From the Department of Pharmacological and
Physiological Sciences and the ¶ Department of Molecular
Microbiology and Immunology, Saint Louis University,
Saint Louis, Missouri 63104
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ABSTRACT |
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The ability of cAMP response-element binding
protein (CREB)-binding protein (CBP) to function as a co-activator for
a number of transcription factors appears to be mediated by its ability to act as a histone acetyltransferase and through its interaction with
a number of other proteins (general transcription factors, histone
acetyltransferases, and other co-activators). Here we report that CBP
also interacts with a novel ATPase termed
Snf2-Related CBP
Activator Protein (SRCAP). Consistent with this
activity, SRCAP contains the conserved ATPase domain found within
members of the Snf2 family. Transfection experiments demonstrate
that SRCAP is able to activate transcription when expressed as a
Gal-SRCAP chimera and that SRCAP also enhances the ability of CBP to
activate transcription. The adenoviral protein E1A was found to disrupt interaction between SRCAP and CBP possibly representing a mechanism for
E1A-mediated transcriptional repression.
CREB1-binding protein
(CBP) has been found to function as a co-activator for a growing number
of sequence specific transcription factors including CREB, the STATs,
and the nuclear receptors (1-5). Binding studies have identified
several regions of CBP that interact with general transcription factors
such as TBP, TFIIB, and RNAP II (2, 6-8), suggesting it functions as a
co-activator in part by recruiting these proteins to the promoter. CBP
has also been shown to have intrinsic histone acetyltransferase (HAT)
activity and to bind to several proteins with HAT activity (P/CAF,
ACTR, NCoA-1). This suggests that CBP alone, or acting in conjunction with these proteins, functions as a co-activator by stimulating remodeling of chromatin (9-12). This is supported by the work of Korus
et al. (13) who demonstrate that several transcription factors have a specific requirement for the HAT activity of NCoAs, P/CAF, and CBP for activation of transcription. The adenoviral protein
E1A also binds CBP but represses the ability of CBP to function as a
co-activator for CREB as well as a number of other transcription
factors (4, 5, 14, 15). This appears to be due in part to the ability
of E1A to prevent binding of P/CAF and P/CIP to the C-terminal end of
CBP. E1A also binds to the N-terminal end of CBP and suppresses the
ability of a Gal-CBP-(1-450) chimera to activate transcription.
Although P/CAF also binds to this same region, competition between
P/CAF and E1A binding has not been demonstrated (5).
Deletion of amino acids 1-460 abolishes the ability of CBP to serve as
a co-activator for CREB and STAT-1 but not for other transcription
factors such as the retinoic acid receptor (5, 6). In
"squelching-type" assays, overexpression of CBP amino acids 1-460
has also been found to block the ability of full-length CBP to activate
CREB-mediated transcription (5). Studies from several laboratories
indicate that this region of CBP contacts proteins, including TBP and
P/CAF, which may be involved in the activation of transcription (5, 6).
Microinjection studies support such a role for P/CAF by demonstrating
that antibodies against P/CAF block the ability of a pGal-CBP-(1-450)
chimera to activate transcription (13).
We have previously used studies with Gal-CBP chimeras to more precisely
localize the transcription activation domain within the N-terminal end
of CBP to amino acids 227-460 (5). To identify proteins that contact
this region of CBP, we have utilized a yeast two-hybrid screen. Using
this approach, we have identified a novel member of the Snf2
family of proteins and have termed it SRCAP (Snf2
Related CBP Activator
Protein).
SRCAP contains the conserved ATPase domain found within other
Snf2 family members, and here we demonstrate that immunopurified SRCAP functions as an ATPase. Results of co-transfection experiments indicate that the Gal-SRCAP chimera can activate transcription of a
Gal-CAT reporter gene and that SRCAP can also enhance the ability of
CBP to activate transcription. This suggests that in some circumstances
SRCAP may function as a co-activator. The interaction of CBP and SRCAP
is blocked by the adenoviral protein E1A, suggesting a novel mechanism
for E1A-mediated transcriptional repression.
Plasmids--
To generate the plasmids
pGal-CBP-(227-410), pGal-CBP-(280-460), and
pGal-CBP-(227-460), PCR was used to generate a cDNA fragment encoding CBP amino acids 227-460, 280-460, and 227-410, which contain 5' XbaI and 3' BamHI sites. These were
subcloned into the plasmid pRSV Gal-(1-147) (6) digested with
XbaI and BamHI. To generate the plasmid pVP16,
PCR was used to generate a cDNA fragment that encoded a methionine
followed by amino acids 412-491 of VP16 (16) and which contained a 5'
XbaI site and a 3' EcoRI site. This was subcloned
into the plasmid pcDNA 3.1 (Invitrogen) digested with
XbaI and EcoRI. The plasmid pVP16-clone 11 was
generated by subcloning the clone 11 cDNA insert, obtained by
EcoRI and BamHI digestion of the pGAD clone 11, into EcoRI- and BamHI-digested pVP16. The pAS1
CBP-(227-460) plasmid was made using PCR to generate a cDNA
fragment encoding amino acids 227-460 of CBP, which contained EcoRI restriction sites at the ends. This was subcloned into
the EcoRI site of the plasmid, pAS1. The plasmid pAS1 and
the HeLa pGAD library were a gift of Paul MacDonald, St. Louis
University, St. Louis MO. The pGal-VP16 chimera was a gift of R. Maurer, Oregon Health Sciences University, Portland, OR. The
pGal-SRCAP-(1275-2971) plasmid was generated by subcloning the
cDNA insert encoding SRCAP amino acids 1275-2971 obtained by
NheI and BamHI digestion of the plasmid
SRCAP-(1275-2971) (described below) into pRSV Gal-(1-147) digested
with XbaI and BamHI.
Library Screen--
S. cerevisiae reporter host
strain HF7c was co-transfected with the plasmid pAS1 CBP-(227-460) and
the pGAD-HeLa library using a yeast transfection kit
(CLONTECH) and grown as described (17) in the
presence of 10 mM 3-amino-1,2,4-triazole. The plasmid
corresponding to clone 11 was isolated and sequenced by the dideoxy
sequencing method. The cDNA corresponding to the AB0002307 sequence
was generated by PCR. Briefly, complimentary PCR primers located at the
beginning of the AB0002307 sequence and spaced about 1-kb apart were
used to screen a human SKN plasmid library (Gift of S. Amara, Vollum Institute, Portland, OR). These primers were designed to introduce restriction enzyme sites that allow assembly of the full-length AB0002307 cDNA without introducing changes in the amino sequences. The most 5'-primer also encoded an initiator methionine and a consensus
Kozak sequence. Following restriction digestion, the cDNA fragments
were subcloned into the plasmid pcDNA 3.1 and named SRCAP-(1275-2971). The clone 11 cDNA was used to screen a cDNA plasmid library by homology. Using this approach, a series of overlapping cDNAs were obtained that extended the SRCAP coding sequences to 9126 base pairs. The sequences of all the clones were
confirmed on both strands using an ABI automated DNA sequencer.
Transfections--
HeLa cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum, 100 mg/ml penicillin, and 100 mg/ml streptomycin and were seeded at 1 × 105 cells per 3.5-cm dish 18 h prior to
transfection. Each transfection utilized 200-300 ng of the pGAL-CAT
reporter (pGAL4/E1b TATA) plasmid (6) and the indicated plasmids. The
LipofectAMINE transfection method was used according to the directions
of the manufacturer (Life Technologies, Inc.). Cells were harvested
48 h after transfection and assayed for CAT activity using the
phase-extraction method (6). Results were normalized to protein levels
as determined by Bradford assay (33).
Immunoprecipitation--
For the labeling studies, A549 or 293 cells were incubated for 1 h with methionine/cysteine-deficient
Dulbecco's modified Eagle's medium, 10% fetal bovine serum dialyzed
against phosphate-buffered saline, and penicillin/streptomycin and then
metabolically labeled overnight in 1-ml of deficient Dulbecco's
modified Eagle's medium and 0.25 mCi
35[S]methionine/cysteine (EXPRESS Label, NEN Life Science
Products). Labeled cells were lysed in 1 ml of p300 lysis buffer (18), and the supernatant was incubated with protein A-Sepharose beads and a
monoclonal antibody raised to the C-terminal end of AB0002307 (SRCAP
amino acids 2733-2971). Immune complexes were washed extensively, then
boiled 2 min in 20 µl 2× Laemmli buffer, and analyzed by SDS-polyacrylamide gel electrophoresis. Radiolabeled proteins were
visualized using a PhosphorImager. For the ATPase activity studies,
nuclei of A459 cells were prepared by the method of Dignam et
al. (19). The nuclei were incubated in p300 lysis buffer, centrifuged, and SRCAP immunoprecipitated by addition of anti-AB0002307 monoclonal antibody and protein A beads to the supernatant. In parallel
experiments, control "mock" immunoprecipitations were performed by
addition of protein A-Sepharose beads alone.
ATPase Assay--
Protein A beads containing the SRCAP protein
were further washed with 1.0 M NaCl, 10 mM
Na2PO4, pH 8, 0.5% Triton X-100 to remove
nonspecifically bound proteins and followed by a final wash in ATPase
buffer consisting of 20 mM Tris, pH 7.9, 0.1% Tween 20, 30 mM NaCl, 5 mM MgCl2, 0.5 mM dithiothreitol, and 0.5 mg/ml bovine serum albumin. The
Km for ATP hydrolysis was determined as described by
Cote et al. (20). Briefly, 2-3 µl of SRCAP-protein A
beads were incubated in 20 µl of ATPase buffer containing various amounts of cold ATP (10-300 µM) and 1 µCi
[ A yeast two-hybrid screen was employed to identify cDNA clones
encoding proteins that contact CBP amino acids 227-460. In the initial
screen an excess of fifty clones were obtained, and these were further
analyzed by DNA sequencing. This analysis indicated that clone 11 shared sequence identity with an uncharacterized cDNA reported in
GenBankTM (AB0002307) (Fig.
1A). A BLAST search with the
AB0002307 sequences indicated it shared homology with two of the seven
domains (V and VI) found within the ATPase domain conserved in proteins
of the Snf2 family (Fig. 1A.). Because of this
homology, we decided to test whether the protein encoded by clone 11 interacted with CBP in mammalian cells. For this assay, we constructed
a plasmid encoding a VP16-clone 11 chimera and co-transfected it with
the plasmid encoding the Gal-CBP-(227-460) chimera. In these studies, the VP16-clone 11 chimera consistently increased about 1.5-fold the
ability of the Gal-CBP-(227-460) chimera to activate transcription of
the reporter gene pGal-CAT (data not shown). We reasoned that the small
1.5-fold activation of transcription observed occurred because the
Gal-CBP-(227-460) chimera is a very strong transcriptional activator.
To circumvent this problem, we tested interaction of the VP16-clone 11 chimera with the Gal-CBP-(227-410) and Gal-CBP-(280-460) chimeras,
which contain small deletions that reduce but do not eliminate their
ability to activate transcription. Shown in Fig. 2, co-transfection of the plasmids
encoding the VP16-clone 11 chimera with the plasmid encoding
Gal-CBP-(280-460) activates transcription about 20-fold compared with
that seen with the Gal-CBP-(280-460) chimera alone. Co-transfection of
the plasmid encoding Gal-CBP-(280-460) with the plasmid encoding only
the VP16 activation domain did not activate transcription, indicating
that contact of the VP16-clone chimera with CBP is mediated by the
clone 11 portion. The VP16-clone 11 chimera failed to activate
transcription of the more active Gal-CBP-(227-410) chimera or
Gal-(1-147), indicating that transcriptional activation is not because
of a nonspecific effect.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol). Following a 20-min
incubation at 30 °C, the unhydrolyzed ATP was removed by the
addition of 150 µl of 10 mM phosphoric acid containing
5% activated charcoal (Sigma). The mixture was vortexed and
centrifuged to pellet the charcoal, and the supernatant was removed to
a new tube. An additional 150 µl of the phosphoric acid/charcoal
solution was added, the mixture was vortexed and centrifuged, and the
32Pi in the supernatant was counted. Specific
32Pi released was determined by subtracting the
nonspecific counts obtained from the mock immunoprecipitated protein A
beads. To ensure the ATPase assay was within the linear range, various
amounts of SRCAP-protein A beads (1-6 µl) were assayed as described
in 100 µM cold ATP and 1 µCi
[
-32P]ATP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A schematic of the SRCAP gene and comparison
of SRCAP-conserved ATPase domains to other Snf2-related
genes. A, domains identified within the 2971-amino acid
SRCAP are indicated, including two highly charged domains, the putative
DNA binding domain, the CBP binding domain, and the position of the
regions which make up the conserved ATPase domains (I, Ia-VI). The
position of clone 11 identified by the yeast two-hybrid screen as
interacting with CBP and the position of the AB0002307 cDNA are
shown. B, a comparison of the conserved ATPase domain of
SRCAP to the ATPase domain of several Snf2-related proteins is
shown (22). The position of the amino acids at the C-terminal side of
each conserved domain is indicated.
View larger version (20K):
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Fig. 2.
CBP interacts in HeLa cells with SRCAP.
The interaction of CBP and SRCAP cDNA (encoded by clone 11) was
assessed using a mammalian two-hybrid assay. HeLa cells were
transfected with 300 ng of reporter gene pGAL-CAT and with 600 ng of
either pGal-CBP-(280-460), pGal-CBP-(227-410), or pGal-(1-147) and
600 ng of either pVP16, pVP16-clone 11, or pcDNA 3.1. The relative
CAT enzymatic activity was determined by dividing CAT enzymatic
activity of each sample by the transcriptional activity induced by the
Gal-CBP-(280-460) chimera. Values are the means ± S.E. from
three separate experiments in which each point was performed in
triplicate.
Based on the above results, we decided to clone the remainder of the
AB0002307 cDNA. Using a combination of approaches (PCR and plasmid
library screening), a 9.1-kb cDNA was obtained (Fig. 1A). This included the 5 kb of the AB0002307 cDNA and an
additional 4.1 kb of new sequence at the 5' end of the molecule. Within
the AB0002307 sequence, we found several differences with the reported sequences, including an additional 111-base pair insertion at nucleotide 4128. The 9.1-kb composite sequence contains a continuous open reading frame. It, however, does not contain a termination codon
(in the coding frame), raising uncertainty as to whether the cDNA
clones obtained encode the full-length protein. The presumptive initiator ATG is positioned at nucleotide 217 and is the first ATG in
the open reading that occurs in the context of a consensus Kozak
sequence (21). Using this ATG as a translational start site, a protein
of 2971 amino acids with a predicted molecular weight of 315 kDa is
obtained. This size is consistent with immunoprecipitation studies in
which anti-AB0002307 antibodies detect a protein which migrates slower
in SDS-PAGE gels than CBP (predicted molecular mass of 265 kDa, Fig.
3.).
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A search of the NCBI data base with the AB0002307 sequence indicated that it contains a complete ATPase domain that consists of seven highly conserved regions (I, Ia-VI) which are dispersed over the length of the protein. Based on the homology to Snf2 family members and, as described below, on the ability to enhance CBP-mediated transcription, we have named this protein SRCAP. A comparison of the amino acid sequence of the SRCAP ATPase domain to the amino acid sequence of the ATPase domains found in other members of the Snf2 family is shown in Fig. 1B.
To test whether SRCAP like other members of the Snf2 family
functions as an ATPase, we immunoprecipitated SRCAP protein from nuclear extracts of A549 cells using the anti-AB0002307 monoclonal antibody and protein A-Sepharose beads. Shown in Fig.
4A, incubation of the
SRCAP-protein A beads in ATPase buffer containing 100 µM cold ATP and 1 µCi [-32P]ATP resulted in the release
of 32Pi, indicating the hydrolysis of ATP was
occurring. The specific ATPase activity of SRCAP was determined by
subtracting out the nonspecific ATPase activity that bound to protein A
in the absence of the anti-AB0002307 antibody. As shown, a linear
increase in the specific ATPase activity was observed with increasing
amounts of SRCAP-protein A beads. To determine the
Km for the hydrolysis of ATP by SRCAP, 2 µl of
SRCAP-protein A beads were incubated with 1 µCi
[
-32P]ATP and either 10, 30, 100, or 300 µM cold ATP. A plot of 1/d[Pi]/dT versus 1/[ATP] for three experiments is shown in Fig.
4B and indicated SRCAP has a Km for
hydrolysis of ATP of 66 µM.
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Several members of the Snf2 gene family have been found to
regulate transcription when tethered to a promoter by a heterologous DNA binding domain. This prompted us to ask whether SRCAP was a
transcriptional activator. For these studies, we tested the ability of
a plasmid encoding a Gal-SRCAP-(1275-2971) chimera to activate
transcription of the Gal-CAT reporter gene in HeLa cells. Shown in Fig.
5, this chimera activated transcription
about 12-fold over the level of transcription induced by Gal-(1-147) and to about the same level as that observed with the Gal-CBP-(1-2441) chimera.
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To test whether SRCAP influenced the ability of CBP to activate
transcription, we co-transfected the plasmid encoding SRCAP amino acids
1275-2971 with the plasmid encoding Gal-CBP-(1-2441). Shown in Fig.
6, SRCAP enhanced the ability of the
Gal-CBP to activate transcription about 2.5-fold. This enhancement of
transcription was specific because SRCAP did not enhance the ability of
Gal-VP16 to activate transcription. Co-transfection of the plasmid
encoding the CBP-(1-2441) along with the plasmid encoding the
Gal-SRCAP-(1275-2971) chimera did not result in a further increase in
transcription (data not shown). The inability of exogenously introduced
CBP to activate SRCAP transcription activity suggests that CBP is either not limiting in HeLa cells or that SRCAP does not function to
activate transcription by recruitment of CBP. Consistent with this
latter hypothesis, we have found that a Gal-clone 11 chimera which
encodes the CBP binding domain of SRCAP does not activate transcription
in either HeLa or F-9 cells (data not shown).
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The recent studies which indicate that the adenoviral protein E1A binds
to the N-terminal end of CBP and inhibits the ability of a
Gal-CBP-(1-450) chimera to activate transcription prompted us to
determine whether E1A inhibits binding of SRCAP to CBP (5). Shown in
Fig. 7, transfection of plasmids encoding
wild type E1A decreased transcription stimulated by interaction of the
Gal-CBP-(280-460) with the VP16-clone 11 chimera. In contrast,
co-transfection with a plasmid encoding an E1A mutant (RG2) that does
not bind CBP did not disrupt SRCAP and CBP interaction.
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DISCUSSION |
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In the present paper we report the cloning of SRCAP, a novel member of the Snf2 gene family. The Snf2 family of genes consists of a number of proteins with diverse functions, including remodeling chromatin, regulation of transcription, and DNA repair (reviewed in Ref. 20). A common feature of the Snf2 family of proteins is the presence of a highly conserved domain called the Snf2 domain, which functions as an ATPase (23). This ATPase domain consists of seven highly conserved motifs interspersed with variable spacers of nonconserved sequences. The entire Snf2 domain is contained in about 400 amino acids in most Snf2 family members (Snf2, Iswi, Chd1, Rad16, and Mot-1) and is contained in about 670 amino acids in others (P113 and Hip116) (24). The Snf2 domain of SRCAP is unique in that the ATPase domain is dispersed in a much larger region of about 1465 amino acids (Fig. 1B). Despite the unique spacing between the conserved motifs, the conservation of residues within the ATPase domain is striking. Consistent with this homology, we have found that SRCAP functions as an ATPase with a Km for ATP hydrolysis of 66 µM which is similar to the reported Km for hydrolysis of ATP by yeast Snf2 of 45 µM (20). The ATPase activity of recombinant yeast Snf2 protein (purified from bacteria) has been reported to be stimulated 5-fold by DNA (23). In contrast, the ATPase activity of SRCAP (purified from A549 cells) was not stimulated by DNA (data not shown). This result suggests that the regulation of the ATPase activity of SRCAP and yeast Snf2 may occur through distinct mechanisms. However, we cannot rule out the possibility that the lack of DNA dependence observed for the ATPase activity of SRCAP is because of differences in the protein purification protocols used, e.g. which may allow DNA to co-purify with SRCAP but not yeast Snf2.
The ATPase domain has been shown to be critical for the function of
several Snf2 family members. The yeast Snf2 protein is the prototype of this family. It functions as part of a multiple subunit SWI/SNF complex that appears to alleviate the repression of
transcription of some promoters induced by chromatin through a process
termed chromatin remodeling (reviewed in Ref. 25). Although the
specific mechanisms for chromatin remodeling are not completely known,
it allows the binding of transcription factors to sites previously
inaccessible because of the presence of nucleosomes. Mutant yeast
Snf2 molecules, in which the ATPase function has been deleted,
neither function in chromatin remodeling assays nor are able to
activate transcription (23). ATPase activity is also required for
transcriptional repression mediated by the Snf2 family member
MOT-1 (26), which utilizes ATP to dissociate TBP from DNA. However, as
discussed below, the ATPase activity of SRCAP is not needed for
SRCAP-mediated transcriptional activation. Although this result differs
from that obtained for the yeast Snf2, an analogous finding has
been reported for the human Snf2 protein (hSnf2) (27).
Similar to our studies with SRCAP, when human Snf2
is
tethered to DNA by a heterologous DNA binding domain, the ATPase
function is not required for activation of transcription. However, the
ATPase function is essential for human Snf2
potentiation of
glucocorticoid receptor-mediated transcription (27). These findings
suggest that activation of transcription by human Snf2 (and
perhaps SRCAP) occurs through several distinct mechanisms, one
requiring the ATPase function and one that does not.
How the SRCAP might activate transcription is not yet known. In the
case of human Snf2, deletion of the highly charged domain results in loss of transcriptional activity (27). The C-terminal end of
SRCAP also contains a highly charged domain, made up of clusters of
acidic and basic residues, which may contribute in a similar fashion to
the ability of SRCAP to activate transcription.
Examination of the primary structure of the SRCAP indicates that, in
addition to binding CBP, it also likely binds to DNA. Shown in Fig.
1A, the C-terminal domain contains four copies of the motif
KR(R/K)RGRP(P/R), of which multiple copies are also found in DNA
binding proteins (chromosomal protein D1 and HMG-1), where it is
thought to mediate the binding of these proteins to A-T-rich regions by
contacts in the minor groove of DNA (28, 29). A similar motif is found
in the C-terminal end of human homologs of yeast Snf2
(hSnf2 and
) and within the DNA binding domain of the
Snf2 gene family protein CHD1 (30, 31).
The adenoviral protein E1A exerts several biological activities including transformation of cells and activation or repression of cellular and viral genes (reviewed in Ref. 32). E1A blocks the ability of CBP to function as a co-activator for a number of transcription factors and binds to three distinct sites within CBP: amino acids 1-460, 1805-1891, and 2058-2163 (5, 12, 14, 15). Understanding how association of E1A with CBP alters the ability of CBP to function as a co-activator has come from studies that demonstrate that E1A binding competitively excludes binding of several proteins shown to be critical for CBP co-activator function. Binding of E1A to CBP amino acids 1805-1891 prevents binding of the histone acetyltransferase P/CAF, whereas E1A binding to CBP amino acids 2058-2163 prevents binding of the co-activator P/CIP (5, 12). Although P/CAF also binds to amino acids 1-460 of CBP, competition between P/CAF and E1A binding has not been demonstrated. In our studies, we have found that E1A binding to amino acids 280-460 of CBP excludes the binding of SRCAP to CBP, suggesting that this represents an additional method by which E1A represses the co-activator function of CBP.
The primary structure of SRCAP indicates that it belongs to the
Snf2 family of proteins that function to modify protein-DNA interactions, suggesting that SRCAP plays a similar role. Consistent with this notion, our results indicate that SRCAP interacts with CBP
and influences its ability to activate transcription. Recent studies
have indicated that different promoters which utilize CBP to activate
transcription have different requirements for co-activators. For
example, both P/CAF and P/CIP have been found to be required for
activation of a CRE-reporter gene, whereas P/CIP but not P/CAF is
required for transcription of a GAS-reporter gene (13). It, therefore,
seems likely that SRCAP may function to enhance CBP-mediated
transcription at some but not all promoters.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF143946.
§ The first two authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
314-268-5291; Fax: 314-577-8233; E-mail: Chrivia{at}SLU.edu.
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ABBREVIATIONS |
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The abbreviations used are: CREB, cAMP response-element binding protein; CBP, CREB-binding protein; NCoA, nuclear receptor coactivator; P/CAF, p300/CBP associated factor; SRCAP, Snf2-Related CBP Activator Protein; PCR, polymerase chain reaction; kb, kilobase(s).
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