Previous Article | Next Article 
Molecular and Cellular Biology, June 2001, p. 3876-3887, Vol. 21, No. 12
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.12.3876-3887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
p300 Forms a Stable, Template-Committed Complex
with Chromatin: Role for the Bromodomain
E. Tory
Manning,1
Tsuyoshi
Ikehara,2
Takashi
Ito,2
James T.
Kadonaga,3 and
W. Lee
Kraus1,4,*
Department of Molecular Biology and Genetics, Cornell
University, Ithaca, New York 148531;
Department of Biochemistry, Saitama Medical School,
Morohongo, Iruma-gun, Saitama 350-0495, Japan2;
Section of Molecular Biology, University of California, San
Diego, La Jolla, California 92093-03473; and
Department of Pharmacology, Weill Medical College of
Cornell University, New York, New York 100214
Received 7 November 2000/Returned for modification 22 December
2000/Accepted 16 March 2001
 |
ABSTRACT |
The nature of the interaction of coactivator proteins with
transcriptionally active promoters in chromatin is a fundamental question in transcriptional regulation by RNA polymerase II. In this
study, we used a biochemical approach to examine the functional association of the coactivator p300 with chromatin templates. Using in
vitro transcription template competition assays, we observed that p300
forms a stable, template-committed complex with chromatin during the
transcription process. The template commitment is dependent on the time
of incubation of p300 with the chromatin template and occurs
independently of the presence of a transcriptional activator protein.
In studies examining interactions between p300 and chromatin, we found
that p300 binds directly to chromatin and that the binding requires the
p300 bromodomain, a conserved 110-amino-acid sequence found in many
chromatin-associated proteins. Furthermore, we observed that the
isolated p300 bromodomain binds directly to histones, preferentially to
histone H3. However, the isolated p300 bromodomain does not bind to
nucleosomal histones under the same assay conditions, suggesting that
free histones and nucleosomal histones are not equivalent as binding
substrates. Collectively, our results suggest that the stable
association of p300 with chromatin is mediated, at least in part, by
the bromodomain and is critically important for p300 function.
Furthermore, our results suggest a model for p300 function that
involves distinct activator-dependent targeting and
activator-independent chromatin binding activities.
| |
INTRODUCTION |
Activated transcription of
genes by RNA polymerase II (RNA Pol II) requires the concerted actions
of sequence-specific DNA-binding transcriptional activators, chromatin
remodeling complexes, histone acetyltransferases (HATs), and
coactivators. Together, these factors function to overcome the
transcriptional repression caused by the packaging of genes into
chromatin and to stimulate the activity of RNA Pol II. The initiating
event in the process of activated transcription is the binding of
transcriptional activator proteins to binding sites in the promoters of
target genes. In many cases, the activities of the activator proteins
are regulated by input from cellular signaling pathways (e.g., steroid
hormones and mitogen-activated protein kinase pathways). Once bound to
DNA, activators can recruit chromatin remodeling complexes, HATs, and
coactivators to the promoter, leading to the formation of a stable RNA
Pol II preinitiation complex and, subsequently, transcription
initiation (reviewed in references 3, 19,
23, 30, 34, 50, and
67).
Transcriptional coactivators are a diverse group of factors and
multipolypeptide complexes, some of which possess intrinsic HAT
activity (16, 42, 43, 45, 63, 68). Coactivators with HAT
activity include p300 and the closely related CREB binding protein
(CBP; often referred to with p300 as p300/CBP) (4, 20), as
well as the p300/CBP-associated factor (PCAF), which functions as part
of a multipolypeptide complex (54, 69). In general,
coactivators play one or more of the following roles: (i) they function
as bridging factors to recruit other coactivators to the DNA bound
transcriptional activator (e.g., members of the p160/steroid receptor
coactivator [SRC] family of proteins which recruit p300/CBP to
ligand-activated steroid hormone receptors) (reviewed in reference
43); (ii) they acetylate nucleosomal histones in and
around the promoters of activated genes (e.g., the PCAF complex which
acetylates nucleosomal histones H3 and H4) (58); (iii)
they alter the activities of transcription factors and
chromatin-associated proteins by acetylating them (e.g., acetylation of
p53 by p300/CBP increases the sequence-specific DNA binding of p53)
(22; see references 9, 25, 49, 61 for
additional examples); and (iv) they make contacts with the basal
transcriptional machinery to stimulate the recruitment of RNA Pol II
and the formation of stable transcription preinitiation complexes
(e.g., the multipolypeptide TRAP-DRIP-ARC complex, which can enhance
the transcriptional activity of nuclear receptors and other activators)
(15, 23, 42, 45).
p300 and CBP are large multifunctional coactivators that participate in
all four of these processes. In addition to their intrinsic HAT
activity, p300 and CBP contain distinct domains for binding to
transcriptional activator proteins, other coactivators, and components
of the basal transcriptional machinery (4, 20). They also
contain a single bromodomain, a conserved 110-amino-acid sequence found
in many chromatin-associated proteins, including almost all nuclear
HATs (reviewed in references 28 and 66). The
bromodomain is thought to function as a histone binding motif (28, 66). Recent studies have shown the bromodomain to
have a conserved structure consisting of four antiparallel alpha
helices (a four-helix bundle) with a left-handed twist (12, 27,
57). The multiple distinct domains and functions of p300/CBP are
differentially required for coactivator function with different classes
of transcriptional activator proteins (35, 38, 39).
Although activator-mediated recruitment of coactivators has been well
established, the events occurring before and after recruitment are less
clear. Many fundamental questions regarding multifunctional coactivators such as p300/CBP relate to how their multiple functional activities are coordinated at the promoter during the course of events
leading to transcription initiation. Do coactivators bind to chromatin
prior to targeting to specific promoters by DNA-bound activators? Do
coactivators remain stably associated with a promoter once recruited?
If so, are contacts with a transcriptional activator sufficient to keep
the coactivators associated with the promoter, or are other
protein-protein contacts also required? In this study, we have used a
biochemical approach to address these questions using p300 as a model
multifunctional coactivator. Our results indicate that p300 forms a
stable, template-committed complex with chromatin. We have also found
that p300 binds directly to chromatin and that the binding requires the
bromodomain. Taken together, our results suggest a model for p300
function that involves distinct activator-dependent targeting and
activator-independent chromatin binding activities.
| |
MATERIALS AND METHODS |
Synthesis and purification of recombinant proteins.
Wild-type and bromodomain-deletion-containing
His6-tagged human p300 proteins were synthesized
in Sf9 cells using a baculovirus expression system and purified as
described previously (35-38). His6-tagged NF-
B p65 subunit (44)
was synthesized in Sf9 cells by using a recombinant baculovirus kindly
provided by J. Hiscott (Lady Davis Institute, Montreal, Canada) and
purified essentially as described for His6-tagged
p300 (36). Purified Sp1 was obtained from Promega
(Madison, Wis.). FLAG-tagged human PCAF was synthesized in Sf9 cells by
using a recombinant baculovirus kindly provided by Y. Nakatani
(Dana-Farber Cancer Institute) and purified as described for
FLAG-tagged human estrogen receptor (36).
DNA templates.
The wild-type human interferon regulatory
factor 1 (IRF-1) template in pUC118 contains 1.3 kb of the native gene,
which includes the core promoter and the upstream regulatory region
with the NF-B and Sp1 binding sites (6, 60). The IRF+30
construct contains a 30-bp insert in the IRF-1 promoter located 44 bp
downstream of the transcription start site and is otherwise identical
to the wild-type IRF-1 construct.
Chromatin assembly and in vitro transcription reactions.
Chromatin assembly reactions were performed with a chromatin assembly
extract derived from Drosophila embryos (S190) (5, 31,
36-38). The transcriptional activator proteins NF-B p65 and
Sp1 were added during the chromatin assembly reactions at concentrations of 200 and 30 nM, respectively. p300 was added at a
concentration of 50 nM to the chromatin templates, where indicated,
after the assembly reactions were complete. The reaction mixtures were
incubated for an additional 30 min at 27°C after the p300 was added
to allow the interaction of the p300 with the chromatin templates prior
to the addition of the HeLa cell nuclear extracts for transcription.
In vitro transcription reactions were performed with HeLa cell nuclear
extracts that were prepared essentially by the method of Dignam et al.
(13) with slight modification (37). The
template competition experiments were set up as indicated in Fig. 1 and described below and were analyzed in single-round transcription reactions as described previously (36), except that
transcription preinitiation complexes were formed for 45 min at 27°C
after the addition of the HeLa nuclear extract. The RNA products from
the in vitro transcription reactions were analyzed by primer extension analysis (36). All reactions were performed in duplicate,
and each experiment was performed a minimum of three separate times to
ensure reproducibility. The data were analyzed and quantified with a
PhosphorImager (Molecular Dynamics).
p300-chromatin interaction assays.
One 15-cm diameter dish
of Sf9 cells was infected with recombinant baculovirus for the
expression of wild-type or bromodomain-deletion-containing His6-tagged human p300 protein (38).
After 3 days of infection, the p300 proteins were purified as described
previously (36) but were left bound to the
nickel-nitrilotriacetic acid (NTA) resin. The p300-bound resin, or
resin similarly treated with uninfected Sf9 cell extract, was mixed
with 200 µl of chromatin prepared using S190 (consisting of 1 µg of
pUC118 plasmid DNA and 1.4 µg of core histones) and 300 µl of
buffer R (10 mM HEPES [pH 7.6], 10 mM KCl, 1.5 mM
MgCl2, 10% glycerol, 2 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT]) containing 0.5% NP-40 and 10 mM imidazole. The resin was then incubated with the chromatin for 2 h at 4°C with gentle mixing. After the incubation, the
resin was washed twice with 1 ml of buffer R containing 0.5% NP-40 and 10 mM imidazole. The material bound to the p300 was eluted in successive 100-µl volumes of buffer R containing 0.5% NP-40, 10 mM
imidazole, and NaCl (0.2, 0.4, 0.6, or 0.8 M). The final elution, which
contained 0.25 M imidazole and no NaCl, was used to elute the p300
itself from the resin. The various fractions were deproteinized with
proteinase K, extracted with phenol-chloroform, ethanol precipitated, and subjected to 1% agarose gel electrophoresis in 1×
Tris-borate-EDTA with ethidium bromide staining for DNA analysis.
Glycerol gradient analyses.
Chromatin was assembled using
the S190 extract as described above or by salt dialysis essentially as
described previously (29). Prior to use in the glycerol
gradient analyses, the salt-dialyzed chromatin was run on a linear
15-to-40% glycerol gradient to purify the slower-migrating chromatin,
which has a lower density of nucleosomes (i.e., not closely packed)
relative to the faster-migrating chromatin (26). For the
experiments whose results are shown in Fig. 4B, a volume of
S190-assembled chromatin containing 2.0 µg of plasmid DNA (pGIE0) and
2.8 µg of purified Drosophila core histones was incubated
with 2.0 µg of purified p300 for 1 h at 25°C. A corresponding control sample that lacked the chromatin but contained the p300 was set
up in buffer R. For the experiments whose results are shown in Fig. 5,
a volume of purified salt-dialyzed chromatin containing 1.6 µg of
plasmid DNA (pGIE0) and 1.0 µg of purified Drosophila core
histones (or an equivalent amount of the DNA alone) was incubated in
various combinations with 0.3 µg of purified p300 in 200 µl of
buffer G (20 mM HEPES [pH 7.6], 75 mM KCl, 0.1 mM EDTA, 10%
glycerol, 0.01% NP-40, 2 mM PMSF, 1 mM DTT) for 1 h at 25°C. In
both cases, after the incubation, the reaction products were applied to
4-ml linear 15-to-40% glycerol gradients prepared in buffer G. The
gradients were centrifuged at 60,000 rpm in a Beckman SW60 rotor for
4 h at 4°C. When the run was complete, 350-µl (for Fig. 4B) or
400-µL (for Fig. 5) fractions were removed from the top down for each
gradient. Fifty microliters of each fraction was extracted with
phenol-chloroform, ethanol precipitated, and subjected to 1% agarose
gel electrophoresis in 1× Tris-borate-EDTA with ethidium bromide
staining for DNA analysis. The remaining portion of each fraction was
precipitated with 25% trichloroacetic acid, run on 6 or 18%
acrylamide-sodium dodecyl sulfate (SDS) gels for p300 and core
histones, respectively, transferred to nitrocellulose, and analyzed by
Western blotting using an alkaline phosphatase detection system (for
Fig. 4B) or a 125I-labeled-protein A detection
system (for Fig. 5). The 125I-protein A blots
were analyzed with a phosphorimager (Fuji).
Histone acetylation reactions and assays.
Large-scale
reactions to generate acetylated histones for the binding assays and
for assembly into chromatin by salt dialysis were set up using core
histones purified from 0- to 12-h Drosophila embryos. Note
that these histones are hypoacetylated (essentially unacetylated), as
determined by Triton-acid-urea gels (M. Levenstein and J. Kadonaga,
unpublished data). About 110 µg of core histones was incubated with 1 µg of purified recombinant p300 or PCAF in 1× HAT buffer (50 mM
Tris · HCl [pH 8], 0.1 mM EDTA, 150 mM NaCl, 0.2 mM PMSF, 0.5 mM DTT) in the presence of 1 mM acetyl coenzyme A (acetyl-CoA) for 30 min at 30°C. The final concentrations of histones, p300, and PCAF in
the reaction mixtures were about 50 µM, 25 nM, and 100 nM, respectively.
To test the extent of histone acetylation, parallel reactions were
performed under similar conditions, replacing 10% of the
cold
acetyl-CoA with [
3H]acetyl-CoA (New England
Nuclear). The test samples were divided
into three portions. The first
portion was used to determine the
total counts per minute in the
reaction mixture by liquid scintillation
counting. The second was used
to analyze the incorporation of
[
3H]acetyl-CoA
into the histones by trichloroacetic acid precipitation,
coupled to a
filter binding assay and liquid scintillation counting.
The third was
analyzed on 18% acrylamide-SDS gels stained using
Coomassie brilliant
blue R-250, with subsequent fluorography.
After fluorography, the
individual histone bands were excised
and subjected to liquid
scintillation counting. Using the information
from these assays, as
well as the known total concentration of
acetyl-CoA in the reactions,
the number of acetyl groups added
per histone was
calculated.
p300 bromodomain-histone interaction assays.
Fusions of
glutathione-S-transferase (GST) with fragments of p300 were
expressed in Escherichia coli and purified using standard glutathione-agarose chromatography. The fragments of p300 contained the
CREB interaction domain (KIX; residues 568 to 828) (10), the bromodomain (residues 1047 to 1157) (28), and the
SRC interaction domain (SID; residues 2021 to 2156)
(32). The purified proteins were left bound to the resin,
adjusted to equal resin and protein amounts per unit of volume, and
stored at 4°C for subsequent in vitro binding assays. The binding
assays were performed using the hypo- or hyperacetylated
Drosophila core histones described above or the same
histones assembled into chromatin by salt dialysis (29).
For the binding assays, equal volumes of resin (15 µl) containing
about 1.5 µg of GST fusion protein were incubated with 2.8
µg of
hypo- or hyperacetylated
Drosophila core histones (or with
salt-dialyzed chromatin containing the same amount of histones)
in 400 µl of binding buffer (10 mM HEPES [pH 7.6], 200 mM NaCl,
10 mM KCl,
1.5 mM MgCl
2, 10% glycerol, 0.05% NP-40, 2 mM
PMSF,
1 mM DTT) for 2 h at 4°C with gentle mixing. After the
incubation,
the resin was washed three times in ice-cold wash buffer
(10 mM
HEPES [pH 7.6], 400 mM NaCl, 10 mM KCl, 1.5 mM
MgCl
2, 10% glycerol,
0.1% NP-40, 2 mM PMSF, 1 mM DTT). After the last wash, the wash
buffer was aspirated completely
from the resin and the resin was
resuspended in 20 µl of 1× SDS gel
loading solution. The samples
were boiled, and 10-µl aliquots were
run on an 18% polyacrylamide-SDS
gel stained with Coomassie brilliant
blue R-250.
| |
RESULTS |
p300 forms a stable, template-committed complex with
chromatin.
In previous studies, we observed that p300 acts
effectively as a transcriptional coactivator in vitro with chromatin
templates but not with nonchromatin templates (37, 38). To
investigate further the basis for this effect, we examined whether p300
interacts or associates with chromatin in a functionally important
manner. To this end, we carried out template competition experiments
(Fig. 1A). In these studies, we used two
nearly identical DNA templates which are responsive to the
transcriptional activator proteins NF-B and Sp1, as well as the
coactivator p300. They are IRFwt, which is the wild-type version of the
IRF-1 promoter (from 
1312 to +38 relative to the transcription start
site), and IRF+30, which is identical to IRFwt except that it has a
30-bp insert downstream of the core promoter (Fig. 1A, top). The
transcript derived from IRF+30 is 30 nt longer than the transcript
produced from IRFwt, and thus, the two transcripts can be clearly
distinguished by primer extension analysis.
View larger version (30K):
[in this window]
[in a new window]
|
FIG. 1.
p300 forms a stable, template-committed complex
with chromatin. (A) (Top) Schematic diagram of the templates IRFwt and
IRF+30. Ovals and rectangles represent binding sites for Sp1 and
NF-B, respectively, and are not drawn to scale. (Bottom)
Outline of the template competition experiments, which are described in
the text. p300 was added to the chromatin templates at a concentration
of 50 nM, which corresponds to approximately one p300 polypeptide per
nucleosome prior to the addition of the challenge template or one p300
polypeptide per two nucleosomes after the addition of the challenge
template. (B) p300 forms a template-committed complex with chromatin.
Template competition experiments were carried out as shown in panel A
(bottom). Briefly, the two templates were separately assembled into
chromatin in the presence of NF-B p65 and Sp1. After completion of
chromatin assembly, purified p300 was added to one template (p300
Before) but not the other, and the samples were incubated for 30 min to
allow the interaction of p300 with the template. Next, the two
templates were mixed and incubated for an additional 30 min to allow
the potential exchange of p300 from one template to the other. In some
instances, p300 was added at the time of template mixing as a control
or reference (p300 After). The amount of transcription from each
template was analyzed in single-round transcription assays with a HeLa
cell nuclear extract. The RNA products were analyzed by primer
extension analysis with the radiolabeled IRF primer shown in panel A
(top) (IRF Primer*). (C) p300 remains functionally active throughout
the template competition experiments. Template competition experiments
were carried out as described for panel B, except that one chromatin
assembly reaction mixture lacked template DNA (mock assembly). The
transcription products from single-round transcription assays were
analyzed by primer extension analysis.
|
|
In the template competition experiments (Fig.
1A, bottom), IRFwt and
IRF+30 were separately assembled into chromatin using
a
Drosophila embryo-derived chromatin assembly extract (S190)
in the presence of the NF-
B p65 subunit and Sp1. After the
completion
of chromatin assembly, purified recombinant human p300 was
added
to one template (template 1) but not to the other (template 2),
and the samples were incubated for 30 min to allow the interaction
of
p300 with template 1. Next, the two templates were mixed and
incubated
for an additional 30 min to allow the possible exchange
of p300 from
one template to the other. In some cases, as a control,
p300 was added
after the mixing of the two templates. The amount
of transcription from
each template was analyzed in single-round
transcription assays with a
HeLa cell nuclear
extract.
The results of template competition experiments with IRFwt and IRF+30
are shown in Fig.
1B. When IRF+30 was used as template
1 and IRFwt was
template 2, commitment of p300 to IRF+30 was seen
(i.e., p300 remained
associated with IRF+30 and activated its
transcription fourfold
more strongly than IRFwt [Fig.
1B, lane
2]). Alternatively,
when IRFwt was template 1 and IRF+30 was template
2, commitment of p300
to IRFwt was observed (Fig.
1B, lane 3).
Control experiments confirmed
that transcription from either IRFwt
or IRF+30 was dependent on
exogenously added, purified p300 (Fig.
1B, compare lane 1 with lanes 4 and 5). Since it was possible
that the apparent template commitment was
due to the loss of p300
activity during the first 30 min incubation
prior to the addition
of template 2, we carried out an additional
control experiment
in which a mock chromatin assembly reaction mixture
(i.e., complete
chromatin assembly reaction mixture minus DNA) was used
instead
of template 1. This experiment revealed that more than 75% of
p300 coactivator activity remained after the first 30 min of incubation
(Fig.
1C, compare lanes 2 and 3). Together, these results suggest
that
p300 can form a template-committed complex with chromatin
during
transcriptional
activation.
Time course and activator independence of p300 template
commitment.
To investigate further the template commitment by
p300, we examined the time course for the stable association of p300
with the chromatin templates. As shown in Fig.
2, the extent of commitment of p300 to
template 1 (i.e., the template to which p300 was first added) increased
as the amount of time before the addition of template 2 increased. For
example, when the preincubation time was 5 min, template 2 was
activated to about 75% of the level of template 1. When the
preincubation time was increased to 45 min, template 2 was activated to
about 30% of the level of template 1, indicating a greater commitment
of p300 to template 1 with longer preincubation time. These results
illustrate the time-dependent nature of the association and commitment
of p300 with chromatin templates.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 2.
Time-dependent commitment of p300 with chromatin. A time
course of p300 commitment with chromatin templates is shown. IRF+30
(template 1) and IRFwt (template 2) were assembled into chromatin in
the presence of NF-B p65 and Sp1. The amount of transcription from
each template was analyzed in single-round transcription assays with a
HeLa cell nuclear extract as described for Fig. 1. The RNA products
were analyzed by primer extension analysis. Each point is the mean from
two independent determinations. Similar results were obtained when
IRFwt was used as template 1 and IRF+30 was used as template 2.
|
|
Activator-dependent targeting is one mechanism thought to be involved
in directing coactivators to specific promoters. Thus,
we tested
whether the p300 template commitment required the presence
of the
activator proteins (i.e., NF-
B p65 and Sp1). In control
reactions,
no transcription was observed when the activator proteins
were left out
of the reaction mixtures, even in the presence of
exogenously added
p300 (Fig.
3, compare lanes 2 and 3 for
IRFwt
and lanes 4 and 5 for IRF+30). Thus, the transcriptional activity
of the test promoters was dependent on both the activators and
p300.
Surprisingly, when template 1 lacked activator proteins,
commitment of
p300 to that template was still observed, as indicated
by a failure of
p300 to fully activate template 2 even in the
absence of transcription
from template 1 (Fig.
3, lanes 2 and
4). For example, even though no
transcription from IRFwt was observed
in the absence of activators when
p300 was added, commitment of
p300 to the IRFwt template was still
observed, as indicated by
the failure of p300 to substantially activate
the IRF+30 challenge
template which contained activator proteins (Fig.
3, lane 2).
These results indicate that p300 can commit to a chromatin
template
even in the absence of transcriptional activator proteins.
Furthermore,
they suggest that activator-dependent recruitment and
template
commitment are distinct activities of p300. Collectively, the
results from the template competition experiments indicate that
there
is a stable, activator-independent association of p300 with
chromatin.
View larger version (37K):
[in this window]
[in a new window]
|
FIG. 3.
The formation of a template-committed complex by p300
does not require the presence of a transcriptional activator. p300
template competition experiments were carried out in the presence or
absence of transcriptional activators (NF-B p65 and Sp1) using the
experimental scheme described for Fig. 1. The amount of transcription
from each template was analyzed in single-round transcription assays
with a HeLa cell nuclear extract, and the RNA products were analyzed by
primer extension analysis.
|
|
p300 binds directly and stably to chromatin.
Three lines of
evidence suggested to us that p300 might bind to chromatin. First, the
results from our template competition experiments are consistent with a
binding process. Second, p300 has HAT activity, which presumably
requires the binding of p300 to histones. Last, p300 contains a
bromodomain, a protein-protein interaction motif thought to be
important for binding to the amino-terminal tails of histones (reviewed
in reference 66). To determine if p300 can bind to
chromatin, we performed the assay shown in Fig. 4A. In the assay, full-length
His6-tagged p300 was immobilized on nickel-NTA
resin and incubated with chromatin assembled using the S190 extract
(Fig. 4A, top). The chromatin was not purified, and thus the binding
reaction mixtures contained all of the S190 extract proteins. The resin
was washed and elutions were performed using increasing amounts of
NaCl. The binding of chromatin to the immobilized p300 was assessed by
assaying for the presence of DNA in the elutions. As shown in Fig. 4A,
a considerable amount of chromatin binding to the p300-containing resin
was observed, in contrast to the control resin lacking p300, with the
peak occurring in the 0.4 and 0.6 M NaCl fractions.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 4.
Full-length p300 binds to chromatin. (A) Immobilized
p300 binds to S190-assembled chromatin. (Top) Schematic representation
of the p300-chromatin interaction assay. (Bottom) Results of a
representative assay performed as follows. Full-length, wild-type,
His6-tagged p300 was immobilized on nickel-NTA resin and
incubated with chromatin assembled using S190 as described in Materials
and Methods. After the incubation, the resin was washed and the bound
material was eluted using buffers containing increasing amounts of
NaCl. The final elution contained 0.25 M imidazole to elute the p300
itself from the resin. The various fractions were deproteinized and
subjected to agarose gel electrophoresis with ethidium bromide (EtBr)
staining for DNA analysis. Twenty percent of the input from each
experiment was loaded. (B) p300 cofractionates with S190-assembled
chromatin on glycerol gradients. Purified p300 was incubated with or
without S190-assembled chromatin for 1 h at 25°C. After the
incubation, each reaction was run on a linear 15-to-40% glycerol
gradient. Fractions from the gradients were analyzed for p300 and core
histones by Western blotting and for DNA by agarose gel electrophoresis
with ethidium bromide staining. Note that the antihistone antiserum
used in this experiment preferentially detects H2A and H2B.
|
|
To confirm this result using a different approach, we assayed the
ability of p300 to bind to and cofractionate with S190-assembled
chromatin on a linear 15-to-40% glycerol gradient. After
centrifugation,
fractions were removed and analyzed for p300 and core
histones
by Western blotting with appropriate antibodies and for DNA by
agarose gel electrophoresis with ethidium bromide staining. As
shown in
Fig.
4B (top), p300 sedimented in the top half of the
gradient
(fractions 2 to 8) when analyzed in the absence of chromatin.
However,
when preincubated with S190-assembled chromatin, a portion
of the p300
(about 10%) cofractionated with the chromatin (i.e.,
histones and
DNA) near the bottom of the gradient (fractions 12
and 13). Together,
the results in Fig.
4 indicated that p300 can
bind to chromatin, but
due to the use of unpurified chromatin,
we could not determine if the
binding was direct or
indirect.
To determine if p300 can bind directly to chromatin, we assayed the
ability of purified p300 to cofractionate with salt-dialyzed
chromatin,
prepared using core histones and plasmid DNA only,
on a glycerol
gradient (Fig.
5). p300 was incubated
with salt-dialyzed
chromatin or an equivalent amount of plasmid DNA
alone and was
then subjected to centrifugation on a glycerol gradient
with subsequent
analysis for p300, histones, and DNA as described
above. When
loaded on the gradient alone, p300 was found as a peak in
fraction
2 (Fig.
5A). In contrast, when p300 was preincubated with
salt-dialyzed
chromatin, the peak of p300 appeared in fraction 5, coincident
with the chromatin (i.e., histones and DNA) (Fig.
5B),
indicating
a direct interaction between p300 and the chromatin. No
interactions
between p300 and DNA alone were observed in this assay
(Fig.
5C).
(Note that the differences between the sedimentation
profiles
in Fig.
4B and
5 are due to the use of crude versus purified
chromatin,
as well as differences in the amounts of p300 in the
reaction
mixtures; see Materials and Methods.) Together, the results
from
Fig.
4 and
5 indicate that p300 can bind directly and stably to
chromatin.
View larger version (26K):
[in this window]
[in a new window]
|
FIG. 5.
p300 cofractionates with salt-dialyzed chromatin on
glycerol gradients. Salt-dialyzed chromatin or an equivalent amount of
DNA alone was incubated with purified p300 for 1 h at 25°C.
After the incubation, each reaction was run on a linear 15-to-40%
glycerol gradient. Fractions from the gradients were analyzed for p300
and core histones by Western blotting and for DNA by agarose gel
electrophoresis with ethidium bromide staining.
|
|
The p300 bromodomain is required for the interaction of p300 with
chromatin.
Previous reports have suggested that the bromodomain is
important for the interaction of HAT proteins (e.g., Gcn5, PCAF, and TAFII250) with the amino-terminal tails of
histones (12, 27, 56, 57). To determine if the p300
bromodomain is required for the binding of p300 to chromatin, we
performed an assay similar to the one shown in Fig. 4A. In this case,
we compared the chromatin-binding ability of wild-type p300 with that
of a mutant p300 lacking the bromodomain (Fig.
6A). As shown in Fig. 6B, deletion of the
bromodomain dramatically reduced the ability of p300 to bind chromatin.
This was not due simply to instability of the mutant p300 protein as indicated by SDS-polyacrylamide gel electrophoresis (PAGE) with samples
of the p300 resins pre- and postincubation (Fig. 6C). These results
indicate that an intact bromodomain is required for the binding of p300
to chromatin.
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 6.
The p300 bromodomain is required for the interaction of
p300 with chromatin. (A) Schematic diagrams of wild-type (Wt) p300 and
a bromodomain-deletion-containing p300 mutant (p300  Bromo). The
locations of the CREB binding site, the bromodomain, the
acetyltransferase domain (AT), the glutamine-rich region (Q-rich), and
the SRC binding site of p300 are shown. (B) The p300 bromodomain is
required for the binding of p300 to chromatin. The interaction of
chromatin with immobilized wild-type or bromodomain-deletion-containing
p300 was assayed as described for Fig. 4A. The DNA component of the
bound chromatin was resolved on agarose gels with ethidium bromide
(EtBr) staining. Twenty percent of the input from each experiment was
loaded on the gel. (C) The wild-type and p300 Bromo proteins are
stable during the interaction assay. Portions of the p300 proteins
immobilized on the resin were analyzed before and after the incubation
with chromatin by SDS-PAGE analysis with subsequent staining using
Coomassie brilliant blue R-250.
|
|
The isolated p300 bromodomain can bind to free histones but not to
nucleosomal histones.
To determine if the isolated p300
bromodomain can bind to histones as reported previously for other
bromodomain-containing proteins (12, 27, 56, 57), we
performed a series of in vitro interaction assays. GST fusions with
fragments of p300 (the bromodomain, the CREB interaction domain
[KIX], or the SID) (Fig. 7A) were
expressed in E. coli and purified using
glutathione-agarose resin (Fig. 7B). The GST resins were incubated with
native Drosophila core histones (which are hypoacetylated as
determined by Triton-acid-urea gel analysis [Levenstein and Kadonaga,
unpublished]), and the specifically bound material was assayed by
acrylamide-SDS gel analysis with subsequent staining using Coomassie
blue. In this assay, the binding of all four core histones, but
preferentially histone H3, to the GST-p300 bromodomain fusion was
observed (Fig. 7C, top, lane 3). In contrast, no binding of the
histones to GST alone or to the GST fusions containing the p300 KIX or
SID fragments was observed (Fig. 7C, top, lanes 2, 4, and 5). These
results indicate that the isolated p300 bromodomain can bind to core
histones and that it preferentially binds to histone H3.
However, our results cannot distinguish between the weak binding of
histones H2A, H2B, and H4 directly to the GST-p300 bromodomain fusion
and their weak association indirectly via interactions with histone H3
(Fig. 7C, top, lane 3).
View larger version (35K):
[in this window]
[in a new window]
|
FIG. 7.
The p300 bromodomain preferentially binds to histone H3
as free histones, but it does not bind to nucleosomal histones. (A)
Schematic diagrams of wild-type p300 and fragments of p300 used as GST
fusions. The locations of the CREB binding site, the bromodomain, the
acetyltransferase domain (AT), the glutamine-rich region (Q-rich), and
the SRC binding site of p300 are shown. (B) Purification of GST fusion
proteins. Fusions of GST with the fragments of p300 indicated in panel
A were expressed in E. coli, purified using standard
glutathione-agarose chromatography, and left bound to the resin.
Aliquots of each resin were analyzed by SDS-PAGE with subsequent
staining using Coomassie brilliant blue R-250. (C) The p300 bromodomain
preferentially binds to histone H3 as free histones. Equal volumes of
resin containing equal amounts of GST fusion protein were incubated
with purified native Drosophila core histones (which are
hypoacetylated; see Materials and Methods) or the same histones
acetylated with purified recombinant p300 or PCAF. After the
incubation, the resins were washed three times and the bound proteins
were analyzed by SDS-PAGE with subsequent staining using Coomassie
brilliant blue R-250. Twenty percent of the input from each experiment
was loaded. (D) Control experiment showing the extent of acetylation of
the Drosophila core histones used for panels C and E. Acetylation reactions similar to those used to generate the acetylated
histones for panels C and E were performed in the presence of a known
concentration of [3H]acetyl-CoA. The reaction products
were analyzed by filter binding assays (not shown), as well as by
SDS-PAGE with subsequent fluorography or staining using Coomassie
brilliant blue R-250 (right panel). The filter binding assays and the
fluorography were used to calculate the number of acetyl groups incorporated per
histone, plotted as the means plus the range for two separate
determinations (left panel). Note the observable shift in the mobility
of the hyperacetylated histones (most evident with histone H3 plus
p300) to a faster-migrating species on both the fluorogram and the
Coomassie-stained gel (marked by dots and arrows). (E) The isolated
p300 bromodomain does not bind to nucleosomal histones. Binding assays
were performed as described for panel C using salt-dialyzed chromatin
in place of the free histones for lanes 3 through 8. The salt-dialyzed
chromatin was assembled from purified native Drosophila
core histones or the same histones acetylated with purified recombinant
p300 or PCAF before chromatin assembly. Note that the salt-dialyzed
chromatin input contained the same amount of histones as used for panel
C.
|
|
Previous studies have suggested a role for acetylation in the binding
of histones to bromodomains (
12,
27,
57). Thus,
we
analyzed whether preacetylating the histones with p300 or PCAF
would
affect their binding to the p300 bromodomain. Large-scale
histone
acetylation reactions were set up using native
Drosophila core histones to generate material for the binding assays.
Smaller-scale
reactions were performed in parallel in the presence of
[
3H]acetyl-CoA under similar conditions to
monitor the extent of
acetylation by filter binding assays (data not
shown) and fluorography
(Fig.
7D, right). p300 added about four acetyl
groups per molecule
of histone H3 and H4 and about one acetyl group per
molecule of
histone H2A and H2B, while PCAF added about one acetyl
group per
molecule of histone H3 and less than one acetyl group per
molecule
of H2A, H2B, and H4 (Fig.
7D, left). These results are in good
agreement with the results of a previous study showing that, with
free
histones, recombinant p300 preferentially acetylates H3 and
H4 at
multiple sites, while recombinant PCAF preferentially acetylates
H3 at
a single site (
58). In the in vitro binding assays, the
acetylated histones (preferentially H3) bound to the isolated
p300
bromodomain (Fig.
7C, lanes 8 and 13) but did not do so more
strongly
than the hypoacetylated histones (Fig.
7C, compare lanes
8 and 13 with
lane 3). Thus, two different histone acetylation
patterns, namely,
those generated by p300 and PCAF, do not affect
the binding of histones
to the p300
bromodomain.
Finally, we examined the ability of the isolated p300 bromodomain to
bind directly to chromatin. Preparations of histones
similar to those
used for the binding experiments in Fig.
7C were
assembled into
chromatin by salt dialysis. The binding of the
salt-dialyzed chromatin
to the GST-p300 bromodomain fusion was
assayed under the same
conditions used for the free histones in
Fig.
7C (i.e., the same
buffer, salt, and detergent conditions,
as well as the same total
amount of histones in the reaction mixtures).
Under these conditions,
we observed no binding of chromatin to
the isolated p300 bromodomain
(Fig.
7E, compare lanes 2 and 4),
even when the histones used to
assemble the chromatin were preacetylated
by p300 or PCAF (lanes 6 and
8). Similar results were obtained
using S190-assembled chromatin or
mononucleosomes (data not shown).
Together, the results in Fig.
6B and
7E suggest that the p300
bromodomain is necessary, but not sufficient,
for the binding
of p300 to
chromatin.
| |
DISCUSSION |
In this study, we examined the functional association of the
coactivator p300 with chromatin templates. We observed that p300 forms
a stable, template-committed complex with chromatin during transcriptional activation. These results led us to examine the binding
of p300 to chromatin. In doing so, we found that p300 binds directly to
chromatin and that the binding requires the bromodomain. In addition,
we observed that the isolated p300 bromodomain binds directly to
histones, preferentially to histone H3, but does not bind to chromatin.
Thus, the bromodomain is necessary, but not sufficient, for the binding
of p300 to chromatin. Considering these results together with our
previous results showing that the bromodomain is functionally important
for p300 HAT and coactivator activities (38), we conclude
that the stable association of p300 with chromatin is mediated, at
least in part, by the bromodomain and is critical for p300 function.
Functional significance of p300-chromatin interactions and template
commitment.
Current models of coactivator function suggest that
sequence-specific DNA-binding transcriptional activator proteins, such as NF-B, target coactivators, such as p300/CBP, to promoters via
direct protein-protein interactions. Indeed, the p65 subunit of NF-B
has been found to bind directly to p300/CBP, and this interaction has
been shown to be critical for the enhancement of NF-B activity by
p300 (47, 71). In Fig. 3, we have shown that the formation
of a stable, template-committed complex of p300 with chromatin
templates does not require the presence of a transcriptional activator
protein. Transcriptional activation by p300, however, does require an
activator. Thus, activator-mediated targeting and template commitment
are distinct activities that contribute to transcriptional activation.
What might be the role of p300-chromatin interactions and template
commitment during transcriptional activation? Interactions
between p300
and chromatin prior to activator-mediated targeting
might represent the
initial association of p300 with chromatin.
Such interactions would
allow DNA-bound activators to recruit
p300 from chromatin, rather than
from solution, and target it
to specific promoters. Alternatively, the
initial interactions
could allow p300 to preacetylate the chromatin
template, allowing
more efficient binding of the transcriptional
activator to the
chromatin. These distinct possibilities will need to
be explored
in more
detail.
Interactions between p300 and chromatin after activator-mediated
targeting could stabilize the association of p300 at transcriptionally
active promoters. For example, in a previous study using the estrogen
receptor as a transcriptional activator, we showed that although
p300
does not function to enhance transcription reinitiation,
it does act
cooperatively with activators to enhance initiation
in each round of
transcription through multiple successive rounds
(
37). The
formation of a stable, template-committed complex
of p300 with
chromatin at the promoter through multiple rounds
of transcription
could facilitate this activity. Indeed, we have
observed p300 template
commitment through three rounds of NF-
B-Sp1-mediated
transcription in multiple-round transcription experiments (i.e.,
without Sarkosyl [data not shown]). Additionally, interactions
between p300 and chromatin after activator-mediated targeting
could
allow the continued presence of p300 at promoters in the
absence of
ongoing DNA binding by a transcriptional activator.
Such a result has
been observed at the
Saccharomyces cerevisiae HO promoter
using chromatin immunoprecipitation assays (
11).
Components of the SWI/SNF chromatin remodeling complex and the
Spt-Ada-GcnS acetyltransferase (SAGA) coactivator complex, both
of which have bromodomain-containing subunits (
28), were
found
to associate with the HO promoter even after a transient
association
of the Swi5 transcriptional activator protein subsided
(
11).
Taken together, our results suggest that distinct
activator-dependent
targeting and activator-independent chromatin
binding activities
can contribute to the function of p300 as a
coactivator at promoters
in
chromatin.
Role of the bromodomain in the function of p300 and other
chromatin-associated factors.
We have shown previously that the
bromodomain is critical for at least two aspects of p300 activity,
namely, the efficient acetylation of nucleosomal histones and the
activation of transcription (38). With regard to p300 HAT
activity, it is likely that the bromodomain participates in the
recognition and binding of the nucleosomal substrate. However, although
the bromodomain is required for the binding of p300 to chromatin, it is
not sufficient (Fig. 6B and 7E). Thus, other domains of p300 must also
contribute to this activity. The HAT domain is a likely candidate. This
is supported by previous studies demonstrating that fragments of
p300/CBP containing the HAT domain retain at least some ability to
acetylate nucleosomal histones (1, 55), an activity that
requires binding of the substrate.
With regard to p300 transcriptional activity, the bromodomain is likely
to have two roles, both involving the binding of nucleosomal
histones.
First, as described above, the bromodomain is required
for efficient
p300 HAT activity with nucleosomal substrates. Since
the p300 HAT
activity is required for full transcriptional activity
(
38), the bromodomain contributes to p300 transcriptional
activity,
at least in part, by supporting p300 HAT activity. In fact,
we
have found that the transcriptional phenotype of the p300
bromodomain
mutant shown in Fig.
6 is similar to that of a p300 HAT
mutant
(
38). Second, the bromodomain is important for the
stable interaction
of p300 with chromatin, thus contributing to its
ability to form
a template-committed complex with chromatin. As
described above,
this could contribute to p300's ability to associate
with chromatin
prior to activator-mediated targeting and to enhance
transcription
initiation by stable association with the promoter
through multiple
rounds of
transcription.
Bromodomains have also been identified in numerous other
chromatin-associated factors, including
S. cerevisiae Gcn5
(yGcn5),
a transcription-related HAT protein. Interestingly, the
bromodomain
of yGcn5 plays roles in the activity of the yeast SAGA
complex,
a Gcn5-containing coactivator complex (
21),
similar to those
of the bromodomain in p300. For example, a recent
study showed
that purified SAGA complex containing wild-type yGcn5, but
not
a bromodomain deletion mutant of yGcn5, was able to acetylate
nucleosomal histones in vitro (
62). Interestingly, the HAT
activity
of another
S. cerevisiae Gcn5-containing complex,
Ada (
21),
was found to be unaffected by deletion of the
yGcn5 bromodomain
(
62). Further analysis revealed that a
specific transcriptional
defect for the
HIS3 gene was
similar in yeast strains harboring
mutations in the yGcn5 bromodomain
or HAT domain, although the
transcriptional defect was not as severe as
we have observed for
the p300 bromodomain mutant in our studies
(
62). Other studies
with
S. cerevisiae have
shown that deletion of the yGcn5 bromodomain
results in minor growth
defects and reduced activation by weak
transcriptional activators
(
8,
18,
46). Thus, with respect
to histone acetylation and
transcriptional activation, the bromodomains
of human p300 and yGcn5
appear to have similar roles, suggesting
an evolutionarily conserved
function for this motif. The role
of the bromodomain in the activities
of other chromatin-associated
proteins seems to vary depending on the
protein. In many cases,
bromodomains are required for full activity of
the proteins that
contain them (
2,
7,
14,
48,
65) and in
other cases
they are not (
15,
17,
41,
62). Together, the
available
data suggest that bromodomains play essential roles in the
activities
of many, but not all, of the proteins that contain
them.
The bromodomain as a histone-interacting domain.
The
biochemical function of the bromodomain had remained elusive since it
was first identified as a conserved sequence motif found in human,
Drosophila, and yeast proteins (24). However, recent studies have begun to elucidate the biochemical function of the
bromodomain as a histone-interacting domain. Results from a number
of recent studies suggest that the bromodomains from various factors
(e.g., Gcn5, PCAF, and TAFII250) can bind
directly to histones (12, 27, 56, 57). Those studies were
performed using short polypeptides representing sequences from the
amino-terminal tails of the histones. Here we show that the isolated
p300 bromodomain can bind directly to purified, native, full-length
histones (Fig. 7C). Furthermore, we have demonstrated a critical role
for the bromodomain in the binding of p300 to chromatin (Fig. 6B),
although the isolated p300 bromodomain is unable to bind to nucleosomes under our assay conditions (Fig. 7E). Thus, bromodomains likely serve
as histone-interacting domains, contributing to chromatin binding, in
most of the proteins in which they are found.
In our studies (Fig.
7C), we observed no effect of histone acetylation
on the binding of the p300 bromodomain to free or nucleosomal
histones.
These results are in contrast to the results of three
recent reports
suggesting that histone acetylation plays an important
role in the
binding of the amino-terminal tail of histone H4 to
the bromodomains of
human PCAF (
12), human TAF
II250
(
27),
and yGcn5 (
57). These contrasting
results may reflect differences
in the patterns of acetylation,
intrinsic differences among the
four different bromodomains used in the
studies, the use of full-length
histones versus short polypeptides from
the histone tails, or
differences in the methodologies used. With
respect to the first
possibility, we tested histones acetylated with
only two different
HATs, namely p300 and PCAF. It is possible that a
particular pattern
of acetylation is required to enhance the binding of
histones
to a bromodomain and that the patterns of acetylation
generated
by p300 or PCAF are not recognized by the p300 bromodomain.
Alternatively,
our results might indicate that the p300 bromodomain
binds to
a region other than the amino-terminal tails of the histones.
Additional studies will be required to explore these
possibilities.
Multiple p300 interactions at transcriptionally active
promoters.
Our results indicate that the binding of p300/CBP to
chromatin via its bromodomain is critically important for directing the stable association of p300 with transcriptionally active promoters in
chromatin, as well as for p300 HAT and coactivator functions. However,
other interactions involving p300 also seem to play a role. They
include stable binding to transcriptional activators, contacts with
components of the basal transcriptional machinery, and indirect
association with chromatin via histone-binding proteins. For example,
p300/CBP makes multiple contacts within the beta interferon
enhanceosome (33, 47, 71). In addition, p300/CBP has been
shown to interact with components of the basal transcriptional machinery, including RNA polymerase-containing complexes (51, 52), TFIIB (40, 53), and TBP (64).
Furthermore, recent studies have indicated that p300/CBP can interact
with histone binding proteins, such as RbAp48 and nucleosome assembly
protein 1 (59, 70). It is likely that multiple distinct
interactions such as those listed above serve to stabilize the
association of p300/CBP with transcriptionally active promoters in vivo
after activator-dependent targeting and activator-independent chromatin binding have occurred. Such interactions could also contribute to the
stable commitment of p300 to a chromatin template and enhanced transcription initiation through multiple rounds of transcription.
| |
ACKNOWLEDGMENTS |
This work was supported by a Career Award in the Biomedical
Sciences from the Burroughs Wellcome Fund and a grant from the National
Institutes of Health (DK58110) to W.L.K, a grant from the National
Institutes of Health (GM46995) to J.T.K, and a grant from the Japanese
Society for the Promotion of Science (JSPS) Research for the Future
Program to T.I.
We thank Kathy Lee, Edwin Cheung, and Erik Andrulis for critical
reading of the manuscript. We are grateful to John Hiscott for the p65
baculovirus; Y. Cha Henderson, A. Deisseroth, and T. Burke for the
hIRF-1 constructs; and Pat Nakatani for the PCAF baculovirus.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Genetics, Cornell University, 465 Biotechnology Building, Ithaca, NY 14853. Phone: (607) 255-6087. Fax: (607) 255-6249. E-mail: wlk5{at}cornell.edu.
| |
REFERENCES |
| 1.
|
Bannister, A. J., and T. Kouzarides.
1996.
The CBP co-activator is a histone acetyltransferase.
Nature
384:641-643[CrossRef][Medline].
|
| 2.
|
Barlev, N. A.,
V. Poltoratsky,
T. Owen-Hughes,
C. Ying,
L. Liu,
J. L. Workman, and S. L. Berger.
1998.
Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the Ku-DNA-dependent protein kinase complex.
Mol. Cell Biol.
18:1349-1358[Abstract/Free Full Text].
|
| 3.
|
Berk, A. J.
1999.
Activation of RNA polymerase II transcription.
Curr. Opin. Cell Biol.
11:330-335[CrossRef][Medline].
|
| 4.
|
Blobel, G. A.
2000.
CREB-binding protein and p300: molecular integrators of hematopoietic transcription.
Blood
95:745-755[Free Full Text].
|
| 5.
|
Bulger, M., and J. T. Kadonaga.
1994.
Biochemical reconstitution of chromatin with physiological nucleosome spacing.
Methods Mol. Genet.
5:241-262.
|
| 6.
|
Burke, T. W., and J. T. Kadonaga.
1997.
The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila.
Genes Dev.
11:3020-3031[Abstract/Free Full Text].
|
| 7.
|
Cairns, B. R.,
A. Schlichter,
H. Erdjument-Bromage,
P. Tempst,
R. D. Kornberg, and F. Winston.
1999.
Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains.
Mol. Cell
4:715-723[CrossRef][Medline].
|
| 8.
|
Candau, R.,
J. X. Zhou,
C. D. Allis, and S. L. Berger.
1997.
Histone acetyltransferase activity and interaction with ADA2 are critical for GCN5 function in vivo.
EMBO J.
16:555-565[CrossRef][Medline].
|
| 9.
|
Chen, H.,
R. J. Lin,
W. Xie,
D. Wilpitz, and R. M. Evans.
1999.
Regulation of hormone-induced histone hyperacetylation and gene activation via acetylation of an acetylase.
Cell
98:675-686[CrossRef][Medline].
|
| 10.
|
Chrivia, J. C.,
R. P. Kwok,
N. Lamb,
M. Hagiwara,
M. R. Montminy, and R. H. Goodman.
1993.
Phosphorylated CREB binds specifically to the nuclear protein CBP.
Nature
365:855-859[CrossRef][Medline].
|
| 11.
|
Cosma, M. P.,
T. Tanaka, and K. Nasmyth.
1999.
Ordered recruitment of transcription and chromatin remodeling factors to a cell cycle- and developmentally regulated promoter.
Cell
97:299-311[CrossRef][Medline].
|
| 12.
|
Dhalluin, C.,
J. E. Carlson,
L. Zeng,
C. He,
A. K. Aggarwal, and M.-M. Zhou.
1999.
Structure and ligand of a histone acetyltransferase bromodomain.
Nature
399:491-496[CrossRef][Medline].
|
| 13.
|
Dignam, J. D.,
R. M. Lebovitz, and R. G. Roeder.
1983.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:1475-1489[Abstract/Free Full Text].
|
| 14.
|
Du, J.,
I. Nasir,
B. K. Benton,
M. P. Kladde, and B. C. Laurent.
1998.
Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essential ATPase in RSC and differs from Snf/Swi in its interactions with histones and chromatin-associated proteins.
Genetics
150:987-1005[Abstract/Free Full Text].
|
| 15.
|
Elfring, L. K.,
C. Daniel,
O. Papoulas,
R. Deuring,
M. Sarte,
S. Moseley,
S. J. Beek,
W. R. Waldrip,
G. Daubresse,
A. DePace,
J. A. Kennison, and J. W. Tamkun.
1998.
Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2.
Genetics
148:251-265[Abstract/Free Full Text].
|
| 16.
|
Freedman, L. P.
1999.
Multimeric coactivator complexes for steroid/nuclear receptors.
Trends Endocrinol. Metab.
10:403-407[CrossRef][Medline].
|
| 17.
|
Gansheroff, L. J.,
C. Dollard,
P. Tan, and F. Winston.
1995.
The Saccharomyces cerevisiae SPT7 gene encodes a very acidic protein important for transcription in vivo.
Genetics
139:523-536[Abstract].
|
| 18.
|
Georgakopoulos, T.,
N. Gounalaki, and G. Thireos.
1995.
Genetic evidence for the interaction of the yeast transcriptional co-activator proteins GCN5 and ADA2.
Mol. Gen. Genet.
246:723-728[CrossRef][Medline].
|
| 19.
|
Glass, C. K., and M. G. Rosenfeld.
2000.
The coregulator exchange in transcriptional functions of nuclear receptors.
Genes Dev.
14:121-141[Free Full Text].
|
| 20.
|
Goodman, R. H., and S. Smolik.
2000.
CBP/p300 in cell growth, transformation, and development.
Genes Dev.
14:1553-1577[Free Full Text].
|
| 21.
|
Grant, P. A.,
L. Duggan,
J. Cote,
S. M. Roberts,
J. E. Brownell,
R. Candau,
R. Ohba,
T. Owen-Hughes,
C. D. Allis,
F. Winston,
S. L. Berger, and J. L. Workman.
1997.
Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex.
Genes Dev.
11:1640-1650[Abstract/Free Full Text].
|
| 22.
|
Gu, W., and R. G. Roeder.
1997.
Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.
Cell
90:595-606[CrossRef][Medline].
|
| 23.
|
Hampsey, M., and D. Reinberg.
1999.
RNA polymerase II as a control panel for multiple coactivator complexes.
Curr. Opin. Genet. Dev.
9:132-139[CrossRef][Medline].
|
| 24.
|
Haynes, S. R.,
C. Dollard,
F. Winston,
S. Beck,
J. Trowsdale, and I. B. Dawid.
1992.
The bromodomain: a conserved sequence found in human, Drosophila and yeast proteins.
Nucleic Acids Res.
20:2603[Free Full Text].
|
| 25.
|
Herrera, J. E.,
K. Sakaguchi,
M. Bergel,
L. Trieschmann,
Y. Nakatani, and M. Bustin.
1999.
Specific acetylation of chromosomal protein HMG-17 by PCAF alters its interaction with nucleosomes.
Mol. Cell. Biol.
19:3466-3473[Abstract/Free Full Text].
|
| 26.
|
Ito, T.,
M. Bulger,
M. J. Pazin,
R. Kobayashi, and J. T. Kadonaga.
1997.
ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor.
Cell
90:145-155[CrossRef][Medline].
|
| 27.
|
Jacobson, R. H.,
A. G. Ladurner,
D. S. King, and R. Tjian.
2000.
Structure and function of a human TAFII250 double bromodomain module.
Science
288:1422-1425[Abstract/Free Full Text].
|
| 28.
|
Jeanmougin, F.,
J. M. Wurtz,
B. P. Le Douarin,
P. Chambon, and R. Losson.
1997.
The bromodomain revisited.
Trends Biochem. Sci.
22:151-153[CrossRef][Medline].
|
| 29.
|
Jeong, S. W.,
J. D. Lauderdale, and A. Stein.
1991.
Chromatin assembly on plasmid DNA in vitro. Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5.
J. Mol. Biol.
222:1131-1147[CrossRef][Medline].
|
| 30.
|
Kadonaga, J. T.
1998.
Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines.
Cell
92:307-313[CrossRef][Medline].
|
| 31.
|
Kamakaka, R. T.,
M. Bulger, and J. T. Kadonaga.
1993.
Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly.
Genes Dev.
7:1779-1795[Abstract/Free Full Text].
|
| 32.
|
Kamei, Y.,
L. Xu,
T. Heinzel,
J. Torchia,
R. Kurokawa,
B. Gloss,
S. C. Lin,
R. A. Heyman,
D. W. Rose,
C. K. Glass, and M. G. Rosenfeld.
1996.
A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors.
Cell
85:403-414[CrossRef][Medline].
|
| 33.
|
Kim, T. K.,
T. H. Kim, and T. Maniatis.
1998.
Efficient recruitment of TFIIB and CBP-RNA polymerase II holoenzyme by an interferon-beta enhanceosome in vitro.
Proc. Natl. Acad. Sci. USA
95:12191-12196[Abstract/Free Full Text].
|
| 34.
|
Kornberg, R. D., and Y. Lorch.
1999.
Chromatin-modifying and -remodeling complexes.
Curr. Opin. Genet. Dev.
9:148-151[CrossRef][Medline].
|
| 35.
|
Korzus, E.,
J. Torchia,
D. W. Rose,
L. Xu,
R. Kurokawa,
E. M. McInerney,
T. M. Mullen,
C. K. Glass, and M. G. Rosenfeld.
1998.
Transcription factor-specific requirements for coactivators and their acetyltransferase functions.
Science
279:703-707[Abstract/Free Full Text].
|
| 36.
|
Kraus, W. L., and J. T. Kadonaga.
1999.
Ligand- and cofactor-regulated transcription with chromatin templates, p. 167-189.
In
D. Picard (ed.), Steroid/nuclear receptor superfamily: a practical approach. Oxford University Press, Oxford, England.
|
| 37.
|
Kraus, W. L., and J. T. Kadonaga.
1998.
p300 and estrogen receptor cooperatively activate transcription via differential enhancement of initiation and reinitiation.
Genes Dev.
12:331-342[Abstract/Free Full Text].
|
| 38.
|
Kraus, W. L.,
E. T. Manning, and J. T. Kadonaga.
1999.
Biochemical analysis of distinct activation functions in p300 that enhance transcription initiation with chromatin templates.
Mol. Cell. Biol.
19:8123-8135[Abstract/Free Full Text].
|
| 39.
|
Kurokawa, R.,
D. Kalafus,
M. H. Ogliastro,
C. Kioussi,
L. Xu,
J. Torchia,
M. G. Rosenfeld, and C. K. Glass.
1998.
Differential use of CREB binding protein-coactivator complexes.
Science
279:700-703[Abstract/Free Full Text].
|
| 40.
|
Kwok, R. P.,
J. R. Lundblad,
J. C. Chrivia,
J. P. Richards,
H. P. Bachinger,
R. G. Brennan,
S. G. Roberts,
M. R. Green, and R. H. Goodman.
1994.
Nuclear protein CBP is a coactivator for the transcription factor CREB.
Nature
370:223-226[CrossRef][Medline].
|
| 41.
|
Laurent, B. C.,
I. Treich, and M. Carlson.
1993.
The yeast SNF2/SWI2 protein has DNA-stimulated ATPase activity required for transcriptional activation.
Genes Dev.
7:583-591[Abstract/Free Full Text].
|
| 42.
|
Lemon, B. D., and L. P. Freedman.
1999.
Nuclear receptor cofactors as chromatin remodelers.
Curr. Opin. Genet. Dev.
9:499-504[CrossRef][Medline].
|
| 43.
|
Leo, C., and J. D. Chen.
2000.
The SRC family of nuclear receptor coactivators.
Gene
245:1-11[CrossRef][Medline].
|
| 44.
|
Lin, R.,
D. Gewert, and J. Hiscott.
1995.
Differential transcriptional activation in vitro by NF-kappa B/Rel proteins.
J. Biol. Chem.
270:3123-3131[Abstract/Free Full Text].
|
| 45.
|
Malik, S., and R. G. Roeder.
2000.
Transcriptional regulation through mediator-like coactivators in yeast and metazoan cells.
Trends Biochem. Sci.
25:277-283[CrossRef][Medline].
|
| 46.
|
Marcus, G. A.,
N. Silverman,
S. L. Berger,
J. Horiuchi, and L. Guarente.
1994.
Functional similarity and physical association between GCN5 and ADA2: putative transcriptional adaptors.
EMBO J.
13:4807-4815[Medline].
|
| 47.
|
Merika, M.,
A. J. Williams,
G. Chen,
T. Collins, and D. Thanos.
1998.
Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription.
Mol. Cell
1:277-287[CrossRef][Medline].
|
| 48.
|
Muchardt, C., and M. Yaniv.
1993.
A human homologue of Saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor.
EMBO J.
12:4279-4290[Medline].
|
| 49.
|
Munshi, N.,
M. Merika,
J. Yie,
K. Senger,
G. Chen, and D. Thanos.
1998.
Acetylation of HMG I(Y) by CBP turns off IFN beta expression by disrupting the enhanceosome.
Mol. Cell
2:457-467[CrossRef][Medline].
|
| 50.
|
Myer, V. E., and R. A. Young.
1998.
RNA polymerase II holoenzymes and subcomplexes.
J. Biol. Chem.
273:27757-27760[Free Full Text].
|
| 51.
|
Nakajima, T.,
C. Uchida,
S. F. Anderson,
C. G. Lee,
J. Hurwitz,
J. D. Parvin, and M. Montminy.
1997.
RNA helicase A mediates association of CBP with RNA polymerase II.
Cell
90:1107-1112[CrossRef][Medline].
|
| 52.
|
Nakajima, T.,
C. Uchida,
S. F. Anderson,
J. D. Parvin, and M. Montminy.
1997.
Analysis of a cAMP-responsive activator reveals a two-component mechanism for transcriptional induction via signal-dependent factors.
Genes Dev.
11:738-747[Abstract/Free Full Text].
|
| 53.
|
O'Connor, M. J.,
H. Zimmermann,
S. Nielsen,
H. U. Bernard, and T. Kouzarides.
1999.
Characterization of an E1A-CBP interaction defines a novel transcriptional adapter motif (TRAM) in CBP/p300.
J. Virol.
73:3574-3581[Abstract/Free Full Text].
|
| 54.
|
Ogryzko, V. V.,
T. Kotani,
X. Zhang,
R. L. Schlitz,
T. Howard,
X. J. Yang,
B. H. Howard,
J. Qin, and Y. Nakatani.
1998.
Histone-like TAFs within the PCAF histone acetylase complex.
Cell
94:35-44[CrossRef][Medline].
|
| 55.
|
Ogryzko, V. V.,
R. L. Schiltz,
V. Russanova,
B. H. Howard, and Y. Nakatani.
1996.
The transcriptional coactivators p300 and CBP are histone acetyltransferases.
Cell
87:953-959[CrossRef][Medline].
|
| 56.
|
Ornaghi, P.,
P. Ballario,
A. M. Lena,
A. Gonzalez, and P. Filetici.
1999.
The bromodomain of Gcn5p interacts in vitro with specific residues in the N terminus of histone H4.
J. Mol. Biol.
287:1-7[CrossRef][Medline].
|
| 57.
|
Owen, D. J.,
P. Ornaghi,
J.-C. Yang,
N. Lowe,
P. R. Evans,
P. Ballario,
D. Neuhaus,
P. Filetici, and A. A. Travers.
2000.
The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p.
EMBO J.
19:6141-6149[CrossRef][Medline].
|
| 58.
|
Schiltz, R. L.,
C. A. Mizzen,
A. Vassilev,
R. G. Cook,
C. D. Allis, and Y. Nakatani.
1999.
Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates.
J. Biol. Chem.
274:1189-1192[Abstract/Free Full Text].
|
| 59.
|
Shikama, N.,
H. M. Chan,
M. Krstic-Demonacos,
L. Smith,
C.-W. Lee,
W. Cairns, and N. B. La Thangue.
2000.
Functional interaction between nucleosome assembly proteins and p300/CREB-binding family coactivators.
Mol. Cell. Biol.
20:8933-8943[Abstract/Free Full Text].
|
| 60.
|
Sims, S. H.,
Y. Cha,
M. F. Romine,
P. Q. Gao,
K. Gottlieb, and A. B. Deisseroth.
1993.
A novel interferon-inducible domain: structural and functional analysis of the human interferon regulatory factor 1 gene promoter.
Mol. Cell. Biol.
13:690-702[Abstract/Free Full Text].
|
| 61.
|
Soutoglou, E.,
N. Katrakili, and I. Talianidis.
2000.
Acetylation regulates transcription factor activity at multiple levels.
Mol. Cell
5:745-751[CrossRef][Medline].
|
| 62.
|
Sterner, D. E.,
P. A. Grant,
S. M. Roberts,
L. J. Duggan,
R. Belotserkovskaya,
L. A. Pacella,
F. Winston,
J. L. Workman, and S. L. Berger.
1999.
Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction.
Mol. Cell. Biol.
19:86-98[Abstract/Free Full Text].
|
| 63.
|
Struhl, K., and Z. Moqtaderi.
1998.
The TAFs in the HAT.
Cell
94:1-4[CrossRef][Medline].
|
| 64.
|
Swope, D. L.,
C. L. Mueller, and J. C. Chrivia.
1996.
CREB-binding protein activates transcription through multiple domains.
J. Biol. Chem.
271:28138-28145[Abstract/Free Full Text].
|
| 65.
|
Syntichaki, P.,
I. Topalidou, and G. Thireos.
2000.
The Gcn5 bromodomain co-ordinates nucleosome remodelling.
Nature
404:414-417[CrossRef][Medline].
|
| 66.
|
Winston, F., and C. D. Allis.
1999.
The bromodomain: a chromatin-targeting module?
Nat. Struct. Biol.
6:601-604[CrossRef][Medline].
|
| 67.
|
Workman, J. L., and R. E. Kingston.
1998.
Alteration of nucleosome structure as a mechanism of transcriptional regulation.
Annu. Rev. Biochem.
67:545-579[CrossRef][Medline].
|
| 68.
|
Xu, L.,
C. K. Glass, and M. G. Rosenfeld.
1999.
Coactivator and corepressor complexes in nuclear receptor function.
Curr. Opin. Genet. Dev.
9:140-147[CrossRef][Medline].
|
| 69.
|
Yang, X. J.,
V. V. Ogryzko,
J. Nishikawa,
B. H. Howard, and Y. Nakatani.
1996.
A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A.
Nature
382:319-324[CrossRef][Medline].
|
| 70.
|
Zhang, Q.,
N. Vo, and R. H. Goodman.
2000.
Histone binding protein RbAp48 interacts with a complex of CREB binding protein and phosphorylated CREB.
Mol. Cell. Biol.
20:4970-4978[Abstract/Free Full Text].
|
| 71.
|
Zhong, H.,
R. E. Voll, and S. Ghosh.
1998.
Phosphorylation of NF-kappa B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300.
Mol. Cell
1:661-671[CrossRef][Medline].
|
Molecular and Cellular Biology, June 2001, p. 3876-3887, Vol. 21, No. 12
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.12.3876-3887.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hou, T., Ray, S., Lee, C., Brasier, A. R.
(2008). The STAT3 NH2-terminal Domain Stabilizes Enhanceosome Assembly by Interacting with the p300 Bromodomain. J. Biol. Chem.
283: 30725-30734
[Abstract]
[Full Text]
-
Durant, M., Pugh, B. F.
(2007). NuA4-Directed Chromatin Transactions throughout the Saccharomyces cerevisiae Genome. Mol. Cell. Biol.
27: 5327-5335
[Abstract]
[Full Text]
-
Fukuda, H., Sano, N., Muto, S., Horikoshi, M.
(2006). Simple histone acetylation plays a complex role in the regulation of gene expression.. Brief Funct Genomic Proteomic
5: 190-208
[Abstract]
[Full Text]
-
Kim, M. Y., Woo, E. M., Chong, Y. T. E., Homenko, D. R., Kraus, W. L.
(2006). Acetylation of Estrogen Receptor {alpha} by p300 at Lysines 266 and 268 Enhances the Deoxyribonucleic Acid Binding and Transactivation Activities of the Receptor. Mol. Endocrinol.
20: 1479-1493
[Abstract]
[Full Text]
-
Nandiwada, S. L., Li, W., Zhang, R., Mueller, D. L.
(2006). p300/Cyclic AMP-Responsive Element Binding-Binding Protein Mediates Transcriptional Coactivation by the CD28 T Cell Costimulatory Receptor. J. Immunol.
177: 401-413
[Abstract]
[Full Text]
-
Kumar, J. P., Jamal, T., Doetsch, A., Turner, F. R., Duffy, J. B.
(2004). CREB Binding Protein Functions During Successive Stages of Eye Development in Drosophila. Genetics
168: 877-893
[Abstract]
[Full Text]
-
Dornan, D., Shimizu, H., Burch, L., Smith, A. J., Hupp, T. R.
(2003). The Proline Repeat Domain of p53 Binds Directly to the Transcriptional Coactivator p300 and Allosterically Controls DNA-Dependent Acetylation of p53. Mol. Cell. Biol.
23: 8846-8861
[Abstract]
[Full Text]
-
Georges, S. A., Giebler, H. A., Cole, P. A., Luger, K., Laybourn, P. J., Nyborg, J. K.
(2003). Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity That Is Chromatin Dependent and Histone Tail Independent. Mol. Cell. Biol.
23: 3392-3404
[Abstract]
[Full Text]
-
Lee, K. C., Li, J., Cole, P. A., Wong, J., Kraus, W. L.
(2003). Transcriptional Activation by Thyroid Hormone Receptor-{beta} Involves Chromatin Remodeling, Histone Acetylation, and Synergistic Stimulation by p300 and Steroid Receptor Coactivators. Mol. Endocrinol.
17: 908-922
[Abstract]
[Full Text]
-
Deng, Z., Chen, C.-J., Chamberlin, M., Lu, F., Blobel, G. A., Speicher, D., Cirillo, L. A., Zaret, K. S., Lieberman, P. M.
(2003). The CBP Bromodomain and Nucleosome Targeting Are Required for Zta-Directed Nucleosome Acetylation and Transcription Activation. Mol. Cell. Biol.
23: 2633-2644
[Abstract]
[Full Text]
-
Whitehouse, I., Stockdale, C., Flaus, A., Szczelkun, M. D., Owen-Hughes, T.
(2003). Evidence for DNA Translocation by the ISWI Chromatin-Remodeling Enzyme. Mol. Cell. Biol.
23: 1935-1945
[Abstract]
[Full Text]
-
An, W., Roeder, R. G.
(2003). Direct Association of p300 with Unmodified H3 and H4 N Termini Modulates p300-dependent Acetylation and Transcription of Nucleosomal Templates. J. Biol. Chem.
278: 1504-1510
[Abstract]
[Full Text]
-
Remboutsika, E., Yamamoto, K., Harbers, M., Schmutz, M.
(2002). The Bromodomain Mediates Transcriptional Intermediary Factor 1alpha -Nucleosome Interactions. J. Biol. Chem.
277: 50318-50325
[Abstract]
[Full Text]
-
Suganuma, T., Kawabata, M., Ohshima, T., Ikeda, M.-A.
(2002). Growth suppression of human carcinoma cells by reintroduction of the p300 coactivator. Proc. Natl. Acad. Sci. USA
99: 13073-13078
[Abstract]
[Full Text]
-
Asahara, H., Tartare-Deckert, S., Nakagawa, T., Ikehara, T., Hirose, F., Hunter, T., Ito, T., Montminy, M.
(2002). Dual Roles of p300 in Chromatin Assembly and Transcriptional Activation in Cooperation with Nucleosome Assembly Protein 1 In Vitro. Mol. Cell. Biol.
22: 2974-2983
[Abstract]
[Full Text]
-
Cheung, E., Zarifyan, A. S., Kraus, W. L.
(2002). Histone H1 Represses Estrogen Receptor {alpha} Transcriptional Activity by Selectively Inhibiting Receptor-Mediated Transcription Initiation. Mol. Cell. Biol.
22: 2463-2471
[Abstract]
[Full Text]
-
Heinlein, C. A., Chang, C.
(2002). Androgen Receptor (AR) Coregulators: An Overview. Endocr. Rev.
23: 175-200
[Abstract]
[Full Text]
-
Kalkhoven, E., Teunissen, H., Houweling, A., Verrijzer, C. P., Zantema, A.
(2002). The PHD Type Zinc Finger Is an Integral Part of the CBP Acetyltransferase Domain. Mol. Cell. Biol.
22: 1961-1970
[Abstract]
[Full Text]
-
Sterner, D. E., Wang, X., Bloom, M. H., Simon, G. M., Berger, S. L.
(2002). The SANT Domain of Ada2 Is Required for Normal Acetylation of Histones by the Yeast SAGA Complex. J. Biol. Chem.
277: 8178-8186
[Abstract]
[Full Text]
-
Georges, S. A., Kraus, W. L., Luger, K., Nyborg, J. K., Laybourn, P. J.
(2002). p300-Mediated Tax Transactivation from Recombinant Chromatin: Histone Tail Deletion Mimics Coactivator Function. Mol. Cell. Biol.
22: 127-137
[Abstract]
[Full Text]
Top
Abstract
Introduction
