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Mol Cell Biol, April 1998, p. 2218-2229, Vol. 18, No. 4
Department of
Biochemistry1 and
Department of
Experimental Oncology,2 St. Jude Children's
Research Hospital, Memphis, Tennessee 38105
Received 21 November 1997/Returned for modification 30 December
1997/Accepted 19 January 1998
The Ets-1 transcription factor plays a critical role in cell growth
and development, but the means by which it activates transcription are
still unclear (J. C. Bories, D. M. Willerford, D. Grevin, L. Davidson, A. Camus, P. Martin, D. Stehelin, F. W. Alt, and J. C. Borles, Nature 377:635-638, 1995; N. Muthusamy, K. Barton, and
J. M. Leiden, Nature 377:639-642, 1995). Here we show that Ets-1
binds the transcriptional coactivators CREB binding protein (CBP) and
the related p300 protein (together referred to as CBP/p300) and that
this interaction is required for specific Ets-1 transactivation functions. The Ets-1- and c-Myb-dependent aminopeptidase N (CD13/APN) promoter and an Ets-1-dependent artificial promoter were repressed by
adenovirus E1A, a CBP/p300-specific inhibitor. Furthermore, Ets-1
activity was potentiated by CBP and p300 overexpression. The
transactivation function of Ets-1 correlated with its ability to bind
an N-terminal cysteine- and histidine-rich region spanning CBP residues
313 to 452. Ets-1 also bound a second cysteine- and histidine-rich
region of CBP, between residues 1449 and 1892. Both Ets-1 and CBP/p300
formed a stable immunoprecipitable nuclear complex, independent of
DNA binding. This Ets-1-CBP/p300 immunocomplex possessed histone
acetyltransferase activity, consistent with previous findings that
CBP/p300 is associated with such enzyme activity. Our results indicate
that CBP/p300 may mediate antagonistic and synergistic interactions
between Ets-1 and other transcription factors that use CBP/p300 as a
coactivator, including c-Myb and AP-1.
The c-Ets-1 (Ets-1) transcription
factor is the cellular counterpart of the v-ets
proto-oncogene product originally described as part of the tripartite
Gag-Myb-Ets fusion protein from the E26 avian leukemia virus (45,
73). Ets-1 is expressed predominantly in B and T cells of adult
mice, where it is critical for T- and B-cell function and development
(12, 50). Ets-1 often cooperates with other transcription
factors, including AP-1 (74, 78) and c-Myb (21,
66), and can be inhibited by MafB (67); however, its
mode of transactivation remains unclear.
The Ets family of transcription factors consists of about 30 members
characterized by the highly conserved Ets DNA binding domain
(73). Outside of this domain, Ets proteins are more diverse, with the exception of the Ets-1 and Ets-2 subfamily, for example (76). Ets-1 can occur in two alternatively spliced variants, p54 (54 kDa) and p68 (68 kDa), that differ in their N termini (73). p68 Ets-1 is present only in birds and reptiles, while p54 Ets-1 is more widely distributed among vertebrates and is the form
expressed in mammals (2, 3). In addition to the Ets domain,
Ets-1 and Ets-2 have similarity in the Pointed domain, so named for the
Drosophila Ets protein Pointed, which cooperates with c-Jun
and Ras in Drosophila eye development (18, 57, 72). The Pointed domain spans about 100 amino acids (aa) in the
N-terminal half of Ets-1 and lacks transactivation function when fused
to a heterologous DNA binding domain, but it is important for
synergistic activity with AP-1 and Ras in mammalian cells (38,
73-75, 78). Deletion analysis indicates that Ets-1 contains an
activation domain between the Pointed domain and the Ets domain at the
C terminus (73). Moreover, p68 Ets-1 and Ets-2 compete for a
limiting factor in transcription activation experiments, suggesting
that they have a common coactivator (73), although it is
still unclear whether p54 Ets-1 uses the same coactivator as p68 Ets-1
or Ets-2.
A growing number of transcription factors, including c-Myb and the AP-1
components Fos and Jun, use the CREB binding protein (CBP) and the
related p300 protein (together referred to as CBP/p300) to mediate the
transactivation of RNA polymerase II (34). CBP/p300 may also
act as a common mediator of synergistic and antagonistic interactions
between these factors and others that bind CBP/p300 (36, 51,
55). Physical contact between the transactivation domains and
CBP/p300 appears to be necessary, but not always sufficient, to
stimulate transcription (70). Although it is unclear how these protein-protein interactions lead to transactivation, one suggestion is that CBP/p300 acts an adaptor between the activation domain and general transcription initiation factors such as TFIID and
TFIIB, or possibly RNA polymerase II (1, 37, 39).
Alternatively, the recruitment of CBP/p300 itself may be responsible
for transactivation (56). Indeed, CBP/p300 has intrinsic
histone acetyltransferase (HAT) activity that could potentially
activate chromatin-repressed promoters and enhancers by acetylation of
histone N-terminal lysine residues or other proteins involved in
transcription (11, 56). The importance of correctly
regulated CBP-associated HAT activity in tissue-specific transcription
is underscored by the t(8;16)(p11;p13) translocation in acute myeloid
leukemias, which fuses a putative acetyltransferase to the N terminus
of CBP, presumably leading to deregulation of CBP-associated HAT
(13).
Here we show that Ets-1 binds CBP and the related p300 and that this
association mediates Ets-1 transactivation potential. Because Ets-1
often requires other CBP/p300 binding transcription factors to
transactivate target genes, these coactivators may also be critical for
mediating Ets-1-dependent transcriptional synergism.
Antibodies.
Specific antisera were purchased from Santa Cruz
Biotechnology. The CBP/p300 cocktail consisted of equal parts of the
following antisera: CBP (A-22), CBP (C-20), and CBP (451) [CBP (451)
also recognizes p300]. A-22 was used for the CBP N-terminus-specific antiserum. The p300-specific cocktail consisted of equal parts of p300
(N-15) and p300 (C-20) antisera. The 5614 and 5729 antisera were
described previously (37) and were raised against
glutathione S-transferase (GST)-CBP fusion proteins
containing CBP residues 455 to 679 and 1 to 117, respectively (a gift
from M. Montminy). The Ets-1-specific antisera, anti-Ets-1(N) and
anti-Ets-1(C), are Ets-1 (N-276) and Ets-1 (C-20), respectively. The
monoclonal antibodies that recognize the Gal4 DNA binding domain (DBD)
and E1A were RK5C1 and M73, respectively. Typically, 1 µg of each antiserum was used for each immunoprecipitation. The normal rabbit serum (NRS; Sigma) control was used at 5 µl per immunoprecipitation (1 µl for coimmunoprecipitations used in the HAT assays). Preblocked antisera were produced by incubating antisera with a 10-fold mass excess of antigenic peptide (Santa Cruz Biotechnology) for 3 h at
room temperature or overnight at 4°C. Immunoblots were developed by
enhanced chemiluminescence (Amersham).
Plasmids and transient-transfection assays.
KG1a
myeloblastic cells (ATCC CCL 246.1) were grown and electroporated with
5 µg of reporter plasmid, 5 µg of Gal fusion protein expression
plasmid, 25 ng of cytomegalovirus (CMV) E1A vectors (where applicable),
and 4 µg of Rous sarcoma virus (RSV)
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Role for CREB Binding Protein and p300
Transcriptional Coactivators in Ets-1 Transactivation
Functions
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-galactosidase (-gal) or
MAP1-SEAP internal control reporter plasmids, as previously described
(66). Larger amounts of CMV E1A sometimes led to a general
repression of reporters and expression vectors. The cells were
harvested after about 16 h, and enzyme assays were performed to
assess reporter gene expression. Reporter gene-derived chloramphenicol acetyltransferase (CAT) or luciferase activity was normalized to
-gal activity derived from the RSV -gal or Renilla
luciferase derived from pRL-TK (Promega) or to secreted alkaline
phosphatase from MAP1-SEAP internal transfection control reporter
plasmids. F9 cells were transfected in 35-mm wells with Superfect
(Qiagen) or Lipofectamine (Gibco BRL), with 1 µg of reporter, 1 µg
of pEVRFO-Ets-1 or pEVRFO, 8 µg of CMVp300 or 1 µg of pRC/RSVmCBP
HA-RK (RSV CBP) or 1 µg of RSV CBP 1-1285, and 50 ng of pRL-TK.
Equal molar amounts of a CMV plasmid religated without the
NotI-HindIII insert were used as controls in
the p300 experiments, with the total amount of DNA balanced with
pBluescript SKII (Stratagene). F9 luciferase assays were normalized to
Renilla luciferase derived from pRL-TK.
2-36 E1A (69) were gifts from
Bob Rooney. RSV CBP 1-1285 is pRC/RSV-CBP (39) with CBP
codon 1286 mutated from GAG to TAG. CMVp300 was constructed by
inserting the HindIII-NotI fragment from
pCMVp300 (22) into pCMV (Clontech). RSV CBP2 was
constructed by inserting a HindIII-XbaI
fragment containing mouse CBP sequences that lacked aa 739 to 2394 into
the RSV expression vector pGR. Gal-Ets 2-440 was constructed by
inserting a BamHI-SpeI fragment encoding mouse
p54 Ets-1 aa 2 to 440 from pEVRFO-Ets1 into the Gal4 fusion vector pM2
cut with BamHI and XbaI (53, 64).
Gal-Ets 2-165 was made by cutting pEVRFO-Ets1 with SphI, blunting with T4 DNA polymerase, and isolating the Ets-1 fragment released after BamHI digestion. This fragment was ligated
into pM2, which had been cut with HindIII, blunted with
Klenow fragment, and digested with BamHI. Gal-Ets 2-129 was
constructed by inserting the BamHI-XbaI fragment
from pEVRFO-Ets-1 into pM2. Gal-Ets 2-155, 2-177, 2-194, and 2-210
were constructed by inserting Ets-1 PCR fragments into pM2. Gal-Ets
166-194, 177-194, and 178-210 were made by PCR
site-directed mutagenesis (32). GST-CBP fusion proteins were
produced from the following pGEX-based vectors (Pharmacia): GST-CBP
553-679 (GST-KIX S/B) was a gift from M. Montminy (58); pGEX-2T-CBP 1-1891 has a mouse CBP BamHI-SmaI
fragment from pRC/RSV-CBP cloned into pGEX-2T cut with BamHI
and SmaI (39); pGEX-3X-CBP 1891-2441 contains a
SmaI-EcoRI CBP fragment from pRC/RSV-CBP cloned
into pGEX-3X cut with SmaI and EcoRI;
pGEX-4T-2-CBP 1-117 was made by cutting pRC/RSV-CBP with
NcoI, blunting with Klenow fragment, digesting with
BamHI, and ligating into pGEX-4T-2 digested with
BamHI and SmaI; pGEX-4T-2-CBP 1-141 was made by
digesting pRC/RSV-CBP with ApaI, blunting with T4 DNA
polymerase, digesting with BamHI, and ligating into
pGEX-4T-2 cut with BamHI with SmaI; pGEX-4T-2-CBP
1-270 was made by digesting pRC/RSV-CBP with KpnI, blunting
with T4 DNA polymerase, cutting with BamHI, and ligating into pGEX-4T-2 cut with BamHI and SmaI;
pGEX-4T-2-CBP 1-312 was made by digesting pRC/RSV-CBP with
EcoRV and BamHI and cloning into pGEX-4T-2 cut
with BamHI and SmaI; pGEX-2T-CBP 1-452 was made
by digesting pGEX-2T-CBP 1-1892 with EcoRI and religating; pGEX-3X-CBP 313-452 was constructed by isolating the
EcoRV-EcoRI fragment from pRC/RSV-CBP and
ligating it into pGEX-3X cut with SmaI and EcoRI;
and pGEX-3X-CBP 357-452 was made by isolating the
PvuII-EcoRI fragment from CMV CBP2 and ligating
it into pGEX-3X cut with SmaI and EcoRI
(17).
Coimmunoprecipitations. Jurkat or KG1a cells (0.8 × 108 to 1 × 108 cells) were harvested, washed once with short-term labeling medium (phosphate- or methionine-free RPMI 1640, 5% dialyzed fetal bovine serum), resuspended at 5 × 106 cells/ml in this medium, and incubated for 20 min at 37°C to deplete intracellular pools of methionine or phosphate. The cells were then incubated in 20 ml of fresh short-term labeling medium containing 0.18 mCi of [35S]methionine per ml (for 3 h) or 0.15 mCi of [32P]orthophosphate per ml (for 2 h). The cells were harvested and washed twice with 20 ml of ice-cold phosphate-buffered saline, and nuclear extracts were prepared as previously described (65), except that the buffers also contained 0.5 µg of leupeptin per ml (and sometimes 10 ng of calyculin per ml), and used for coimmunoprecipitation. The first immunoprecipitation was performed by adding antiserum and 50 µl of protein A-Sepharose (50% slurry) to about 80 to 100 µl of nuclear extract diluted with 3 volumes of ice-cold PC+100 buffer (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 5 mM MgCl2, 0.1% Nonidet P-40, 20% glycerol, 0.01% bovine serum albumin [BSA], 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg of leupeptin per ml) followed by rotation at 4°C for 3 h (58). The first immunoprecipitation pellet was washed twice with 0.5 ml of ice-cold PC+100 buffer, and antigens were released by boiling for 2 min in 100 µl of boiling buffer (20 mM Tris-HCl [pH 8.0], 0.5% sodium dodecyl sulfate [SDS], 1 mM dithiothreitol). For experiments showing a primary immunoprecipitate of Ets-1 and CBP/p300, PC+100 containing 400 mM KCl (PC+400) and 1% BSA was used in the immunoprecipitation, and for the four subsequent 1-ml washes, PC+400 containing 0.01% BSA was used. After centrifugation, the supernatant was removed, diluted with 4 volumes of RIPA buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate), and used in a second immunoprecipitation with 50 µl of antibody-protein A-Sepharose (50% slurry) at 4°C for 3 h or overnight. The immunocomplex was washed three times with 0.5 ml of RIPA buffer (containing 0.1% SDS), and the pellet was boiled in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Samples were sometimes normalized for counts per minute before SDS-PAGE (large format for examining primary immunoprecipitates, which were not normalized) and fluorography of the dried gel.
Coimmunoprecipitation HAT assays. Nuclear extracts from nonlabeled KG1a or Jurkat cells were prepared as described above. Extracts were precleared by adding protein A-Sepharose and NRS, and the supernatants were used for immunoprecipitations, as detailed above. Each immunoprecipitation experiment was performed in quadruplicate, with each immunocomplex washed three times with ice-cold PC+100 buffer. HAT assays were performed as previously described by Bannister and Kouzarides (11), except that phosphocellulose filters were washed with the buffer used by Ogryzko et al. (56). HAT assays based on histones or BSA control were performed in duplicate with four separate immunoprecipitations for each antiserum. Background counts per minute were determined from HAT assays performed with two NRS immunoprecipitations without additional protein substrates. The average background counts per minute (usually about 100) was subtracted from the experimental values.
Coimmunoprecipitation of GST-Ets-1 and CBP 1-714. 35S-labeled CBP 1-714 protein was produced with a coupled reticulocyte lysate system (Promega), using CMV CBP2 plasmid cut with SphI (17). GST-Ets-1 fusion protein (GST-Ets 2-440) was eluted from glutathione-agarose (GSH beads; Sigma) after expression in Escherichia coli. Coimmunoprecipitation was carried out by incubating about 0.5 µg of GST-Ets 2-440 and 1 µl of CBP 1-714 in 30 µl of PC+100 buffer for 20 min at room temperature. After the addition of antiserum, the immunocomplexes were washed and analyzed by SDS-PAGE followed by fluorography.
Coimmunoprecipitation of Ets-1 and CBP 739-2394.
Jurkat
cells (45 × 106 to 50 × 106 cells)
were electroporated with 50 µg of the RSV CBP2 plasmid at 960 µF
and 250 V. Then 180 × 106 to 200 × 106 electroporated cells were pooled and labeled overnight
in 60 ml of methionine-free RPMI 1640 supplemented with 10% regular RPMI 1640, 10% fetal bovine serum, and 0.12 mCi of
[35S]methionine per ml, before preparation of nuclear
extracts and coimmunoprecipitation.
GST pull-down assays. GST fusion proteins were purified from E. coli, and GST pull-down assays were performed as previously described (58). GSH beads were preblocked for nonspecific protein interactions by using NRS and two washes with PC+100 buffer (58). In vitro-transcribed and -translated [35S]methionine-labeled protein (2 to 4 µl) was added to the GSH beads, and the mixture was stirred for 30 min. The GSH beads were washed three to four times and boiled in 20 µl of 2× SDS gel sample buffer before SDS-PAGE was performed. Quantitation was performed with Molecular Dynamics Storm.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Transactivation of the Ets-1- and c-Myb-dependent CD13/APN promoter
requires CBP/p300.
Transcriptional activation of the
CD13/APN gene in hematopoietic cells of the myeloid lineage
depends upon c-Myb and Ets-1 binding sites in the promoter
(66). Since c-Myb has been shown to use CBP as a
coactivator, we sought to determine if CBP/p300 was necessary for
c-Myb/Ets-1 synergism on the CD13/APN promoter (20, 55). To
this end, we used the adenovirus 12S E1A protein, which specifically
binds the CH3 region of CBP/p300 and inhibits associated transactivator
function (5, 44). 12S E1A repressed CD13/APN promoter
activity about sixfold in transiently transfected myeloblastic KG1a
cells (Fig. 1A). By contrast, an E1A
mutant incapable of binding CBP/p300 (2-36 E1A) had no effect
on CD13/APN promoter activity (Fig. 1A) (69). Thus, CBP/p300
appears to be required for activation of the CD13/APN promoter in
myeloid cells. To ascertain if E1A was targeting the Ets-1 component of CD13/APN promoter activity, we tested this promoter when the c-Myb binding site was mutated (66). Although mutation of the
c-Myb binding site reduced CD13/APN activity about 40-fold, the
residual Ets-1-dependent activity was further repressed about 5-fold by 12S E1A (Fig. 1B), suggesting that E1A indeed targets Ets-1
transactivation function.
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Ets-1), p300 (CMVp300), or empty vector (CMV). Luciferase
activity derived from the CD13/APN reporter was normalized to
Renilla luciferase derived from the internal control
reporter pRL-TK. (G) Full-length CBP potentiates Ets-1 activity in F9
cells. Transfections were performed as in panel F, except that
full-length CBP (RSV CBP) was compared to a CBP expression vector
(RSV CBP 1-1285) containing a nonsense mutation at CBP codon 1286. Results are means and standard errors (n 
2).
CBP/p300 is necessary for Ets-1-mediated transactivation.
12S
E1A-mediated repression of the CD13/APN promoter lacking c-Myb binding
sites suggested that Ets-1 also uses CBP/p300 as a coactivator. To more
rigorously test this prediction, we assessed the effects of 12S E1A on
the transactivation potential of a Gal-Ets 2-440 fusion protein
containing the Gal4 DNA binding domain (Gal DBD) fused to mouse Ets-1
residues 2 to 440 (wild-type Ets-1 is 1 to 440); this strategy allows
one to determine Ets-1 transactivation function without interference
from endogenous Ets proteins. Gal-Ets 2-440 transactivated a reporter
gene containing five Gal4 binding sites (G5B CAT) about 33-fold more
efficiently than did Gal DBD in the presence of 2-36 E1A (Fig. 1C)
or empty expression vector (data not shown). However, Gal-Ets 2-440
was inhibited approximately fourfold by 12S E1A, indicating that Ets-1
requires CBP/p300 as a coactivator in KG1a cells. To control for the
specificity of 12S E1A-dependent inhibition of Gal-Ets 2-440, we also
used a CBP-independent activator, Gal-CREB 160-284, which fuses the
Gal DBD to the glutamine-rich activation domain of CREB, termed Q2 (Q2
binds the TFIID component dTAF110 [16, 25, 61, 77]). Gal-CREB 160-284 stimulated the reporter about 12-fold compared to Gal
DBD in KG1a cells, but, in contrast to Gal-Ets 2-440, its activity was
stimulated about 2.5-fold by 12S E1A, demonstrating the specificity of
12S E1A repression on Gal-Ets 2-440 activity (Fig. 1C). The divergent
effects of 12S E1A on Gal-Ets 2-440 and Gal CREB 160-284 suggested
that E1A was not repressing Gal-Ets 2-440 by lowering its expression
(both Gal fusion proteins are expressed from similar simian virus 40 early-promoter/ori-driven expression vectors). We confirmed this by
comparing Gal-Ets 2-440 expression in KG1a cells cotransfected with
12S E1A or 2-36 E1A (Fig. 1D). Furthermore, the 2-36 E1A
protein was expressed comparably to 12S E1A in KG1a cells (Fig. 1E),
consistent with the notion that the ability of E1A to inhibit Ets-1 is
dependent on E1A N-terminal residues implicated in binding CBP/p300
(69).
CBP/p300 and Ets-1 associate in nuclear extracts. Inhibition of Gal-Ets 2-440 activity by 12S E1A, and synergism between Ets-1 and CBP/p300, indicated that Ets-1 may interact with CBP/p300 in the nucleus. We tested this possibility by coimmunoprecipitating Ets-1 and CBP/p300 with an Ets-1 N-terminus-specific antiserum [anti-Ets-1(N)] from nuclear extracts prepared from Jurkat T cells labeled with [35S]methionine (Fig. 2A). To confirm the SDS-PAGE positions of Ets-1 and CBP/p300 following a primary immunoprecipitation we performed in parallel two sequential immunoprecipitations, the first under mild buffer conditions followed by antigen release and the second under more stringent conditions (Fig. 2A, lane 7 for Ets-1 and lanes 9 and 10 for CBP/p300 [note the broad p300 band]). Bands corresponding to CBP/p300 and Ets-1 were present in the primary immunoprecipitation with anti-Ets-1(N) (lane 3), but not when this antiserum was preblocked with an excess of peptide antigen (lane 2). Interestingly, an Ets-1 C-terminus-specific antiserum [anti-Ets-1(C)] that was more efficient at immunoprecipitating Ets-1 was noticeably less proficient at coimmunoprecipitating CBP/p300 (lane 1). This suggests that in nuclear extracts the anti-Ets-1(C) antiserum disrupts an Ets-1-CBP/p300 complex or preferentially immunoprecipitates Ets-1 that is not complexed with CBP/p300. Although anti-Ets-1(N) and a p300 N-terminus-specific antiserum [anti-p300(N)] appear to immunoprecipitate similar amounts of p300 (compare lanes 3 and 5), this is probably due to the inability of the commercial antisera to immunoprecipitate more than a small fraction of CBP/p300 in extracts (15a). The presence of CBP/p300 in the Ets-1 immunoprecipitate was confirmed by immunoprecipitation of KG1a nuclear extracts followed by immunoblotting with the CBP/p300-specific antisera 5614 and 5729 (Fig. 2B). We also observed Ets-1 in immunoprecipitates with antisera 5614 and 5729 (but not NRS), suggesting that CBP/p300 antisera could also coimmunoprecipitate Ets-1 and CBP/p300 (data not shown). Thus, anti-Ets-1(N) specifically recognized a complex containing Ets-1 and CBP/p300 in KG1a myeloblastic and Jurkat T-cell nuclear extracts. Together, these results suggest that the amount of Ets-1 complexed with CBP/p300 is variable, depending on the cell type and probably other factors.
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Ets-1
(N), Ets-1-CBP/p300 immunocomplexes have HAT activity. Recently, CBP and p300 have been shown to have intrinsic HAT activity and to bind the HAT protein PCAF, suggesting that HAT activity should be present in the Ets-1-CBP/p300 immunocomplex (11, 56, 79). To confirm this prediction, we performed immunoprecipitations with KG1a (Fig. 2D) and Jurkat (Fig. 2E) nuclear extracts with anti-Ets-1(N) and CBP N-terminus-specific antiserum [anti-CBP(N)], followed by HAT assays of the immunocomplexes with [3H]acetyl coenzyme A and BSA or histones as protein substrates. The anti-Ets-1(N) and anti-CBP(N) immunocomplexes contained acetyltransferase activities that were specific for histones, while the NRS control showed little activity. Similar results were obtained with p300-specific antiserum (data not shown). Moreover, preblocking anti-Ets-1(N) and anti-CBP(N) with their respective antigenic peptides significantly reduced HAT activity, showing that the presence of HAT correlates with the presence of an Ets-1-CBP/p300 complex and CBP in the respective immunocomplexes (Fig. 2D and 2E). Intriguingly, the similarity in HAT activity between the Ets-1 and CBP/p300 immunoprecipitations could be due to intrinsic differences in HAT function between an Ets-1-CBP/p300 complex and a CBP or p300 complex without Ets-1. While other factors such as inefficient immunoprecipitation or HAT inhibition by CBP or p300 antibodies may also account for this observation, we are investigating if Ets-1 modulates HAT activity as part of its transactivation functions.
Ets-1-CBP/p300 association is independent of DNA binding. Our coimmunoprecipitation data suggested that Ets-1 binds CBP/p300 by protein-protein interactions. To rule out an indirect association between Ets-1 and CBP/p300 through a contaminating nucleic acid "link" between the Ets-1 DBD domain and a DNA binding CBP/p300 complex, we used ethidium bromide to disrupt protein-DNA interactions (30, 40). Immunoprecipitations with Ets-1 antiserum (and control antisera and washes) were performed with buffer containing 50 µg of ethidium bromide per ml, a concentration that preferentially inhibits DNA-protein interactions (30, 40). The Ets-1-CBP/p300 complex coimmunoprecipitated from Jurkat nuclear extracts was insensitive to ethidium bromide (Fig. 3A, compare lanes 3 and 11), suggesting that Ets-1 and CBP/p300 interact through protein-protein contacts. Similar results were obtained with KG1a cell nuclear extracts (Fig. 3B). Although the Ets-1-CBP/p300 coimmunoprecipitation was unaffected by ethidium bromide, several other bands were lost (Fig. 3A, compare lanes 1 to 6 with lanes 9 to 14), showing that ethidium bromide did affect other proteins associated with the immune complex. Consistent with the results in Fig. 2, addition of anti-Ets-1(N) antigenic peptide blocked coimmunoprecipitation of Ets-1 and CBP/p300 (Fig. 3, compare lanes 2 and 5 and lanes 10 and 13 for Ets-1; compare lanes 3 and 6, lanes 11 and 14, and lanes 18 and 19 for CBP/p300). Thus, the physical association of Ets-1 with CBP/p300 in nuclear extracts appears to be independent of DNA binding.
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Ets-1 associates with the N terminus of CBP.
To define regions
of CBP necessary for Ets-1 interactions, we performed
coimmunoprecipitations in vitro with GST-Ets 2-440 fusion protein
purified from E. coli and in vitro-translated CBP containing
residues 1 to 714. CBP 1-714 was specifically coimmunoprecipitated with two different Ets-1-specific antisera only in the presence of
GST-Ets 2-440 (Fig. 4A). This suggested
that CBP residues 1 to 714 were sufficient to bind GST-Ets 2-440 and
that other cellular factors, unless present in the reticulocyte lysate,
were not necessary to mediate the Ets-1-CBP interaction. To confirm
this result by using nuclear extracts, we transiently transfected
Jurkat cells with a plasmid expressing a mutated CBP gene lacking
residues 739 to 2394 (CBP 739-2394; wild-type CBP is 1 to 2441)
(17). Consistent with the in vitro data,
coimmunoprecipitation with a nuclear extract from these cells showed
that CBP 739-2394 and endogenous CBP/p300 coimmunoprecipitated with
anti-Ets-1(N) (Fig. 4B, lanes 11 and 13). However,
anti-Ets-1(C) was much less efficient at coimmunoprecipitating
CBP/p300 and CBP 739-2394 from Jurkat nuclear extracts (Fig. 4B,
lane 10), even though it could coimmunoprecipitate CBP 1-714 in vitro
(Fig. 4A, lane 6). Therefore, there are differences in CBP-Ets-1
interactions in nuclear extracts from those in more purified systems.
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Ets-1 binds two regions of CBP. To determine the Ets-1 binding region within the first 714 residues of CBP, we performed GST `pull-down' assays with in vitro-translated Ets-1 and GST-CBP fusion proteins purified from E. coli. About 25% of the input Ets-1 was retained on GST-CBP 1-452 or GST-CBP 313-452 prebound to GSH beads (Fig. 5A), localizing the interaction domain to CBP 313-452. By contrast, Ets-1 did not bind other GST fusion proteins containing the CBP N-terminal 312 residues (Fig. 5A). Nor did GST-CBP 1892-2441 (Fig. 5B) or GST-CBP 553-679 (data not shown) bind Ets-1, in contrast to GST-CBP 357-452, further localizing an Ets-1 interaction domain on CBP to residues 357 to 452 (Fig. 5B, lane 12). CBP 357-452 sequences include the cysteine- and histidine-rich CH1 region, which has been proposed to contain the TAZ motif (transcriptional adaptor putative zinc fingers; Cys-X4-Cys-X8-His-X3-Cys) (60). The ability of Ets-1 to bind the CBP CH1 region indicated that it may also bind to another CBP cysteine- and histidine-rich region with TAZ motifs (CH3) (60). Since CH3 approximately spans CBP residues 1676 to 1849, we tested whether Ets-1 would bind a GST-CBP protein containing CBP residues 1 to 8 fused to residues 1459 to 1892. Figure 5C shows that Ets-1 was bound by GST-CBP 1-8+1459-1892 to approximately the same extent as by GST CBP 313-452 and GST CBP 357-452. Thus, Ets-1 can bind at least two separate regions of CBP that are rich in cysteines and histidines and have TAZ motifs.
|
Ets-1 transactivation function correlates with binding to CBP
residues 1 to 714.
To more precisely correlate Ets-1 sequences
that bind CBP 1-714 and also activate transcription, we performed
pull-down assays with GST-Ets-1 fusion proteins and transactivation
assays with the cognate Gal-Ets-1 fusion genes in KG1a cells. Figure
6A and B shows the
results of a typical pull-down assay; the results of four independent
experiments are summarized in Fig. 6C. Figure 6C also summarizes the
relative activities of the Gal-Ets-1 fusions from three experiments.
GST-Ets-1 proteins fell into three classes based on their ability to
bind CBP 1-714 (Fig. 6C): weak binders bound less than 6% of that
seen with GST-Ets 2-440 (GST, GST-Ets 2-129, and GST-Ets 2-153);
moderate binders bound about 30 to 60% of that seen with GST-Ets
2-440 (GST-Ets 2-177, GST-Ets 166-194, GST-Ets 178-194, and
GST-Ets 178-210); and a strong binder bound about 150% of that
seen with GST-Ets 2-440 (GST Ets 2-210). This grouping correlated
well with the activities of the Gal-Ets-1 fusions (Fig. 6C): poor
activators had less than about 2.5% of the activity of Gal-Ets 2-440
(Gal DBD, Gal-Ets 2-129, and Gal-Ets 2-153); moderate activators had
between 9 and 17% of Gal-Ets 2-440 (Gal-Ets 2-165, Gal-Ets 2-177,
Gal-Ets 166-194, Gal-Ets 178-194, and Gal-Ets 178-210); and
strong activators had about 130 to 150% the activity of Gal-Ets 2-440
(Gal-Ets 2-194 and Gal-Ets 2-210). Similar amounts of GST-Ets-1
fusion proteins were used (Fig. 6B), and the Gal-Ets-1 fusions were
expressed comparably (Fig. 6D and E). Thus, it appears that Ets-1
residues between 153 and 210 are necessary for full activity in KG1a
cells and efficient binding to CBP 1-714 in vitro.
|
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our findings indicate that Ets-1 physically interacts with two distinct but similar regions of CBP, CH1 (aa 357 to 452) and CH2/CH3 (aa 1459 to 1892). Mapping the sequences of Ets-1 that bind the CH1 region revealed that the amino-terminal half of Ets-1 (aa 1 to 210), including the Pointed domain (aa 33 to 130) and a portion of the activation domain (aa 131 to 270) (75), is sufficient for strong interaction and that residues between 153 and 210 are necessary for optimal Ets-1-CH1 binding. The Pointed domain is not sufficient, and it is unclear if it is necessary, for binding. Moreover, the binding efficiency of Ets-1 to the CH1 region qualitatively correlated with its ability to transactivate Gal4 promoter constructs (Fig. 6), demonstrating that the Ets-1-CBP/p300 interactions are functionally significant. Testing of Ets-1 internal deletions and truncations in this region suggests that there is not a singular compact CBP N-terminus-binding motif but that multiple residues may contribute to binding and transactivation. Whether Ets-1 transactivation functions also correlate with binding to the CBP carboxyl-terminal CH2/CH3 region remains to be shown, but the presence of the TAZ motif in both the CH1 and CH3 regions suggests that Ets-1 interacts similarly with both of these regions (60). We do not know if Ets-1 affects the binding of the coactivator accessory factors RNA helicase A, TFIIB, or PCAF to the CH3 region. It is also possible that the Ets-1-CH2/CH3 interaction will show a stronger quantitative correlation between binding and transactivation than that seen with Ets-1-CH1, although in the latter instance the lack of a strict quantitative linear relationship may be due to comparisons of protein-protein interactions in vivo and in vitro. It is also conceivable that binding of CBP/p300 by Ets-1 is not sufficient for transactivation, which would be consistent with an exacting quantitative comparison of the transactivation and Ets-1-CBP 1-714 binding assays.
Functional synergism between Ets-1 and c-Myb was first noted with the tripartite Gag-Myb-Ets fusion protein isolated from the E26 avian leukemia virus, where expression of the fusion protein or coexpression of individual c-Myb and Ets-1 proteins results in higher rates of transformation than those seen upon expression of either protein alone (26, 48). In addition, c-Myb and Ets-1 can act synergistically to transactivate the myeloid cell-specific CD13/APN promoter (66), and Gag-Myb-Ets activates many promoters, including mim-1 (21). These examples of functional synergy suggest that Ets-1 and c-Myb may also interact physically. However, aside from the Gag-Myb-Ets protein, such interactions have been difficult to demonstrate (21, 28, 47-49), indicating an additional level of complexity and perhaps involving a contribution from accessory or bridging molecules. CBP/p300 fits the requirements for such a bridging molecule. Indeed, preliminary experiments suggest that Ets-1 mutants deficient for binding the CBP CH1 region are also impaired in their ability to synergize with c-Myb on the CD13/APN promoter in C33A cells (data not shown). c-Myb binds to CBP residues 590 to 699 within the KIX domain (20, 55), a region physically distinct from sites that bind Ets-1 (Fig. 5), raising the possibility of a ternary complex of Ets-1-CBP/p300-c-Myb. Transcriptional synergism could therefore result from increases in CBP/p300 binding affinity and concomitant contact with the basal transcriptional machinery or from alteration of CBP/p300-associated enzymatic activities. However, even in the absence of ternary-complex formation, CBP/p300 could mediate c-Myb/Ets-1 cooperation through other mechanisms, such as kinetic synergism, whereby Ets-1 and c-Myb would sequentially act upon CBP/p300 (31). Studies investigating these possibilities are under way.
The concurrent binding of c-Myb and Ets-1 by CBP/p300 may also explain other attributes of the Gag-Myb-Ets fusion protein. Gag-Myb-Ets represses transactivation by the retinoic acid receptor (RAR) and thyroid hormone receptor (TR) (62), which also bind and require CBP/p300 to function (19, 36). Gag-Myb-Ets would be predicted to bind CBP/p300, since the CBP/p300-binding regions of c-Myb and Ets-1 are both present in the oncogene (42, 54), which might sequester CBP/p300 or impede its binding to RAR and TR. In addition, expression of Gag-Myb-Ets overcomes the inhibition of AP-1 activity by RAR and TR (62), and since the AP-1 components Jun, Fos, and JunB also interact with CBP/p300 (10, 41, 57), Gag-Myb-Ets may block RAR and TR binding to CBP/p300, thus allowing AP-1 to act productively. Indeed, competition for limiting CBP has been invoked to explain cross-talk among various signaling pathways as well as the multiple developmental anomalies associated with Rubinstein-Taybi syndrome (33, 36, 51, 59).
Direct demonstration of the functional necessity of CBP/p300 for
transactivation is not always straightforward due to a lack of CBP and
p300 mutant cell lines. Another option is to test if CBP/p300 can
augment transcription factor activity. Indeed, we observed a
synergistic effect of CBP and p300 on Ets-1 activity in F9 cells (Fig.
1), the modest nature of which may be due to the low activity of Ets-1
or to the reporter/cell system used. However, this is not uncommon,
since several other studies have noticed similarly weak (2.5-fold or
less) effects of CBP/p300 on CREB (7, 52), MyoD
(23), E47 (23), c-Jun (68), p65 (27), and Sap-1a (35). In other systems, effects
larger than fourfold can be observed (9, 29), sometimes
after cotransfection of large amounts of CBP or p300 expression vector
(39, 44). Alternatively, the ability of the 12S E1A protein
to inhibit CBP-dependent transcriptional activity depends on its
ability to bind CBP/p300, providing a reliable indicator of
CBP/p300-dependent transactivation (5, 44). Consistent with
a role for functional CBP/p300 in Ets-1 transactivation function, 12S
E1A specifically inhibited both CD13/APN promoter and Gal-Ets 2-440
activity (Fig. 1A to C), while the 12S 2-36 E1A mutant was not
inhibitory to reporter levels. However, in accord with other systems
(7), overexpression of CBP or p300 in KG1a cells does not
significantly augment Gal-Ets 2-440 activity or CD13/APN promoter
activity (data not shown), possibly because Ets-1 activity in KG1a
cells is not limited by CBP/p300 or perhaps requires additional
factors. Interestingly, Gal-Ets 2-440, which showed CBP/p300-dependent
transcriptional activation in KG1a myeloid cells, was virtually
inactive in Jurkat T cells (data not shown), although we could clearly
show an Ets-1-CBP/p300 complex in both of these cell types. Thus,
Ets-1-CBP/p300 binding appears not to be sufficient for Ets-1
activation. This observation may also explain why Ets-1 and c-Myb
transcriptional synergy does not occur on every promoter containing
c-Myb and Ets sites (66). Perhaps additional requirements
for c-Myb and Ets-1 cooperation are needed, including binding-site
geometry and/or other promoter- or cell-type-specific considerations.
In this regard, phosphorylation of Ets-1 Thr38 does not appear to be
necessary for binding to the CBP CH1 region, even though there are
reports that Thr38 is critical for Ets-1 transactivation function
(75, 78). One possibility is that Thr38 phosphorylation is
required in addition to CBP/p300 binding for transactivation to occur.
Indeed, this could explain why we could coimmunoprecipitate
Ets-1-CBP/p300 from Jurkat cells where Gal-Ets 2-440 was
inactive. Alternatively, Thr38 phosphorylation may not be required for
all Ets-1 transactivation functions; in this light, one of us
(L.H.S.) has observed that mutation of Thr38 to Ala has no effect
on Ets-1 transactivation or cooperation with c-Myb on the CD13/APN
promoter (data not shown). The Gal-Ets-1 system will facilitate our
analysis of this intriguing component of transcription factor
interaction and tissue specific-gene regulation.
We also showed a physical interaction between Ets-1 and CBP/p300 in nuclear extracts and with semipurified proteins, supporting the notion that direct contact between Ets-1 and CBP/p300 is required for transcriptional activity. Previously, CREB and CBP interactions were demonstrated by a mammalian cell two-hybrid protein interaction assay (17). We were unable to demonstrate an Ets-1-CBP interaction by this method for several cell types, including Jurkat and KG1a cells (data not shown), although it is not uncommon for results from two-hybrid assays to disagree with protein binding data derived by other methods (4).
The results from the coimmunoprecipitation assays argue strongly that p300 and probably CBP are complexed with Ets-1 in the nucleus. Because our CBP/p300 antiserum cocktail recognized both CBP and p300, we cannot be certain that CBP is complexed with Ets-1 in these assays. However, the likelihood of such complexes appears high, based on our in vitro CBP-Ets-1 binding data (Fig. 4 and 5). It should also be noted that two protein species were usually present in the CBP/p300 control immunoprecipitations from Jurkat nuclear extracts (Fig. 2C, lane 15), consistent with the multiple CBP and/or p300 species observed by other workers (8, 37). The difference between these species is unclear because the cocktails we used included both N- and C-terminus-specific CBP and p300 antisera, precluding the identification of specific epitopes present in each band. The more slowly migrating species is generally more abundant in the Ets-1 coimmunoprecipitations, perhaps indicating that this protein is preferentially associated with Ets-1 in these extracts. This observation will form the basis for further studies.
In a broader context, our findings suggest that CBP/p300 interactions may play a role in the unique transforming properties of the Gag-Myb-Ets oncoprotein. Furthermore, since CBP and p300 integrate multiple intracellular signals for transcription (19, 33, 34, 36, 51) and since Ets-1 functions primarily in combination with other transcription factors (14, 15, 24, 43, 63, 66, 67, 74, 78), it follows that CBP/p300 is an important mediator of synergistic and antagonistic interactions between Ets-1 and its numerous transcription factor partners. Not surprisingly, some of these Ets-1-cooperating factors also bind CBP/p300; one example is AP-1, which synergizes with Ets-1 and Ras on specific promoters (74, 78). In a similar context, CBP/p300 has recently been implicated in the mediation of Ras-dependent AP-1/Ets-2 synergy (33). It is also intriguing to speculate that CBP/p300 may be the target of the Ets-1 repressor MafB (67).
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ACKNOWLEDGMENTS |
|---|
We thank Xiaoying Wang, Mike Long, and Geli Gao for technical assistance; Marc Montminy for the gift of GST-KIX (S/B) plasmid and CBP antisera; Richard Goodman and Roland Kwok for pRC/RSV mCBP HA-RK; Bob Rooney for the E1A expression vectors; and Barbara Graves for the Ets-1 mutant constructs. We also thank David Shapiro, Barbara Graves, and John Cleveland for helpful comments on the manuscript.
This work was supported by NIH grants CA70909 (to L.H.S.) and RO1 CA76385 (to P.K.B.), National Cancer Institute Cancer Center Support (CORE) grant P30 CA21765, and the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children's Research Hospital.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN, 38105. Phone: (901) 495-2522. Fax: (901) 525-8025. E-mail: paul.brindle{at}stjude.org.
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Materials & Methods
Results
Discussion
References
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Mol Cell Biol, April 1998, p. 2218-2229, Vol. 18, No. 4
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