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Mol Cell Biol, June 1998, p. 3596-3603, Vol. 18, No. 6
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
p300/CREB Binding Protein-Related Protein p270 Is a Component
of Mammalian SWI/SNF Complexes
Peter B.
Dallas,1
Ian Wayne
Cheney,1,
Da-Wei
Liao,1
Valerie
Bowrin,1
Whitney
Byam,1
Stephen
Pacchione,1
Ryuji
Kobayashi,2
Peter
Yaciuk,3 and
Elizabeth
Moran1,*
Fels Institute for Cancer Research and
Molecular Biology, Temple University School of Medicine, Philadelphia,
Pennsylvania 191401;
Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York
117242; and
St. Louis University
School of Medicine, St. Louis, Missouri 631043
Received 14 August 1997/Returned for modification 2 October
1997/Accepted 11 March 1998
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ABSTRACT |
p300 and the closely related CREB binding protein (CBP) are
transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and
associated histone acetyltransferase activity, suggesting that
chromatin modification is an essential part of their role in regulating
transcription. Detailed characterization of a panel of antibodies
raised against p300/CBP has revealed the existence of a 270-kDa
cellular protein, p270, distinct from p300 and CBP but sharing
at least two independent epitopes with p300. The subset of
p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative
molecular masses ranging from 44 to 190 kDa. Purification and analysis
of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of
this complex.
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INTRODUCTION |
The cellular protein p300 is a
direct target of the transforming functions of the adenovirus E1A gene
(19, 51) and as such is implicated in the regulation of both
cell cycle-specific and tissue-specific gene expression (18, 27,
38, 43, 46, 52, 54). p300 is highly homologous (about 64%
identical) to the cyclic AMP response element binding protein
(CREB) coactivator, CBP (CREB binding protein) (5, 11, 17, 30,
34). Both p300 and CBP are present in intracellular complexes
with the TATA binding protein (TBP) (1, 13). Both act as
cofactors for p53 (6, 21, 33, 44) and nuclear hormone
receptors (9, 24, 28). Both also contain intrinsic and
associated histone acetyltransferase (HAT) activity (39,
53), suggesting that chromatin modification is an essential part
of their role in regulating transcription.
A recent detailed characterization of a panel of antibodies raised
against a mixture of native p300 and CBP revealed the existence of a
270-kDa cellular protein, distinct from p300 and CBP but sharing at
least two independent antigenic determinants with p300 (13).
Four of the eleven antibodies in the panel recognize p270. The subset
of p300/CBP-derived antibodies that recognizes p270 consistently
coprecipitates a series of cellular proteins with relative
molecular masses ranging from 44 to 190 kDa. Typical of these is the
antibody designated NM1, whose immunoprecipitation pattern is shown in
Fig. 1. TBP-specific antibodies
coprecipitate a subset of these proteins including p300, CBP, and
the phosphoprotein species indicated in Fig. 1 as p64 and p59
(1, 13). Because the TBP-specific antibodies do not
coprecipitate all of the p300 family-associated proteins, it is
likely that the array of proteins seen in Fig. 1 represents more
than one intracellular complex.
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FIG. 1.
Immune complexes precipitated by a
p300/CBP/p270-reactive antibody. 35S- or
32P-labeled 293 cell lysates were immunoprecipitated with
monoclonal antibody NM1 under nondenaturing conditions ( ). To
distinguish associated from cross-reactive species, half of the complex
was denatured (+) by boiling in SDS and reprecipitated with fresh NM1
in conditions where only proteins directly recognized by the
antibodies are recovered. The proteins from each
immunoprecipitation were separated by electrophoresis and visualized by
autoradiography. The positions of the associated protein species
are shown on the right; the positions of p300, CBP, and p270 are shown
on the left. The 35S panel is lightly exposed to enhance
the resolution of p300, CBP, and p270. Only the bands corresponding to
p300, CBP, and p270 are recovered by the antibody after the complex has
been denatured.
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We have now identified four of the remaining p300/CBP/p270-associated
proteins as members of another important cellular complex: the
mammalian SWI/SNF complex. The 190-kDa protein visible in the
p300-related complex is BRG1, the human homolog of the yeast transcriptional activator, SWI2/SNF2. The 170- and 155-kDa species are
the recently identified BRG1-associated factors, BAF-170 and BAF-155, both homologs of yeast SWI3 (50). A component
of the 44-kDa band is the previously identified
BRG1/hbrm-associated factor, hSNF5 (INI-1)
(36). Reciprocal precipitation of the immune complex using
antibodies raised against BAF-155 reveals p270 to be the p300-related
protein in direct association with the mammalian SWI/SNF complex.
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MATERIALS AND METHODS |
Cell culture.
HeLa cells (American Type Culture Collection)
were cultured in Dulbecco's modified Eagle's medium containing 5%
fetal bovine serum (Research Sera, Summit Biotechnology) and
supplemented with 50 penicillin (IU/ml) and streptomycin (50 µg/ml).
The cells were grown at 37°C in a humidified atmosphere of 95% air
and 5% CO2.
Protein purification and peptide sequencing.
A preparative
NM1 immune precipitation from a lysate of approximately 2 × 109 HeLa cells was separated by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE), and the proteins
in the immune complex were visualized by staining with Coomassie Blue-G
(Sigma). The gel fragments containing the p270, p190, p170, and p155
bands were excised and digested with lysylendopeptidase
(Achromobacter protease I; Wako). Peptide fragments were
extracted from the gel, separated by high-pressure liquid
chromatography using a Vydac C18 column and were
sequenced by automated sequencers (Applied Biosystems models 470, 473, and 477).
Antibodies.
The generation of monoclonal antibodies to
p300/CBP (NM1 and NM11) and the reactivity of NM1 with p270 have been
described elsewhere (13). A peptide corresponding to
residues 2 to 15 from the amino-terminal sequence of BRG1 reported by
Khavari et al. (29) was synthesized and used to raise rabbit
immune sera specific for BRG1. A similar antibody to BRG1 (residues 2 through 15) was also generated in BALB/c mice. Rabbit antipeptide
antibodies to BAF-170 and BAF-155 were developed by using keyhole
limpet hemocyanin-conjugated peptides corresponding to residue
positions 15 to 27 of the p170.K47 peptide and residues 592 to 605 of
the p155.K31 sequences shown in Fig. 3. An antipeptide antibody
specific for p270, showing no cross-reactivity with p300 or CBP, was
developed in a similar manner from p270-specific peptide sequence
(peptide sequence, RITATMDDMLL). All rabbit polyclonal sera were
produced by CoCalico Biologicals (Reamstown, Pa.). The simian virus 40 T-antigen-specific antibody 419 (22) and the TBP-specific
monoclonal antibody SL8 (42) were provided by Ed Harlow and
Nouria Hernandez, respectively. The BRG1- and INI-1-specific polyclonal
antibodies (50) used for Fig. 5 were provided by G. Crabtree.
Immunoprecipitations.
32P- or
35S-labeled or unlabeled total cell lysates were
immunoprecipitated with antibody and protein A-Sepharose CL-4B
beads (Pharmacia) for 1.5 h at 4°C as described previously
(49, 52). Immune complexes were washed four times with E1A
lysis buffer (49) and then either boiled off the beads in
SDS in preparation for SDS-PAGE or used in further enzymatic
assays. Sequential immunoprecipitations were performed as
described by Dallas et al. (13).
Immunoblotting.
Total cell lysates were immunoprecipitated
as described above and then separated through an SDS-7.5%
polyacrylamide gel. After electrophoresis, proteins were blotted
onto Immobilon P membranes (Millipore) by using standard procedures
(23). The membrane was blocked with 5% nonfat milk in
either Tris-buffered saline (20 mM Tris [pH 7.5], 150 mM NaCl) for
Western blots using alkaline phosphatase-conjugated secondary antibody
or PBST (0.2% Tween in phosphate-buffered saline) for enhanced
chemiluminescence detection using horseradish peroxidase-conjugated
secondary antibody. Chemiluminescence detection was performed with
enhanced chemiluminescence reagents (Amersham) according to the
manufacturer's guidelines.
HAT assays.
Immune complexes were prepared as described
above from 2.5 mg of total cell lysate but were treated additionally
with ethidium bromide (12.5 µg/ml, final concentration) for 15 min at
4°C and then cleared by centrifugation at 3,500 × g
prior to the immunoprecipitation. The ethidium bromide treatment was
used to ensure that the immune complex represents only specific
protein-protein interactions, not nonspecific protein
associations mediated through DNA binding. In most experiments, an
aliquot of the immune complex was reserved for parallel ATPase assays.
The complexes were washed four times in a buffer consisting of 20 mM
sodium phosphate, 250 mM NaCl, 0.1% Nonidet P-40 and 5 mM EDTA (pH
7.0) and were resuspended in HAT buffer (7) with 1.0 mg of
calf thymus histones (Sigma type IIA). HAT assays were performed as
outlined by Brownell and Allis (7) in the presence of
3H-acetyl coenzyme A and scored by detecting acetylation of
histone substrates in a filter binding assay. 3H
incorporation into histone substrate was determined by liquid scintillation counting.
ATPase assays.
Protein extracts were prepared for ATPase
assays as described for the HAT assay. Equal amounts of total cellular
lysate were used for corresponding samples in these two assay systems.
ATPase activity was measured in a polyethyleneimine-cellulose
thin-layer chromatography system as the ability to hydrolyze the
terminal phosphate from [
-32P]ATP. Reactions were
performed essentially as described by Laurent et al. (32)
except that the running buffer was modified to 0.75 M
KH2PO4 (pH 3.5). Reaction conditions consisted
of the immune complex in a 20-µl reaction volume containing 25 mM
Tris (pH 6.9), 10 mM MgCl2, 1 mM dithiothreitol, 0.2 mg of
salmon sperm DNA per ml, and 5 nM [-32P]ATP (3,000 Ci/mmol; NEN). Reactions were incubated at 37°C for the times
indicated and stopped by the addition of EDTA to a final concentration
of 0.025 M. The percent conversion from ATP-bound 32P to
free 32Pi was quantified on a Fuji
phosphoimaging system. Release of 32P-labeled
Pi from the ATP substrate was also detected by
autoradiography.
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RESULTS |
Identification of multiple members of the NM1 immune complex as
BRG1 and BRG1-associated factors.
Figure 1 shows the immune
precipitation pattern obtained with NM1, 1 of 4 monoclonal antibodies
in our panel of 11 raised against p300/CBP that also recognizes p270. A
combination of immunoprecipitation and Western blotting with
p300-specific versus CBP-specific antibodies has confirmed that the
300-kDa band in these immunoprecipitations contains both p300 and CBP,
while the 270-kDa band is distinct from both (13). p270 is
related to p300 at a minimum of two epitopes because the four
p300/CBP-derived antibodies that recognize p270 include ones that are
specific for p300 as well as ones that recognize an epitope shared by
p300 and CBP (13).
Each of the antibodies that recognizes p270 coprecipitates a series of
cellular products. Analysis of
35S-labeled lysates (Fig.
1,
lane 1) reveals coprecipitated species
of 190, 170, 155, 75, 71, 64, 59, 50, and 44 kDa. These are relatively
low percentage gels, designed
to improve the resolution of the
high-molecular-weight species,
and thus do not show products migrating
at less than 40 kDa. We
have examined the complexes at lower-molecular-weight
ranges
and do not find proteins of lower molecular weight
consistently
part of the complex. The only prominent species in the
35S-labeled lysate that do not have
phosphoprotein counterparts
are the products migrating at
71 and 75 kDa (lane 3 compared with
lane 1). Denaturation of the
complexes and reprecipitation with
NM1 shows that only p300, CBP, and
p270 are recognized directly
by the antibody; the remaining species are
associated with the
antibody targets (Fig.
1, lanes 2 and 4).
Because the products in these complexes are likely to represent
important cellular partners of the p300-related proteins,
we have
begun to purify and obtain micropeptide sequence on each
of the
components. The sequence from the protein designated p190
in Fig.
1 reveals that this species is identical or
highly related
to the transcriptional coactivator BRG1.
Eight peptides were obtained
(Fig.
2), and all show a very high degree
of identity to the cDNA-derived
sequence of BRG1 (
29). The
relationship is not limited to any
one domain, as peptides were
recovered corresponding to regions
throughout the BRG1 sequence (Fig.
2A and B). Three of the peptides
(K9,
K16, and K42) were obtained from very limited amounts of
material and
show some mismatches relative to the published BRG1
sequence. We do not
think that these represent alternative forms
of BRG1 because attempts
to isolate products with these sequences
were negative. The presence of
BRG1 specifically is supported
by the identity to BRG1 in the majority
of the peptides, even
in areas where BRG1 sequence differs from that of
other very closely
related human forms such as h
brm which
is 80 to 90% identical
to BRG1 over most of its length
(
10,
37; reviewed in reference
41). The presence of BRG1 in the NM1 complex
is confirmed by
reactivity with antiserum raised against the
BRG1-specific amino-terminal
sequence (Fig.
2C). The BRG1-specific
serum (lane 2), but not
preimmune serum (lane 1), reacts strongly and
specifically in
Western blots with the 190-kDa band immobilized by
electrophoretic
transfer of the NM1 immune complex.
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FIG. 2.
Identification of the 190-kDa protein in NM1
complexes as BRG1. (A) Physical map of identified or predicted domains
of BRG1, adapted from reference 29. BRG1 has a
1,613-amino-acid open reading frame. BRG1, like other members of the
Swi2 family, contains multiple motifs characteristic of helicases and
ATPases, although helicase activity has not been detected in these
proteins. The ATPase/helicase domain spans amino acid residues 774 to 1237, and the bromodomain spans amino acid residues 1448 to 1523. BRG1 also contains a functional retinoblastoma protein binding
motif (LXCXE) encompassing residues 1322 to 1326 (16). (B)
The sequences of eight peptides derived from micropeptide sequencing of
gel-purified p190 isolated from human cells are represented on the top
line of each pair. Dots between residues indicate identity with the
cDNA-derived human BRG1 sequence. Uncertainties in the reading of the
sequencing cycles are indicated by underlining; X indicates an
unreadable cycle. Uncertainties and unreadable cycles were not counted
as identities, although they may be matches. The enzyme used to digest
p190 prior to sequencing (lysylendopeptidase) cuts almost exclusively
at lysine residues, so that each peptide sequence is expected to be
immediately preceded by a lysine (K). As shown in the BRG1 sequence, a
lysine occurs in BRG1 at each position where it is predicted to occur
in p190, increasing the percent identity in the match. R represents the
amino acid number of the initial lysine residue in BRG1 as reported in
Khavari et al. (29). Peptides were recovered from regions
throughout BRG1. (C) NM1-precipitated proteins from HeLa cell
lysates were separated by SDS-PAGE, transferred to nitrocellulose, and
probed by Western blotting with a BRG1-specific amino-terminal
antipeptide antibody (lane 2) or preimmune serum (lane 1).
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BRG1 (
brm-related gene) (
29) is a
mammalian homolog of the
Drosophila brm gene, which is
required during development for
activation of multiple homeotic genes
(
48). Both
BRG1 and
brm are close
homologs of the yeast
SWI2/SNF2 gene (
2,
29,
32),
which regulates the yeast mating-type switch and the switch to
different carbohydrate utilization pathways (
8,
47).
In each
system, the BRG1-related product exists in
complex with a series
of associated proteins (
12,
15,
31,
40,
50). A recent
detailed characterization of mammalian
SWI/SNF complexes (
50)
indicates that they are present in
multiple forms made up of 9
to 12 proteins now designated
BRG1-associated factors (BAFs),
ranging in estimated size from 47 to
250 kDa. These complexes
include species of 155 and 170 kDa (BAF-155
and BAF-170) whose
sequence has been determined by these investigators.
A comparison
of the peptide sequence that we have obtained for the 155- and
170-kDa species in the NM1 complex with the deduced amino acid
sequences reported for BAF-155 and BAF-170 indicates that these
are the
same products (Fig.
3).
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FIG. 3.
The NM1 complex proteins p155 and p170 are BAF-155
and BAF-170. The sequences of two peptides derived from micropeptide
sequencing of gel-purified p155 (K31 and K26) and two peptides derived
from p170 (K43 and K47) are represented on the top line of each pair.
The sequences show near-perfect identity with the deduced amino acid
sequences of BAF-155 and BAF-170, respectively, as reported by Wang et
al. (50). The R value indicates the amino acid position of
the first residue in the deduced BAF sequence. An uncertain reading of
the peptide sequencing cycle is indicated by underlining; an unreadable
cycle is indicated by an X. Identity is indicated by a dot between
residues. An initial K residue, not written in the sequenced peptides,
is inferred from the nature of the enzyme used in the digest (Fig. 2).
A K occurs at each corresponding predicted position in the BAF
proteins. Peptide sequence was obtained as described in the legend
to Fig. 2.
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p270 is a p300-related protein associated with the mammalian
SWI/SNF complex.
A comparison between the higher-molecular-weight
proteins precipitated by BAF-155-specific antibodies and those in
the NM1 immune complex suggests that p270 is the major p300-related
protein in the complex (Fig. 4A). The
two antibodies precipitate similar arrays of proteins.
However, while a band comigrating with p270 is present in the
BAF-155 complex, there is no evidence of species corresponding to p300
or CBP. Sequential immunoprecipitation (Fig. 4B and C) using the
BAF-155 antibody, followed by denaturation and
reimmunoprecipitation with the NM1 antibody, shows that the 270-kDa
species from the BAF-155-specific complex is recovered by NM1 (lane 2),
confirming that this species is the p300-related protein p270.
Neither p300 nor CBP was found in the BAF-155 complex although p300,
CBP, and p270 were all successfully reimmunoprecipitated from the
original NM1 immune complexes (lane 4).
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FIG. 4.
p270 is the p300 family member present in BAF155
complexes. (A) The high-molecular-weight proteins in the
32P-labeled immune complex precipitated with
BAF-155-specific antibodies (lane 2) are compared with the complex
precipitated by the NM1 antibodies (lane 3). The preimmune (Pre-imm)
serum from the BAF-155 rabbit is used as a background control (lane 1).
The positions of p300/CBP, p270, BRG1, BAF-170, and BAF155 in the NM1
complex are indicated. A species comigrating with p270 is seen in the
BAF-155 complex, but there is no detectable appearance of species at
the p300/CBP position. (B) 32P-labeled HeLa cell lysates
were immunoprecipitated with either the BAF-155-specific antipeptide
antibody (Ab), a preimmune control, or the NM1 antibodies and then
denatured and reprecipitated with NM1 to identify the
NM1-reactive species present in each complex (lanes 2 through 4). The
standard NM1 immune complex is shown in lane 1. The NM1 antibody
recovers p300, CBP, and p270 in the NM1 complex (lane 4) but detects
only p270 in the BAF-155 complex (lane 2). (C) Enlargement of the
high-molecular-weight region of lanes 2 through 4 of the gel shown in
panel B to provide better resolution of the p300 and p270 bands.
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We have also generated antipeptide antibodies specific for p270 which
are suitable for Western blots. These antibodies do
not cross-react
with p300 or CBP. Electroblotting the BAF-155
immune complex (Fig.
5)
and probing with the p270-specific antibodies
shows again that p270 is
a component of the mammalian SWI/SNF
complex (Fig.
5B, lane 2). Analysis of the immune
complex precipitated
by the BRG1-specific antibodies (J1) that were
used to define
BAF-155 and BAF-170 (
50) likewise
shows a positive reaction
with p270 antibodies (Fig.
5B, lane 3). When
the immune complexes
are probed with NM-11 (Fig.
5A), a monoclonal
antibody that, in
contrast to NM-1, recognizes p300 and CBP but
not p270 (
13,
14), a positive reaction is seen with
the NM1 complex (lane
4) but not with the BAF-155 or BRG1 complexes
(lanes 2, 3). Antibodies
to BRG1 and BAF-170 indicate the presence of
their respective
proteins in all three complexes, NM1, BRG1, and
BAF-155 (Fig.
5C and D, lanes 2 to 4). (Although BAF-170 appears
as a single
band in
35S- or
32P-labeled images, it consistently shows as a doublet in
silver
stains or Western blots such as in Fig.
5D.) Antibodies to the
BRG1-associated protein hSNF5 (INI-1) also react positively with
all three complexes (Fig.
5E), indicating that hSNF5 is also present
in
the NM1 complex. The hSNF5-reactive band runs just below the
immunoglobulin heavy chain, which can be seen in lanes 2 and 3
of
Fig.
5E, and coincides with the band designated p44 in the
NM1 immune
complex. One additional immune complex, the TBP-specific
complex
brought down by the SL8 monoclonal antibody, was probed
in these
experiments. The TBP complex shows the presence of p300/CBP
(Fig.
5A,
lane 5) as reported previously (
13) but shows no reaction
with p270-specific antibodies (Fig.
5B, lane 5).
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FIG. 5.
Immune complexes probed with antibodies specific for
p300 family members and mammalian SWI/SNF complex components. The
immune complexes precipitated by the series of antibodies indicated
below lanes 1 through 5 were transferred to nitrocellulose and probed
with a panel of antibodies individually specific for p300
family members and mammalian SWI/SNF complex components. The 419 antibody (lane 1) recognizes the simian virus 40 virus T
antigen and serves as a negative control. The BAF-155
antibody (lane 2) is the antipeptide antibody raised against the
K31 peptide sequence from BAF-155, also used for Fig. 4. J1 (lane 3) is
a rabbit polyclonal antibody raised against the C-terminal
portion of BRG1 (50). NM1 (lane 4) is a monoclonal antibody
raised against human p300 that cross-reacts with CBP and p270
(13). SL8 (lane 5) is a monoclonal antibody directed against
human TBP (42). The specifities of the antibodies used
to probe the nitrocellulose blot are indicated at the right. The
p300/CBP-specific antibody (A) is monoclonal antibody NM11,
which does not cross-react with p270 (13, 14). The
p270 antibody (B) is an antipeptide antibody raised against
peptide sequence obtained from purified p270. The BRG1 antibody
(C) is the antipeptide antibody described in the legend to Fig.
2. The BAF-170 antibody (D) is an antipeptide
antibody raised against a portion of the BAF-170 K47 sequence.
(Although BAF-170 appears as a single band in 35S- or
32P-labeled images, it consistently shows as a doublet in
silver stains or Western blots such as in panel D.) The
hSNF5 (INI-1) antibody is a rabbit polyclonal antibody
described by Wang et al. (50).
Membranes used for these Western blots were checked by
autoradiography to verify the efficient transfer of the proteins
prior to antibody probing.
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The NM1 complexes contain HAT and ATPase activities that
segregate with individual complex components.
BRG1 has
multiple motifs characteristic of ATPases (indicated
schematically in Fig. 2), and SWI/SNF complexes contain a
BRG1-associated ATPase activity (31). p300 and CBP have
intrinsic and associated HAT activity (39, 53). The presence
of these activities in NM1 complexes was assayed as shown in Fig.
6 and 7.
HAT assays were performed on immunoprecipitated complexes and scored by
detecting acetylation of histone substrates in a filter binding assay.
NM1 immune complexes contain p300 and CBP and show readily detectable HAT activity (Fig. 6). Also positive in this assay were SL8
(TBP-specific) immune complexes, which contain p300, CBP, and
TAFII250, which has also been found to contain HAT activity
(35). However, the BRG1 complex (BAF-155 antibodies) showed
no HAT activity above the background levels represented by the 419 and
preimmune antibodies.
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FIG. 6.
HAT activity. HAT assays were performed on
immunoprecipitated HeLa cell complexes in the presence of
3H-acetyl coenzyme A and scored by detecting acetylation of
histone substrates in a filter binding assay. Results are represented
as disintegrations/minute per filter.
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FIG. 7.
ATPase activity. Antibody complexes isolated from HeLa
cells in parallel with those used in the HAT assay (Fig. 6) were
assayed for associated ATPase activity. Equal amounts of total cell
protein were immunoprecipitated with the appropriate
antibody. ATPase activity was measured in a thin-layer
chromatography assay as the ability to hydrolyze the terminal phosphate
from [-32P]ATP. The percent conversion from
ATP-bound 32P to free 32Pi
was quantified on a Fuji phosphoimaging system. The results shown
are the averages of six independent experiments.
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ATPase activity was measured in parallel immunoprecipitations as
percent conversion from ATP-bound
32P to free
32P
i in a thin-layer chromatography assay (Fig.
7). The NM1- and
BAF155-derived complexes both show ATPase activity.
The TBP-specific
complex (SL8) does not show ATPase activity above
background levels.
Similarly, the NM11 complex, which contains p300 and
CBP but not
p270 or the associated SWI/SNF complex (
13,
14),
does not
show ATPase activity.
The presence of these activities in NM1 immune complexes and their
segregation into TBP-specific and BAF-155-specific complexes
are
consistent with the interpretation that NM1 immune complexes
represent
multiple distinct complexes involving the direct NM1
targets, p300,
CBP, and p270. The lack of detectable HAT activity
in the SWI/SNF
(BAF-155) complex implies that p270 is not a HAT
like the antigenically
related p300 and CBP.
p270 association with BAF-155 and BAF-170 is not dependent on
BRG1.
Analysis of mammalian SWI/SNF complexes is facilitated by
the availability of the human carcinoma cell line SW13, which is severely deficient in BRG1 expression (16, 37). As expected from the identification of the p190 band in NM1 complexes as BRG1, this
band is greatly reduced in NM1 complexes precipitated from SW13 cells
(Fig. 8A). The lowest-molecular-weight
species, p44, now identified as hSNF5 (INI-1), is also absent from the
SW13 NM1 complex. The BAF-170 and BAF-155 species are still present when the complex is isolated from SW13 cells. The stability of the in
vivo association between p270 and BAF-155 and BAF-170 in the absence of
BRG1 supports the interpretation that p270 is an integral member of
this complex. ATPase activity was compared in NM1 complexes isolated
from HeLa and SW13 cells (Fig. 8B). The background level of ATPase
activity in the reaction is indicated by immunoprecipitation of
HeLa or SW13 cells with the 419 control antibody. NM1 complexes
precipitated from HeLa cells show readily detectable ATPase
activity, while NM1 complexes precipitated from SW13 cells show
no ATPase activity above background levels. In contrast, the loss of
SWI/SNF complex components in SW13 cells has little effect on HAT
activity in the NM1 complexes (Fig. 8C). These results support the
conclusion that BRG1 is in stable intracellular association with p270
and is the source of the ATPase activity in the NM1 complexes.
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FIG. 8.
Analysis of NM1 complexes in BRG1-deficient cells. (A)
Immune complexes. NM1 complexes were precipitated from the
BRG1-deficient cell line SW13 (lane 1) or from HeLa cells (lane 2). The
position of the p190/BRG1 band in the HeLa cell complex is indicated at
the right. In all comparisons between HeLa and SW13 cells, protein
concentrations from each cell lysate were normalized before immune
precipitation. (B) ATPase assay. NM1 complexes isolated from SW13 cells
were assayed for associated ATPase activity in comparison with HeLa
cell-derived complexes. Assays were performed as described for Fig. 7,
and results are expressed as percent conversion from ATP-bound
32P to free 32Pi. The results shown
are the averages of four independent experiments (standard
deviation = 13.7%). Baseline activities in HeLa and SW13 lysates
precipitated with control monoclonal antibody 419 are represented by
circles and triangles, respectively; activity in HeLa-derived NM1
complexes is represented by squares; activity in SW13-derived NM1
complexes is represented by diamonds. (C) HAT assay. HAT activity was
determined as described for Fig. 6, using aliquots of the same lysates
shown in panel B. Antibody 419 was again used as a background
control.
|
|
| |
DISCUSSION |
The monoclonal antibodies that we have raised against p300
identify an immunologically related cell protein, p270, that
shares at least two distinct antigenic determinants with p300
(13). The subset of antibodies that cross-reacts with p270,
exemplified by NM1, recognizes p300, CBP, and p270 directly and
also indirectly coprecipitates a series of at least nine cellular
proteins stably associated with p300, CBP, or p270 in physiological
conditions. These proteins resolve into at least two complexes
involving the p300/CBP/p270 family. One complex contains TBP along with
p300 or CBP (apparently as alternates to TAFII250) in
association with at least two other TBP-associated proteins, p64
and p59 (1, 13). The SL8 (TBP-specific) complex, consistent
with the presence of p300, CBP, and TAFII250, contains HAT
activity. This complex does not contain detectable p270 or ATPase
activity.
A second group of proteins brought down with the NM1 antibodies
contains p270 as an integral component of SWI/SNF complexes. We have
confirmed the presence in the p270-containing complexes of BRG1, the
BRG1-associated factors BAF-155 and BAF-170 (50), and the
BRG1/hbrm-associated factor hSNF5 (INI-1) (36).
Conversely, probes of SWI/SNF complexes isolated through either
BAF-155-specific antibodies or BRG1-specific antibodies confirm the
presence of p270. The identification of p270 as an integral member of
human SWI/SNF complexes is consistent with the presence of a 250-kDa band noted by Wang et al. (50) and designated BAF-250. It is likely that p270 is equivalent to BAF-250.
Both yeast and human SWI/SNF complexes contain a nucleosome remodelling
activity (reviewed in reference 41). Taken
together with the HAT activities associated with p300/CBP (39,
53), the presence of p270 in SWI/SNF complexes suggests that
multiple aspects of chromatin remodelling may be mediated through a
series of proteins with structural homology to p300.
Identification of the precise structural determinants shared by p270
and p300 will likely require a better understanding of the
three-dimensional structure of p270 and p300 than is available now. The
relationship is likely to be limited, as the majority of
antibodies developed against p300 do not cross-react with p270 and
because p270 and p300 show distinguishable activities. The p270-containing complexes do not show HAT activity if p300 and CBP are
not present (Fig. 6). p270 is also not targeted by E1A (not shown) and
is not present in the TBP-specific complexes that contain p300 and CBP
(Fig. 5). We are currently sequencing p270 (12a). The
sequence (represented schematically in Fig.
9) reveals some unique features of p270,
in particular, the presence of a newly recognized DNA binding domain,
termed AT-rich interactive domain (ARID), first identified in the
mouse Bright and Drosophila Dead-Ringer proteins
(20, 26). This feature is also present in yeast Swi1,
suggesting that p270 is the human homolog of this member of the yeast
SWI/SNF complex. p300 does not contain an ARID region. p270 shows some
specific features in common with p300: both contain Q-rich regions
spanning about 200 residues (implicated in transactivation
functions [45]), and both contain potential
nuclear hormone receptor binding motifs, LXXLL (25). These two features are present in SWI1 as well and support the interpretation that p270 is a SWI1 homolog. The LXXLL motifs have not
previously been noted in SWI1, as their potential significance has only
recently become apparent. p270 does not contain long stretches of
sequence homology to p300 like those seen in comparison of p300 and
CBP. However, a filtered BLAST search program (3, 4)
identifies at least four homology regions (I to IV) distinct from the
Q-rich regions or LXXLL motifs. These are amino acid stretches of 22 to
66 residues showing 27 to 46% identity between p300 and p270.
Interestingly, all but one of these regions (IV) are in areas that are
not highly conserved between p300 and CBP. Also interestingly, mapping
of the NM1 epitope in p270 indicates that it is distinct from all of
these regions, implying that the extent of the relationship is greater
than indicated by the degree of linear sequence homology. The NM1
epitope appears to identify a structural epitope shared by p270 and
p300. This antibody reacts with native protein but not with
denatured proteins such as that present in Western blots
(13); its reaction in sequential immunoprecipitations depends on at least partial renaturing of the proteins. There are
at least two NM1-reactive sites in p300, near the amino and carboxy
ends. The NM1 epitope in p270 lies in the middle of the protein.
There is no recognizable linear sequence that is shared by the regions
containing the NM1 epitopes, supporting the idea that this is a
structural determinant. Because the p300/CBP population that we used as
antigen was native, enzymatically active protein, as determined
from the presence of HAT activity in stored immunogen samples, it is
likely that the surface epitopes that served as antigenic determinants
correlate with functional regions, perhaps with cofactor binding sites.
p270 may recruit SWI/SNF complexes to transcription sites in a manner
analogous to p300, bringing histone-modifying activity to such sites.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 9.
Alignment of p300, p270, and SWI1. The linear
amino acid sequences of p300, p270, and SWI1 are represented
schematically. Regions highly conserved between p300 and CBP include
the bromodomain, the HAT domain, and the E1A and P/CAF binding region.
The sequence of p270 is over 90% complete (assuming an estimated size
of 2,000 amino acids [aa]; the uncertain sequence is represented by a
dashed line). p270 does not show HAT activity of E1A binding activity.
There is also no evidence thus far of a bromodomain consensus sequence.
p270 contains an ARID DNA binding region (hatched box), a feature not
present in p300. p270 does show several specific features in common
with p300. Regions of conserved sequence between p270 and p300
(homology regions I to IV) are indicated by solid boxes within the
bars. In addition to the four regions of conserved sequence, p270 and
p300 have in common the presence of Q-rich regions (implicated in
transactivation functions) and LXXLL motifs (implicated in
nuclear receptor binding). p270 and p300 also have in common the
structural epitope recognized by NM1. p270 and SWI1 have an
overall similarity of structure, in that they both contain ARID
regions, and they both have multiple LXXLL motifs as well as Q-rich
regions. SWI1 has an unusual asparagine/threonine-rich stretch near
the N terminus; we do not yet know if this feature also occurs in p270.
Other than these common features, p270 and SWI1 do not show direct
sequence homology.
|
|
| |
ACKNOWLEDGMENTS |
The first three authors contributed equally to this work.
We thank Gerry Crabtree, Nouria Hernandez, and Ed Harlow for generous
gifts of antibodies, and we thank Annette Heagy, Patty Baxter, Steve
Dorfman, and John Gibas for expert technical assistance.
This work was supported by PHS grants CA53592, CA55330 (E.M.), and
CA68066 (P.Y.) from the NIH.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Fels Institute
for Cancer Research and Molecular Biology, Temple University School of
Medicine, 3307 N. Broad St., Philadelphia, PA 19140. Phone: (215)
707-7313. Fax: (215) 707-6989. E-mail:
betty{at}sgi1.fels.temple.edu.
Present address: Canji, Inc., San Diego, CA 92121.
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Abstract
Introduction
