fos (1). The
c-fos promoter contains three major regulatory elements, the
CaCRE, the SRE, and the SIE (2, 3). The Ca2+ and
cAMP-response element (CaCRE) can mediate activation by cAMP-PKA or
Ca2+-calmodulin-dependent kinase signals, which
lead to phosphorylation of the transcription factor CREB on Ser-133
(4). CREB is also targeted by MAPK cascades through the activation of
MAPKAP kinases (5, 6). The v-sis-inducible element (SIE) is
targeted through cytokine- and growth factor-driven STAT1 and -3 activation (7), and their activity can be modulated by the MAPKs.
The serum response element (SRE) on the c-fos promoter alone
is sufficient to confer a signal-dependent activation (8). Genomic footprinting studies have shown that a complex is assembled over this promoter before, during, and after induction (9). A complex
with similar characteristics can be reproduced on the SRE in
vitro by a dimer of serum response factor (SRF) and one molecule
of ternary complex factor (TCF) (10). The TCFs are encoded by a family
of Ets proteins that includes Elk-1, SAP-1a, and a third member
variously called NET, ERP, or SAP-2 (11). Elk-1 and Sap-1a play a key
role in translating signals from kinases into transcriptional
activation. The TCFs represent major nuclear targets for the MAPKs ERK,
p38, and SAPK. The resulting phosphorylation of TCF plays a major role
in the induction of the c-fos gene by a mechanism that
remains to be fully resolved (2).
SRE-driven transcriptional activation appears to involve coactivators
of the CBP/p300-family. CBP has been described to interact with the
C-terminal transactivation domains of the TCFs Elk-1 and Sap-1a and
with full-length SRF (12-14). This involves the CBP region spanning
aa 451-721 for TCF (12, 13) and the N-terminal CBP region
spanning aa 1-1097 for SRF (14). Accordingly, CBP increases
transcriptional activation by Elk-1, SAP-1a, and SRF in transient
transfection assays (12-15). Thus, SRE-driven transcriptional activation appears to involve the recruitment of CBP to the ternary complex, as has also been observed for the CREB·CaCRE and
STAT·SIE c-fos promoter complexes (16-18).
The kinetics of c-fos transcriptional induction suggest that
the recruitment of the coactivator is either a very rapid process or
that the coactivator is already present on the promoter. Here we have
tested the latter possibility, namely that CBP might interact constitutively with the complex assembled over the SRE. We show that
the bromodomain of CBP interacts with the TCF Elk-1 in solution and
generates an Elk-1-dependent quaternary complex in
vitro on the SRE. A similar Elk-1-dependent complex
forms on the E74 site bound directly by Elk-1. Moreover, this novel
complex represses transcriptional activation of SRE and E74 reporter
genes driven by RasV12, whereas activity is restored by including the
C- and N-terminal activation domains of CBP. These data suggest a model where the bromodomain anchors CBP to the SRE via TCF in a higher order
complex. This would facilitate the rapid transcriptional activation of
c-fos mediated by CBP through interaction with
transcription factors targeted by signaling-driven phosphorylation.
Materials--
Restriction enzymes were obtained from Life
Technologies and New England BioLabs. Antibodies against the DNA
binding domain of Gal4 were purchased from CLONTECH
and Santa Cruz Biotechnology, Inc., rat monoclonal antibodies to
hemagglutinin were from Roche Molecular Biochemicals, and the Elk-1
antibody against the Ets domain (aa 1-82) of Elk-1 ( Plasmids--
The Gal4-CBP and GST-CBP subclones have been
previously described (12, 13). The Gal4-CBP-(1-1460) construct was
generated by insertion of the N-terminal portion of CBP into the
SalI and NarI sites of the Gal4-CBP-(1100-1460)
subclone. The Gal4-CBP-(1100-2441) construct was generated by
insertion of the C-terminal fragment of CBP into the BamHI
and NarI sites of the Gal4-CBP-(1100-1460) subclone. The
Elk-1 expression vector used for in vitro transcription (aa
1-428) was previously described (21). The Elk-1 N-terminal (aa 1-308)
and C-terminal aa ( PCR Amplification and Cloning of the CBP Bromodomain--
The
oligos 5'-AGTCGAATTCGAGGAGCTACGCCAGGCACTTATGC-3' (upper strand)
and 5'-AGCTAAGCTTCTAAGACTGCATGACAGGGTCAATT-3' (lower strand) were
used to generate a fragment of CBP spanning the bromodomain (aa
1089-1196) flanked by EcoRI and HindIII
sites to facilitate cloning into expression vectors. The 25-µl PCR
reactions contained 2 ng of CBPwt expression vector, 300 nM of each oligonucleotide, 200 nM dNTPs, 1.5 mM MgCl2, the supplier's reaction buffer
(Promega), and 5 units of Taq polymerase. After 4 min at
95 °C, amplification was carried out for 24 cycles (0.5 min,
95 °C; 0.5 min, 55 °C; 0.5 min, 72 °C). The fragments were
purified using a NucleoSpin extract kit, digested with EcoRI
and HindIII, and then purified from an agarose gel using the
NucleoSpin kit. After cloning into Bluescript KS+ (Stratagene),
positive clones were confirmed by sequencing. The bromodomain-encoding
fragment was recloned into pGEX-2T-6His and clones screened for
expression of the GST-bromo fusion protein, as well as into
pCDNA3.1-FLAG. This vector was used to confirm bromodomain
expression by coupled in vitro transcription translation
(see below).
Purification of Bacterially Expressed Recombinant
Proteins--
Escherichia coli strain BL21(LysE) was
electroporated with the appropriate expression vector. Fresh colonies
were used to inoculate 200-ml bacterial cultures, which were grown at
37 °C, and recombinant protein expression induced in exponential
phase by adding isopropyl thiogalactoside to 0.1 mM. After
1-4 h at 37 °C, bacteria were collected by centrifugation and lysed
in 10 ml of B-Per reagent according to the supplier's recommendations. Insoluble proteins were removed by centrifugation at 27,000 × g for 15 min at 4 °C. The supernatant was brought to 1 mM imidazole and incubated overnight with 50-200 µl of
Talon resin at 4 °C. The mix was poured into a column, the resin was
washed twice with 5 ml of RJD* buffer (10 mM HEPES,
pH 7.9, 5 mM MgCl2, 50 mM NaCl, 17% glycerol, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.05% Nonidet P-40) containing 1 mM
imidazole and freshly added protease inhibitors (2.5 µg/ml aprotinin,
leupeptin, pepstatin, 0.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride). Recombinant His-tagged proteins were eluted with two column volumes of RJD* buffer containing 200 mM imidazole, pH 7.6, and stored in aliquots at
In Vitro Translation--
35S-Labeled Elk-1 proteins
were synthesized from Bluescript KS+ vectors encoding Elk-1-(1-428),
Elk-1-(1-307), or Elk-1-(308-428) by coupled in vitro
transcription/translation in the presence of
L-[35S]methionine (1000 Ci/mmol) using TnT
kits according to the supplier's recommendations. C-terminal truncated
proteins were produced identically, using the Elk-1-(1-428) vector
digested with the following restriction enzymes: XbaI
(Elk-1-(1-428)), BsmI (Elk- 1-(1-374)), ApaI
(Elk-1-(1-253)), StuI (Elk-1-(1-212)), and NspI
(Elk-1-(1-122)).
Pull-down Assays--
Recombinant proteins purified by metal
affinity chromatography were directly bound to glutathione-Sepharose
beads at 4 °C for 1 h. The beads were then washed three times
with a 100-fold excess of RJD*-buffer containing protease inhibitors
and stored short-term at 4 °C. 5 µl of the in vitro
translated Elk-1 proteins described above were added to 10 µl of a
50% slurry of protein-bearing beads in a total volume of 100 µl of
RJD* buffer containing protease inhibitors and incubated with gentle
agitation at 4 °C for 4 h. After three washes with 500 µl of
RJD*, the beads were collected, resuspended in 20 µl of 5× Laemmli
buffer (0.25 M Tris-HCl, pH 6.8, 10% SDS, 10% glycerol,
5% 2-mercaptoethanol, 0.001% bromphenol blue) and denatured for 5 min
at 95 °C, and the bound proteins were analyzed by SDS-polyacrylamide
gel electrophoresis. Proteins were visualized by Coomassie Blue
staining, followed by autoradiography of the dried gels.
Immunoblotting--
Proteins were subjected to electrophoresis
on 6-10% SDS-PAGE minigels. Proteins larger than 100 kDa were
transferred to nitrocellulose membranes by immersion blotting, whereas
smaller proteins were immobilized on either nitrocellulose or
polyvinylidene fluoride membranes by semi-dry transfer. Membranes were
blocked with 5% dry milk in TBST (50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 3 mM KCl, and 0.05% Tween 20) and
then incubated with the indicated primary antibody diluted 1:1000 into
the blocking buffer. After washing in TBST (6 × 5 min), the
membranes were incubated with the appropriate secondary antibody
coupled to horseradish peroxidase diluted in blocking buffer. The
membranes were again washed in TBST as above, and the immune complexes
were visualized by enhanced chemiluminescence.
Transient Transfection and Luciferase Assays--
NIH3T3 cells
were maintained in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin. Cells were seeded to
30% confluency in 6-well plates 16 h prior to transfection. The
medium was changed 1 h before transfection, which was performed using a classical Ca2PO4 protocol (22). The
transfection mixes contained 300 ng of Luciferase reporter construct, 1 µg of Gal4-CBP expression vector, 200 ng of RasV12 expression vector,
5 ng of pSV-Renilla Luciferase expression vector, and 3.5 µg of
pUC18. These optimal values were determined experimentally several
times. Cells were washed in phosphate-buffered salt solution 8-10 h
after transfection and maintained in serum-free DMEM for 40 h.
Cell extracts were prepared, and luciferase activity was determined following the protocol supplied with the Dual Luciferase kit (Promega). Measurements were made in duplicate using a Berthold Lumat
LB950. For protein overexpression, COS-7 cells were transfected
using Transfast (Promega) according to the supplier's protocol, and whole cell extracts were prepared as described (23). Protein concentrations were determined using Bradford's reagent with BSA as a
standard. Protein expression was confirmed using immunoblotting prior
to gel retardation analyses.
Gel Retardation Analysis--
Probes for binding analyses were
prepared from subcloned SRE (20), EL (10), and E74 sequences (21) by
Klenow enzyme-catalyzed end labeling of an
EcoRI-NarI fragment in the presence of 50 µCi of [ Methylation Interference Analysis--
SRE probes were prepared
from the same site described above but cloned into pUC18 at the
XbaI site. After digestion with
HindIII-XmaI, the upper and lower strands were
end-labeled using Klenow enzyme, 300 µM dGTP, and either
50 µCi of [ Footprint Analysis--
Preparative reactions were carried out
as described above but incubated for 30 min at 4 °C. Reactions were
brought to 2.5 mM CaCl2, 7.5 mM
MgCl2 were attained and incubated with 0.01 unit of DNaseI
at 37 °C for 1.5 min. The reaction was terminated by adding
100 µl of 0.01% Sarkosyl, 0.1 M Tris-HCl, pH 8.0, 0.1 M NaCl, 0.02 mM EDTA, 0.03 µg/µl calf
thymus DNA, and 0.075 µg/µl Proteinase K, incubated an additional
15 min at 37 °C followed by 5 min at 95 °C. Labeled DNA was
purified by organic extraction and alcohol precipitation, resuspended
in formamide dye mix, denatured again, and analyzed on 10% sequence
gels as described above.
Expression of Gal4- and GST-CBP Fusion Proteins--
To
investigate the interactions between Elk-1 and CBP in detail, different
subdomains of CBP were fused to the Gal4 DNA binding domain for
expression in mammalian cells and to glutathione transferase to
facilitate purification after overexpression in bacteria (12, 13). The
different expression constructs are presented schematically in Fig.
1A. Immunoblotting with a
Gal4-DNA binding domain antibody showed that the various fusion
proteins were expressed at different levels after transient
transfection of COS-7 cells (Fig. 1B). Similarly, variable
amounts of the various GST fusion proteins were obtained after affinity
purification of bacterially expressed proteins (Fig. 1C,
lower panel). These assays were used to determine the
functional protein concentration for subsequent experiments. It should
be noted that the fusion proteins encoded by Gal4-CBP-(1100-1460) (12,
13) are truncated due to the presence of a stop codon after aa 1286 in
the original CBP expression vector. Therefore, this protein contains
the bromodomain of CBP but not the histone acetyltransferase domain
immediately downstream and will subsequently be labeled
CPB-(1100-1286).
Interaction in Solution between CBP Fusion Proteins and
Elk-1--
We first tested for solution interactions between various
regions of CBP and Elk-1. Recombinant, bacterially produced GST-CBP fusion proteins were immobilized on glutathione-agarose beads and
incubated with 35S-labeled Elk-1 (aa 1-428) produced by
coupled in vitro transcription/translation (Fig.
1C). Two versions of Elk-1 are produced in vitro,
the smaller of which arises from internal initiation of translation at
Met-55 (also see below) (25). Both full-length and N-terminal truncated Elk-1 bound to CBP-(451-721), which has previously been shown to
interact with the C-terminal activation domain of Elk-1 (13). Full-length and N-terminal truncated Elk-1 also interacted with CPB-(1100-1286), which spans the bromodomain, and CBP-(1460-1891), which contains most of the histone acetyl transferase domain. The
increased yield of the truncated version of Elk-1 versus
that of full-length Elk-1, particularly in respect to their relative levels in the reaction input (left lane), suggests that
these two CBP domains bound preferentially to N-terminal truncated
Elk-1 in solution. Neither version of Elk-1 interacted with GST alone (Fig. 1C) or with other domains of CBP (not shown), nor did
we detect any binding of SRF produced in vitro to CBP (not shown).
We then investigated the solution interaction between the bromodomain
of CBP and different portions of Elk-1. To this end, in
vitro translated 35S-labeled full-length (aa 1-428),
N-terminal (aa 1-307), and C-terminal (aa 308-428) Elk-1 were used as
above (Fig. 1D). Similar to full-length Elk-1, the
N-terminal construct gave rise to two translation products in
vitro, unlike the C-terminal construct. This further supports the
notion that the smaller form of Elk-1 arises from internal initiation
of translation and not premature termination. Once again, full-length
Elk-1 bound to the bromodomain of CBP in solution, as did Elk(NT), with
some preference shown for the truncated version (compare bound
lanes to input). CBP-(1100-1286) also interacted with
in vitro-translated C-terminal domain of Elk-1.
Interestingly this interaction appears to be independent of its
activation by phosphorylation, because Elk-(308-428) is not
phosphorylated during in vitro translation. In contrast to
these interactions, none of these proteins bound to GST alone (Fig.
1D). Thus we can detect complexes between multiple domains
of CBP, in particular the bromodomain, and Elk-1 in solution.
To confirm that the bromodomain alone (CBP aa 1089-1196) accounts for
binding to Elk-1, an expression vector for GST-CBP-(1089-1196) was
generated by PCR. This protein showed interactions with Elk(FL) and
Elk(NT) that were indistinguishable from CBP-(1100-1286) (Fig. 1E) and in fact showed binding to progressively truncated
versions of Elk until the region between amino acids 212 and 122 was
deleted (Fig. 1E). This indicates that the region in Elk-1
spanning aa 122 to 212 is important for interaction in solution with
the CBP bromodomain.
The Bromodomain of CBP Forms DNA-dependent Complexes
with Elk-1 on the Consensus Ets-binding Site E74--
The interactions
in solution between the bromodomain of CBP and the TCF Elk-1 led us to
test for similar interactions in the context of DNA-bound complexes,
using the E74 Ets sequence to which Elk-1 binds directly as a monomer
(21). To this end we employed extracts from COS-7 cells, transiently
transfected with expression vectors for different Gal4-CBP fusions, and
analyzed complex formation on a 32P-labeled E74
oligonucleotide. Endogenous Elk-1 forms a characteristic complex in
nontransfected cell extracts and those containing Gal4-CBP-(1-451) (Fig. 2A, lanes 1 and 2). However, cell extracts containing the bromodomain of
CBP (aa 1100-1286) present a novel complex, termed the bromocomplex,
with slightly faster mobility than the Elk-E74 binary complex (Fig.
2A, lane 3).
Because we did not cotransfect the cells with an Elk-1 expression
vector, this complex could reflect direct binding by the bromodomain of
CBP. However, the bromocomplex is sensitive to a limiting concentration
of an Elk-1 Ets-domain antibody but is unaffected by an antibody
specific for SRF (lanes 7 and 8, also see below),
indicating that the complex contains Elk-1 and is not due to direct
binding to the site by the bromodomain. Notably, the addition of
recombinant Elk-(308-428) had no effect on complex formation
(lane 6), suggesting that complex formation involves the
N-terminal domain of Elk-1. Accordingly, GST-Elk1-308 binds to the probe directly and also generates a novel, slower migrating complex with Gal4-CBP-(1100-1286) (lane 5). To confirm this
interpretation, we performed the same test using Elk-(1-253), Elk-(1-212), and Elk-(1-122) produced in vitro by coupled
transcription-translation. Elk-(1-253) and Elk-(1-212), but not
Elk-(1-122), also formed a novel complex that migrated more slowly
than the bromocomplex formed with endogenous Elk-1 (Fig.
2B), as well as much more slowly than the direct complexes
formed by these proteins (not shown) (21). Thus the same domain of
Elk-1 is required for interaction in solution and in the context of DNA
binding. These data suggest that bromocomplex formation is due to
direct interaction between Elk-1 and the bromodomain of CBP, and that
this can be attributed to the first 212 aa of Elk.
The Bromodomain of CBP Forms a Quaternary Complex on the c-fos SRE
Together with Elk-1 and SRF--
Elk-1 can interact with the
c-fos SRE only in the presence of SRF (20), forming a
ternary complex that consists of Elk-1, a dimer of SRF, and the SRE. We
tested whether the bromocomplex could also be observed in this context.
As above, we used extracts from COS-7 cells, transiently transfected
with expression vectors for the different Gal4-CBP fusions, and
analyzed ternary complex formation on the SRE. To better visualize the
TCF complexes, the experiments contained coreSRF, a truncation mutant
that spans aa 90-245 and contains the MADS box domain necessary for
dimerization, DNA binding and ternary complex formation. The
nontransfected extract shows the normal pattern of a coreSRF binary
complex and a slower migrating ternary complex containing Elk-1, the
major species of TCF in these cells (Fig.
3A, lane 1; see
below), as does an extract from cells transfected with
Gal4-CBP-(1-451) (Fig. 3A, lane 2) or
Gal4-CBP-(462-662) (Fig. 3B, even-numbered
lanes). Cell extracts containing the bromodomain of CBP (aa
1100-1286) present, in addition to the ternary complex, a novel faster
migrating bromocomplex (Fig. 3A, lane 3; Fig.
3B, lanes 3, 5, and 7). In contrast, the bromocomplex formed upon adding full-length recombinant SRF migrates more slowly than the ternary complex (not shown), indicating a surprisingly compact structure of the bromocomplex formed
with endogenous Elk-1 and coreSRF. The complex was observed in both
nonstimulated and stimulated extracts, showing that this interaction is
independent of Elk-1 phosphorylation (not shown).
We next characterized the bromocomplex using antibodies specific for
different components of the ternary complex. These reactions contained
extracts from COS-7 cells transfected with Elk-1- and Gal4CBP-(1100-1286) to amplify the ternary- and bromocomplexes. The
bromocomplex is supershifted by an anti-SRF antibody (Fig. 3A, lane 11) and blocked by the Ets
domain-specific Elk-1 antiserum that blocks complex formation (Fig.
3A, lane 12). This indicates that both ternary
complex components contribute to formation of the bromocomplex. Because
antibodies directed against this domain of CBP are not available, we
used a Gal4 antibody to target the CBP component. An antiserum directed
against the DNA binding domain of Gal4 significantly reduces
bromocomplex formation, unlike BSA or another antiserum from the same
source (Fig. 3B, compare lanes 3, 5,
and 7). The Gal4 antiserum also slightly diminishes ternary complex formation but to a lower extent than the bromocomplex (Fig.
3B, upper panel). These data show that the
bromocomplex contains a protein bearing the Gal4 DNA binding domain.
Because the bromocomplex does not form with other Gal4-CBP fusion
proteins, it also requires the bromodomain of CBP. It is unclear why
the Gal4 antiserum does not completely block or supershift the
bromocomplex, although this correlates with our difficulty in using
this antibody for immunodetection of Western blots (not shown).
An excess of purified recombinant GST-Elk-(1-307) drives formation of
several distinct complexes and competes for the bromocomplex formed by
transfected cell extracts (Fig. 3A, lane 9). This
is not the case when adding an excess of GST-Elk-(308-428) or GST alone (Fig. 3A, lanes 8 and 10). These
data indicate that, as with the E74 probe, the bromocomplex requires
the Elk N-terminal domain, and that any complex formed between
GST-Elk-(1-307) and Gal4-CBP-(1100-1286) shows a slower migration
relative to transfected wt Elk-1. We did not detect bromocomplexes in
the absence of coreSRF (not shown), nor did we detect them using a SRE
probe with a mutated TCF-binding site (EL), which nevertheless showed
normal coreSRF·SRE binary complexes (not shown). Thus Elk-1 is
required for the bromocomplex, and the bromodomain of CBP cannot
facilitate and stabilize Elk-1 binding to the SRE in the absence of
SRF.
Methylation Interference and DNA Footprinting Analysis of the
Bromocomplex--
We used methylation interference and DNA
footprinting to determine whether the bromocomplex made protein-DNA
contacts beyond those of the Elk·coreSRF ternary complex, especially
because the Gal4 DNA binding domain antibody partially blocked
bromocomplex formation. For methylation interference, SRE probes with
either the upper or lower strand 32P-labeled were partially
methylated with dimethyl sulfate and then used in preparative band
shift analyses. The DNA was purified from different complexes,
subjected to piperidine cleavage, and then analyzed by denaturing gel
electrophoresis. Somewhat surprisingly, the sensitivity of the
bromocomplex to methylation is indistinguishable from that of the
ternary complex on both the upper and lower strands (Fig.
4). Thus, the bromocomplex shows no
strict requirement for G or A residues outside the TCF binding site.
Moreover, it does not reproduce the pattern observed on the
c-fos AP1-like element, or FAP, in genomic footprints prior
to and during induction (9).
The lack of any apparent difference in methylation pattern led us to
analyze the bromocomplex by DNaseI footprinting. Solution binding
reactions were assembled using SRE probes labeled on the upper or lower
strands, coreSRF, Elk-1, and either Gal4-CBP-(1-451) or
Gal4-CBP-(1100-1286) and subjected to a brief DNaseI digestion, and
the purified DNA was analyzed by electrophoresis on a sequencing gel as
above. The lower strand showed subtle differences in the DNaseI
digestion pattern between the ternary complex and the bromocomplex. A
hypersensitive site in the 3'-half of the SRE appears in the bromocomplex (Fig. 5, right,
filled circle), and increased protection is apparent within
and upstream of the TCF-binding site with the bromodomain relative to
CBP-(1-451) and TCF (Fig. 5, right, arrows). Under these conditions the ternary complex alone does not protect the
lower strand from DNase I but rather appears to increase its sensitivity (compare lanes 2 and 3, lower
strand). No protection is seen with these proteins in the absence of
coreSRF (Fig. 5, right). The upper strand shows the classic
protection pattern conferred by the ternary complex (Fig. 5,
left) (26). Interestingly, adding CBP-(1100-1286), but not
CBP-(1-451), led to increased protection immediately upstream of the
CAGG sequence contacted by TCF (10, 26) and Elk-1 (20). (Fig. 5,
left, arrows). As with the lower strand,
CBP-(1100-1286) and TCF show the same DNase pattern as free DNA (Fig.
5, left, lanes 5 and 6). The
bromocomplex also does not protect the FAP site downstream of the SRE
(Fig. 5, left). Thus, the bromocomplex contains no obvious
direct DNA contacts by the bromodomain, but rather protein-protein
interaction with Elk-1 that seems to enhance Elk-1 binding to its
recognition sequence.
CBP-(1100-1286) Represses SRE-driven Transcriptional Activation in
NIH3T3 Cells--
To investigate the potential functional significance
of the bromocomplex, we measured its effect on SRE-driven reporter
genes in transient transfection assays. In nonstimulated NIH3T3 cells, a 3xSRE luciferase reporter gene shows weak activity that is unaffected by cotransfecting Gal4-CBP-(1100-1286) (not shown). Ras-mediated signaling strongly induces SRE-driven gene expression, thus we tested
the effect of different CBP expression vectors together with activated
Ras (RasV12) on the activity of a 3xSRE luciferase reporter gene in
these cells. The bromodomain strongly diminished SRE activation by
RasV12, an effect not seen with other CBP regions (Fig.
6A). This repressive effect
suggests that the bromodomain of CBP, on its own, can repress SRE
activity under induced conditions. This was not surprising, because the
molecule lacks both the N- and C-terminal activation domains that
potentiate SRE activity in transfected cells (12-14). To test whether
the activation domains could reverse this inhibition, we transfected
the following expression vectors: CBP-(1-1460), which contains, 1) the
N-terminal transactivation domain, 2) the region spanning aa 451-721
that interacts with the TCF C-terminal transactivation domain, 3) the
SRF-interacting module, 4) the bromodomain (but not the histone
acetyltransferase domain immediately downstream) CBP-(1100-2441),
which contains, 1) the bromodomain, 2) the histone acetyl transferase
activity, 3) the C-terminal transactivation domain; CBP-(1-2441), the
full-length CBP protein. These different proteins relieved the
repressive effect of the bromodomain. CBP-(1-1460) showed an
intermediate effect, whereas CBP-(1100-2441) restored reporter
activity to nonrepressed levels. Surprisingly, CBP-(1-2441) was not
significantly more effective than the CBP-(1100-2441) in potentiating
transcriptional activity (Fig. 6A).
We then performed the same experiments with a 4xE74-luciferase
construct to investigate the functional role of the bromocomplex in the
absence of SRF (Fig. 6B). This consensus Ets-binding site is, like the SRE, targeted by Ras-mediated signaling. Because this
reporter shows a high background due to its recognition by endogenous
Ets-proteins (21), we cotransfected an Elk-1 expression vector. The
same pattern was observed as with the SRE. The bromodomain led to a
significant decrease in RasV12-dependent activation, an
effect that again was not seen with other Gal4-CBP fusions. The
addition of the N- or C-terminal transactivation domains of CBP
(Gal4-CBP 1-1286, 1100-2441, and 1-2441) showed essentially the same
relief of repression as with the SRE reporter.
Because the bromocomplex does not form on the SRE mutant EL where the
binding of TCF is abolished, we used an EL-driven luciferase reporter
gene to test for nonspecific inhibition by CBP-(1100-1286) (Fig.
6C). It has been shown that SRF alone can mediate SRE-driven transcriptional activation in a pathway dependent on the Rho/Rac/CDC42 family of small G proteins (27). This reporter gene, as well as one
containing three copies of the EL-SRE, was unaffected by cotransfection
with the CBP-(1100-1286) expression vector, ruling out nonspecific
inhibition. Curiously, the EL reporter was also not activated by the
CBP-(1-1460) expression vector containing the domain described to
interact with SRF (14). Thus, this putative interaction is not
sufficient to mediate transactivation in our conditions. Altogether,
these data further confirm that the bromodomain of CBP is interacting
with the TCF Elk-1 and not directly with SRF.
Elk-1 is strongly phosphorylated by MAPKs, and phosphorylation of
serines 324, 383, and 389 is essential for transactivation (11). It has
previously been shown that CBP-(451-721) interacts with the C-terminal
domain of Elk-1 and that this interaction mediates Elk-1
transactivation (13). Our data suggested that bromocomplex formation
in vitro did not require this interaction nor activated
Elk-1. To test this functionally, we cotransfected the bromodomain, the
4xE74-luciferase reporter gene and an Elk-1 mutant where the three
serine residues have been changed to alanine (Fig. 5D).
Elk3A was still responsive to RasV12, although the level of activation
was lower than wt Elk-1. Notably, the repressive effect of
cotransfecting Gal4-CBP-(1100-1286) was still observed as was the
rescue by the longer versions of Gal4-CBP fusions. Furthermore, the
relative effects were similar to wt Elk-1. These data strongly suggest
that bromodomain-driven interactions are independent of phosphorylation.
Our data present evidence for a novel regulatory complex on the
c-fos SRE. This complex involves the interaction of the
bromodomain of CBP with the ternary complex composed of SRF and TCF.
Notably, these interactions seem to be independent of signaling that
drives SRE activation. These conclusions are based on the following: 1)
The bromodomain of CBP interacts with the C- and N-terminal regions of
the TCF Elk-1 in solution. 2) The bromodomain forms a quaternary
complex on the c-fos SRE together with SRF and Elk-1 in vitro. 3) The DNA-dependent in
vitro interactions can be reconstituted on the Ets protein binding
site E74, where Elk-1 binds in the absence of SRF, suggesting a sole
requirement for TCF in bromocomplex formation. 4) Methylation
interference analysis of the bromocomplex reveals no absolute
requirement for any particular residue, instead suggesting that it
forms over the ternary complex. 5) In transient transfection assays,
the bromodomain of CBP represses SRE and E74 reporter activity in the
absence of the C- and N-terminal activation domains. This repression is
relieved when these domains are added. Moreover, the repression is
dependent on Elk-1.
The rapid signaling-dependent induction of c-fos
transcription in vivo implies a high degree of
compartmentalization of transcription factors/promoter complexes
together with adaptors/coactivators within the nucleus. Our starting
hypothesis was that the coactivator CBP may form a constitutive complex
with the SRE. In our in vitro system, bromocomplex formation
seems to be independent of activation, suggesting that recruitment of
CBP by phosphorylated transcription factors may not be required for the
initial association of CBP with promoters. The constitutive presence of
CBP on the c-fos SRE might explain the stability of the
ternary complex in vivo and the extremely rapid activation
of c-fos transcription after stimulation. This stability has
yet to be seen in vitro where the ternary complex
dissociates rapidly (10).
When formed with coreSRF (aa 90-245), the bromocomplex shows faster
mobility than the ternary complex. Nevertheless, the complex contains
CBP-(1100-1286), Elk-1, and core SRF, suggesting that the quaternary
complex is more compact than the TCF·coreSRF ternary complex. In
contrast, in the presence of full-length SRF, GST-Elk307, or Elk-1 truncation mutants generated in vitro, the
resulting bromocomplex migrates more slowly than does the ternary
complex. In many instances the yield of the bromocomplex is increased
relative to the ternary complex, but the basis for this increased
efficiency is still unclear. The DNase footprints indicate that
protection is increased around the TCF binding site in the
bromocomplex. This suggests tighter binding by TCF in the complex. An
additional component may involve changes in the conformation of the
binding site, because the bromocomplex generates a hypersensitive site 3' in the SRE. These would also aid formation of a tighter,
faster-migrating bromocomplex. Interestingly, this is only observed
with Elk-1 expressed in mammalian cells, not with Elk-1 produced in
bacteria or in vitro, which implies that post-translational
modification contributes to this behavior.
Many factors, such as ATF-6, Phox-1, YY-1, C/EBP The homeoprotein Id also negatively affects TCF function in
vitro and in transfected cells (37). In contrast to the mechanism we propose for the bromocomplex, Id interferes with DNA binding by
Elk-1 and SAP-1a by acting on the Ets DNA binding domain. Similar to
the bromodomain, Id overexpression blocks signaling-driven SRE
activity. On the other hand, coexpression of Elk-1 rescues repression
by Id but not by the bromocomplex (not shown). Thus Id and the
bromodomain appear to repress SRE activity by distinct mechanisms
involving TCF.
Our transient transfection results confirm that the bromocomplex can
form on the c-fos SRE in culture cells. In the absence of
the N- and C-terminal transactivation domains of CBP, the bromodomain repressed SRE- and E74-driven transcription, and this required an
intact Elk-1-binding site on the SRE or coexpression of Elk-1 in the
case of the E74 reporter. Thus, the bromodomain interferes with the
ability of Elk-1 to transactivate. Expressing the bromodomain linked to
the CBP transactivation domains facilitated activation, in agreement
with previous studies (12-14). Janknecht and Nordheim (13) found that
the KIX domain (aa 451-721) interacted with and potentiated
MAPK-driven transactivation by the C-terminal region of Elk-1. Here we
describe another interaction that involves other regions of both
proteins. In particular, the interaction we describe is constitutive
and not dependent upon signaling. It therefore could complement or
synergize with other interactions between these two proteins.
The bromodomain of P/CAF (p300- and CBP-associated factor) has been
described to function as a link between histones and histone acetylases
(38). This raises the possibility that Elk-1 is also a target for these
enzymes and that the interactions described here are dependent on
acetylation. Our preliminary data indicate that endogenous Elk-1 may be
acetylated, which would add a level of regulation independent of
phosphorylation. Indeed, acetylation affects the enhanceosome on
the IFN- We present the following model for the signaling-driven transcriptional
activation of the SRE. The SRE is constitutively occupied by the
ternary complex and CBP in vivo (Fig.
7, upper panel). Signaling-driven phosphorylation of Elk-1, SRF, CREB, and STAT, as well
as CBP itself (13), would drive a conformational change that permits
the transactivation domains of CBP (both N- and C-terminal) to contact
the basal transcription machinery and thus potentiate the initiation of
transcription (Fig. 7, lower panel). The bromocomplex would
then constitute a key intermediate for the rapid induction of
c-fos by a multitude of signals. Furthermore, the notion of a constitutive recruitment of bromodomain-containing coactivators, such
as CBP and p300, to stable promoter complexes may also apply to other
promoters depending on their respective coactivator and function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Ets) and the
SRF antibody (NOP-13) have been described previously (19, 20).
Horseradish peroxidase-coupled secondary antibodies were from
Sigma-Aldrich. Glutathione-Sepharose, poly (dI-dC)·(dI-dC), ECL-plus
nitrocellulose membranes, and the T7 DNA polymerase sequencing kit were
purchased from Amersham Pharmacia Biotech, whereas Talon metal-affinity
resin came from CLONTECH. Transfast transfection
reagent, a TnT-coupled in vitro transcription translation
kit, Taq DNA polymerase, and the Dual Luciferase kit were
purchased from Promega. Radioisotopes, ECL reagents, and polyvinylidene
difluoride were obtained from PerkinElmer Life Sciences. Immobilon-P
polyvinylidene difluoride membrane was from Millipore. DMEM and
Dulbecco's PBS and FBS were purchased from Life Technologies. X-ray
films and intensifying screens were purchased from Kodak, and B-Per
reagent was purchased from Pierce. Nucleobond AX plasmid purification
cartridges and the NucleoSpin extract kit were obtained from
Machery-Nagel, and Jetstar plasmid cartridges came from Q-biogene.
Protease and phosphatase inhibitors were from Sigma; all other
ultra-pure biochemicals were from AppliChem (Darmstadt, Germany).
14-308) expression vectors for in
vitro transcription were generated by digesting the corresponding bacterial expression constructs and subcloning them into the pCal-n vector (Stratagene). The 3xSRE-, 1xEL-, and 3xEL-fos TATA-luciferase reporter genes were kindly provided by G. Bilbe (Novartis Pharma, Basel, Switzerland). The 4xE74-fos TATA-luciferase reporter construct was generated in this vector by inserting an
NotI-SpeI fragment containing four tandem copies
of the E74 sequence (21). The RasV12 and SV40 renilla luciferase
expression vectors were kindly provided by A. Philips (Institut de
Génétique Moléculaire de Montpellier, Montpellier,
France) and a CBPwt expression vector by R. Janknecht
(Department of Biochemistry, Mayo Clinic, Rochester, MN). The original
mouse CBPwt expression vector used for the GST- and
Gal4-CBP constructs (12, 13) contained a stop codon after aa 1286 leading to truncated proteins from the original clones spanning this
region. Therefore the vectors originally described as
GST-CBP-(1100-1460) and Gal4-CBP-(1100-1460) actually express a
protein containing CBP amino acids 1100-1286 and are labeled accordingly. Plasmids were purified using cartridge systems for in vitro manipulations and by double banding in CsCl for transfections.
70 °C.
-32P]dATP (3000 Ci/mmol, 10 µCi/µl) and 300 µM dTTP (23). The labeled fragments were purified from
acrylamide gels by electroelution. The gel retardation experiments were
carried out as previously described (23). In brief, reactions (7.5 µl) contained 4 fmol of labeled probe, a salt/protein/buffer mix, 2.5 µg of poly (dI-dC)·(dI-dC), recombinant coreSRF-(90-245) with SRE
probes, and the proteins indicated in the figures. Where appropriate,
0.5-1 µl of antibodies was added to the reaction before the addition
of protein extracts. After incubation for 30 min at room temperature,
complexes were resolved on a 5% polyacrylamide gel containing 0.5×
TBE at 1 mA/cm for 4 h. Complexes were visualized by
autoradiography and phosphorimaging of the dried gel.
-32P]dATP (3000 Ci/mmol, 10 µCi/µl)
for the HindIII end or 50 µCi of
[
-32P]dCTP (3000 Ci/mmol, 10 µCi/µl) for the
XmaI end. The gel-purified fragment was treated for 3.5 min
at 22 °C with 1% dimethyl sulfate in 60 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. Reactions were stopped as described previously (24), and the probes were subjected to
several rounds of alcohol precipitation. Binding reactions were
prepared and analyzed as for gel retardation analysis except that all
components were increased 5-fold. The complexes were visualized by
autoradiography of the wet gel. Regions of the gel corresponding to
different complexes and free DNA were electrotransferred to DEAE paper
(NA45, Schleicher and Schuell) and eluted in 0.5 M NaCl,
0.5% SDS, 20 mM Tris-HCl, pH 8.0, 0.5 µg/ml Proteinase K
at 65 °C. The eluted fragments were purified by organic extraction followed by ethanol precipitation. The fragments were incubated for 40 min at 95 °C in freshly made 1 M piperidine, and the
piperidine was removed by multiple rounds of lyophilization and
resuspension in H2O. The samples were finally resuspended
in formamide dye mix (0.3% each bromphenol blue and Xylene Cyanol FF;
10 mM EDTA, pH 7.5, 80% deionized formamide), denatured
for 5 min at 95 °C, and analyzed by denaturing electrophoresis in a
10% polyacrylamide-8.5 M urea sequencing gel. The gel was
dried, and radioactivity was visualized by autoradiography at
70 °C with an intensifying screen and phosphorimaging as described above.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The bromodomain of CBP interacts with Elk-1
in vitro. A, schematic diagram of CBP and the
different regions (striped boxes) fused to the Gal4 DNA
binding domain (unfilled box). The different functional
domains of CBP are indicated, as are the proteins described to interact
with them. The numbers correspond to the codon positions in
CBP. B, expression of various Gal4-CBP fusion proteins.
COS-7 cells were transfected with the Gal4-CBP-fusions indicated
above the lanes. 25 µg of whole cell extract were analyzed
by immunoblotting using an antiserum specific for the Gal4 DNA binding
domain (CLONTECH). C, the bromodomain of
CBP interacts with Elk-1 in solution. Pull-down assays were performed
using glutathione-agarose beads bearing GST or the GST-CBP fusion
indicated above the lanes and full-length Elk-1 produced by
in vitro translation
(35S-Elk428). The reactions were separated
on 10% SDS-PAGE, and Elk was detected by autoradiography of the dried
gel (upper panel). An aliquot of the
35S-Elk428 input was loaded as control
(1/20 input, left lane). The lower
panel shows a Coomassie Blue-stained SDS-PAGE of the GST proteins.
D, both the N-terminal and C-terminal domains of Elk-1 bind
to the bromodomain in solution. Reactions were performed and analyzed
as described in C, using glutathione-agarose beads bearing
GST or GST-CBP-(1100-1286). The different versions of
35S-Elk were produced by in vitro translation:
full-length Elk-1 (FL, amino acids 1-428), the N-terminal
307 amino acids of Elk-1 (NT), and the C-terminal
transactivation domain of Elk-1 (CT, amino acids 308-428).
Note that FL and the NT vectors generate
isoforms of Elk that start at Met-1 and at Met-55, as indicated at the
side of each autoradiogram (lower
panels) (25). The upper panel shows the Coomassie
Blue-stained protein gel. E, mapping of the N-terminal
region of Elk-1 that interacts with the CBP bromodomain. Agarose beads
were loaded with GST or GST-CBP-(1089-1196), which contains
exclusively the bromodomain and thus is labeled GST-Bromo.
The 35S-Elk-1 C-terminal truncated proteins are indicated
schematically at the right. The number corresponds to the
C-terminal amino acid. The panels show the appropriate
portion of the autoradiogram of the pull-down assay performed as
described in C. 1/20 input denotes the aliquot of
the 35S-Elk used in each reaction loaded as a control
(left lanes). We note that each mutant also generates a
second isoform that starts at Met-55, which is not shown.
Ets, the Ets DNA binding domain, aa 1-90; SRF,
the B domain that interacts with SRF, aa 148-168; SATAD,
the signaling-activated transactivation domain, aa 308-428.
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Fig. 2.
The bromocomplex forms with Elk-1 alone on
the E74 Ets protein binding site. A, gel retardation
analysis of complexes formed on a 32P-labeled E74 probe by
10 µg of whole cell extract from nontransfected COS-7 cells
(lane 1) or cells transfected with expression vectors for
Gal4-CBP-(1-451) (lanes 2) or Gal4-CBP-(1100-1286)
(lanes 3-8). The following reagents were added as indicated
above each lane: lane 4, 1 µg of purified GST
protein; lane 5, 1 µg of purified GST-Elk307;
lane 6, 1 µg of purified GST-Elk-(308-428); lane
7, 0.5 µl of anti-SRF antiserum; lane 8, 0.5 µl of
anti-Elk Ets domain antiserum. The panel shows the relevant
region of the autoradiogram of the dried polyacrylamide gel. The
bromocomplexes together with the noninduced and induced Elk-1
(P-Elk) complexes are indicated on the left, and
the GST-Elk307 and Bromo-GST Elk307 complexes
are on the right. A nonspecific complex comigrates with the
noninduced Elk-1 complex that is unaffected by the Elk-specific
antiserum. B, gel retardation experiments were performed as
in A, using whole cell extracts from COS-7 cells transfected
with Gal4-CBP-(1100-1286) and Elk-1 C-terminal truncation mutants
Elk253, Elk212, and Elk122 produced
by in vitro translation (see Fig. 1). The panel
shows the relevant region of the autoradiogram of the dried
polyacrylamide gel. The different complexes are indicated on the
right.
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Fig. 3.
The bromodomain of CBP forms a novel complex
with TCF and coreSRF on the c-fos SRE.
A, gel retardation analysis of complexes formed on a
32P-labeled SRE probe by the SRF deletion mutant
coreSRF-(90-245) and 10 µg of whole cell extract from nontransfected
COS-7 cells (lane 1) or cells transfected with expression
vectors for Gal4-CBP-(1-451) (lanes 2 and 4) or
Gal4-CBP-(1100-1286) (lanes 3, 5-12). The
following reagents were added as indicated above each lane:
lanes 6 and 7, 10- and 100-fold excesses of
double-stranded E74-binding site oligonucleotide; lane 8, 1 µg of purified GST protein; lane 9, 1 µg of purified
GST-Elk307; lane 10, 1 µg of purified
GST-Elk-(308-428); lane 11, 0.5 µl of anti-SRF antiserum;
lane 12, 0.5 µl of anti-Elk Ets domain antiserum. The
panel shows the relevant region of the autoradiogram of the
dried polyacrylamide gel. The coreSRF-(90-245)·SRE binary complex,
the TCF·coreSRF-(90-245)·SRE ternary complex and bromocomplexes
are indicated on the left, and the complexes supershifted by
SRF antiserum are on the right (filled circles).
B, the bromocomplex is partially blocked by an antiserum
directed against the Gal4 DNA binding domain. Complexes were assembled
as in A using whole cell extracts from nontransfected COS-7
cells or cells transfected with either Gal4-CBP-(1100-1286) or
Gal4-CBP-(462-662) expression vectors. As indicated above the
lanes, reactions also contained either BSA, anti-Pyk2 antiserum,
or anti-Gal4 DNA binding domain antiserum (Santa Cruz Biotechnology).
The upper panel shows the induced TCF complex from a longer
exposure of the same gel. The identities of the different complexes are
indicated to the left of the autoradiogram.
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Fig. 4.
Methylation interference analysis of binary,
ternary, and bromocomplexes formed on the c-fos
SRE. coreSRF·SRE, TCF·coreSRF·SRE, and
Gal4-CBP-(1100-1286)·TCF·coreSRF·SRE complexes were formed using
partially methylated SRE probes labeled selectively on the upper or
lower strands. The different complexes (see Fig. 2) were purified from
the gel, and their methylation sensitivity was determined by piperidine
cleavage of the isolated DNA and analysis on a 10% sequencing gel. The
identity of the different complexes is shown on the left as
is the probe strand being visualized. The sequence of the SRE probe is
indicated between the two autoradiograms. The
lines indicate the position of the corresponding residue on
the gel, the arrows underlie the dyad-symmetrical core of
SRE, and the asterisks show the two G residues on the upper
strand contacted by TCF, also indicated by TCF above the upper
strand autoradiogram.
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Fig. 5.
DNase I footprint analysis of the
bromocomplex. Binding reactions were assembled on SRE probes
32P-labeled on the upper strand or lower strand as
indicated. The reactions contained the following proteins: none
(lane 1, right and left), coreSRF and
BSA (lane 2, right), coreSRF and Elk-1
(lane 3, right; lane 2,
left), coreSRF Elk-1 and Gal4-CBP-(1-451) (lane
4, right; lane 3, left), coreSRF
Elk-1 and Gal4-CBP-(1100-1286) (lane 4, left;
lane 5, right), Elk-1 (lane 5,
left; lane 6, right),
Gal4-CBP-(1-451) (lane 7, right),
Gal4-CBP-(1100-1286) (lane 6, left; lane
8, right). After a short DNase I digestion, the
fragments were purified and analyzed on a 10% sequencing gel as in
Fig. 4. The positions of the TCF-binding site, SRF-binding site, and
the FAP sequence are shown next to the autoradiograms. The
arrowheads indicate sites protected in the bromocomplex, and
the filled circle indicates the lower strand hypersensitive
site in the bromocomplex.
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Fig. 6.
The bromodomain of CBP represses
RasV12-driven activation of SRE and E74 reporter genes in NIH3T3
cells. An expression vector for activated Ras (RasV12,
200 ng) was cotransfected with different Gal4-CBP expression vectors
(1.5 µg) into NIH3T3 cells together with a luciferase reporter gene
(300 ng) driven by either three copies of the c-fos SRE
(A), four copies of the E74 site (B and
D), or a c-fos SRE mutated in the TCF-binding
site (EL, C). A wt Elk-1 expression vector (50 ng) was cotransfected in B and D, as well as one
for Elk-3A, with three alanine point mutations in the major MAPK
phosphorylation sites, in D. The level of induction was
calculated relative to the activity of the reporter in the absence of
activation. The values represent the mean of duplicate or triplicate
samples and are representative of at least three independent
experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, TFII-I, and ASC-2
have been reported to bind to or increase the efficiency of the
SRF·SRE complex (28-36). The direct DNA requirement of these SRF-dependent interactions remains unclear, but in
transient transfections they have been shown to variously activate or
repress transcriptional activity. In contrast, the bromocomplex
involves solely the TCF component of the ternary complex, because it
showed identical behavior on the E74 site and on the SRE. Apparently,
SRF is necessary for bromocomplex formation, because it is a
prerequisite for ternary complex formation. The functional aspects of
the bromocomplex are thus equally well studied by the SRE as well as
the E74 element. It will be interesting to determine whether SRF
interacting factors that interact with SRF can synergize with CBP in
the regulation of the SRE.
gene, where the protein HMG I(Y) is acetylated
by CBP (39), leading to transcriptional repression. Interestingly, the
SRE has been shown to have a centrally positioned nucleosome adjacent
to the SRE (40), which likely contributes to the higher order structure
of the promoter in vivo and may be target for acetylation.
Our data suggest that the bromodomain could form a constitutive
preinduction complex with the SRE via TCF. This would be consistent
with the genomic footprint where a constitutive
non-signaling-dependent complex is present on the c-fos SRE. The structural changes that occur upon activation
of the c-fos promoter are most probably more complex than
our transient transfection and in vitro binding assays
suggest, considering the geometry of the promoter described by Herrera
and coworkers (40). Nevertheless, our results are a first step toward a
signaling-dependent functional and structural
reconstitution of the c-fos promoter in
vitro.
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Fig. 7.
Model for the signaling-dependent
transcriptional activation from the c-fos SRE.
The SRE is constitutively occupied by the ternary complex and CBP
through interactions between the bromodomain of CBP and TCF.
Signaling-driven phosphorylation of the proteins bound to the
c-fos promoter elements would lead to a conformational
change of CBP through interactions between different domains of CBP and
these factors as described in Fig. 1A. This would allow CBP
to contact the basal transcriptional machinery and potentiate
transcriptional initiation.
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ACKNOWLEDGEMENTS |
---|
We thank our colleagues in the Institut de Génétique Moléculaire de Montpellier, in particular the groups of C. Sardet and J.-M. Blanchard, for their advice and continued critical input during the course of these studies, R. Janknecht for the GST- and Gal4-CBP expression vectors, G. Bilbe and H. Richener for reporter gene constructs, and A. Philips for his invaluable counsel in the art of transient transfection.
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FOOTNOTES |
---|
* This work was supported in part by grants from the French Association pour la Recherche sur le Cancer and Fondation pour la Recherche Medicale, as well as from the Pharma Division of Novartis, S.A.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.
Received support for some research costs from The Danish Research Academy.
§ To whom correspondence should be addressed: Tel.: 33-467-613-667; Fax: 33-467-040-231; E-mail: hipskind@jones.igm.cnrs-mop.fr.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M007824200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; CaCRE, calcium- and cAMP-responsive element; SRE, serum response element; SIE, v-sis-inducible element; PKA, protein kinase A; CREB, cAMP response element binding protein; MAPKAP, mitogen-activated protein kinase-activated protein kinase; STAT, signal transducer and activator of transcription; SRF, serum response factor; TCF, ternary complex factor; ERK, extracellular signal-regulated kinase; SAPK, stress-activated protein kinase; CBP, CREB binding protein; aa, amino acid(s); DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; Sarkosyl, N-lauroyl sarcosine; PCR, polymerase chain reaction; BSA, bovine serum albumin; wt, wild type; coreSRF, SRF-(90-245); FAP, c-fos AP1-like element.
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