From the Laboratoire de Virologie Moléculaire,
Institut de Génétique Humaine,
Institut de
Génétique Moléculaire, Montpellier 34296, France, the
§§ Departement des Maladies Infectieuses,
Institut Cochin, Paris 75014, France, and the ¶ Department of
Biochemistry, The University of Hong Kong, Hong Kong, China
Received for publication, September 18, 2002, and in revised form, October 19, 2002
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ABSTRACT |
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NF- The NF- A key step to controlling NF- The activity of NF- The p65 subunit of NF- Plasmid Constructions and Antibodies Used--
Eukaryotic
expression vectors for T7-I Cell Culture and Immunological Techniques--
Jurkat cells were
cultured in RPMI 1640 GlutamaxI medium (Invitrogen) supplemented with
10% FBS (Invitrogen), penicillin, and streptomycin. HeLa and 293 cells
were propagated in Dulbecco's modified Eagle's medium with 10% FBS.
Transfections were performed using calcium phosphate or, where
indicated, by LipofectAMINE (Invitrogen) according to the
manufacturer's instructions. Amounts of DNA are as indicated in the
figure legends. The total amount of expression vectors was kept
constant by using empty-vector DNA. Where indicated, cells were treated
with LMB (10 nM) overnight and during labeling.
For preparation of cytoplasmic and nuclear extracts, cells were washed
twice in cold phosphate-buffered saline, resuspended in 400 µl of 10 mM Hepes, pH 7.8, 10 mM KCl, 2 mM
MgCl2, 0.1 mM EDTA, 1 mM DTT, and
protease inhibitors, and incubated on ice for 20 min. Nonidet P-40 was
added to the cells to a final concentration of 0.5% followed by mixing
and centrifugation for 30 s at 4 °C. Supernatants
(corresponding to cytoplasmic extracts) were collected, and nuclear
extracts were prepared by resuspending pellets in 50 µl of 50 mM Hepes, pH 7.8, 50 mM KCl, 300 mM
NaCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and
protease inhibitors. Proteins were extracted by agitation for 20 min at
4 °C and clarified by centrifugation. Total cell extracts were
prepared by lysing the cells in Triton buffer (300 mM NaCl,
50 mM Tris, pH 7.5, 0.5% Triton, and protease inhibitors).
Immunoprecipitation and Western blotting were performed as described
previously (24).
For immunofluorescence, HeLa cells were transfected with FLAG-tagged
p65 wild type, p65KK-RR or p65KK-AA. Cells were fixed 24 h after
transfection in a solution containing 4% paraformaldehyde, 0.5% Triton, and 5% FBS. Cells were double-stained with anti-FLAG monoclonal antibody and rabbit polyclonal anti-I Acetylation and Deacetylations Assays--
In vivo
acetylation and deacetylation assays were performed as follows.
Transfected HeLa or 293 cells 24 h post-transfection were washed
three times in phosphate-buffered saline and incubated in media minus
methionine/cysteine supplemented with 2% FBS for 2 h at 37 °C.
Cell cultures were halved and incubated in media containing either
[35S]Met plus Cys at 1 mCi/ml or
[3H]NaAc at 1 mCi/ml for 1 h at 37 °C.
Cells were washed in phosphate-buffered saline and resuspended in lysis
buffer (300 mM NaCl, 50 mM Tris, pH 7.5, 0.5%
Triton X-100, and protease inhibitors).
In vitro assays for protein acetylation were performed using
peptides or GST-p65 fusion protein as described previously (24). For
in vitro deacetylation assays, 293 cells were transfected with FLAG-p65, FLAG-HDAC2, or FLAG-HDAC3 expression plasmids. 24 h
after transfection, FLAG-p65-transfected cells were labeled with
[3H]NaAc for 1 h at 37 °C. Extracts were prepared
and subjected to immunoprecipitation using anti-FLAG antibody. FLAG-p65
was eluted using FLAG peptide overnight at 4 °C. FLAG-HDAC2 and
FLAG-HDAC3 were kept on the beads. The purified FLAG-p65 was incubated
with FLAG-HDAC2 or FLAG-HDAC3 in HDAC buffer (10 mM Tris,
pH 8, 10 mM NaCl, 10% glycerol), in the absence or
presence of 200 mM TSA (Sigma), for 1 h at 37 °C.
Reactions were analyzed by 10% SDS-PAGE. Gels were fixed in 30%
methanol and 10% acetic acid, enhanced, dried and exposed to x-ray
film at EMSA--
EMSA was performed using 104 cpm of
[ Chromatin Immunoprecipitation Assay--
Six 60-mm-diameter
Petri dishes of transfected 293 cells were used per chromatin
immunoprecipitation reaction, performed essentially as described
previously (27). To cleared chromatin extracts, 2 µg of FLAG M2
monoclonal antibody was added. PCR was performed in the presence of
0.11 µCi of [ p65 Is Acetylated on Lysines 122 and 123 by p300 and
PCAF--
p300/CBP and PCAF bind to the p65 but not the p50 subunit of
NF-
Because the acetyltransferase activities of p300/CBP and PCAF have been
shown to be important for transcriptional activation of p65 (19, 20),
we investigated whether p65 may be a substrate for acetylation by these
transcriptional coactivators. p65 was acetylated by both p300 and PCAF
in vitro (Fig. 1B). No acetylation of GST or
GST-p50 was observed (data not shown). Peptide mapping analysis showed
that lysines 122 and 123 within peptide 3 (p3) are the only residues
acetylated by p300 and PCAF in vitro (Fig. 1C).
Thus, GST-p65 wt or a mutant in which Lys-122 and Lys-123 were
mutated to alanine (KK-AA) were used as substrates in in vitro acetylation assays. Substitution of the two lysines
completely abrogated acetylation of p65 by both p300 and PCAF (Fig.
1D). No acetylation of GST-p50 was observed (lanes
3 and 6). Coomassie Blue staining confirmed that
equivalent amounts of proteins were loaded (data not shown). To confirm
that K122/123 were also acetylated in vivo, 293 cells were
transfected with vectors expressing either FLAG-p65 wild type or
mutants where the two acetylated lysines were changed to arginines
(KK-RR) or alanines (KK-AA). Cells were pulse-labeled with either
[3H]NaAc or [35S]Met/Cys, lysed, and
protein was immunoprecipitated using anti-FLAG antibody (Fig.
1E). In vivo acetylation was observed in cells transfected with wild type p65 but not with KK-RR or KK-AA mutants. Metabolic labeling using [35S]methionine plus cysteine
confirmed that p65 wild type and mutant proteins were expressed to
equivalent levels in transfected cells. These results show that p65 is
acetylated at dual lysine residues, Lys-122 and Lys-123, by both p300
and PCAF.
Simultaneous treatment of cells with PMA and TSA enhanced the
acetylation of p65 in vivo (Fig. 1A), implying a
tight regulation of p65 acetylation by deacetylases in vivo.
We first determined the HDACs that interact with p65 in
vivo. Both HDAC2 and HDAC3 interacted with p65, whereas no
interaction was observed between HDAC1 and p65 (Fig.
2A). The presence of HDAC1,
HDAC2, and HDAC3 in the immunoprecipitates is shown (Fig.
2A, lanes 7-9). It was recently reported that
p65 interacts directly with HDAC1 but not HDACs 2 or 3 (30). The
discrepancy between the results obtained by Ashburner
et al. (30) and those shown in Fig. 2A may be due to the different antibodies used.
We next analyzed the ability of HDAC2 and HDAC3 to deacetylate p65
in vitro. FLAG-p65, immunopurified from transfected 293 cells that had been labeled with [3H]NaAc, was incubated
with HDAC2 or HDAC3 in the presence or absence of TSA. HDAC3 but not
HDAC2 was able to deacetylate p65 in vitro (Fig.
2B). Deacetylation of p65 by HDAC3 was inhibited by TSA. Western blot analysis showed that comparable amounts of p65 were present in all samples. Both HDAC2 and HDAC3 deacetylated histone H3
in vitro (lanes 7-9) showing that the purified
HDACs were active. To establish the role of HDAC3 in p65 deacetylation
in vivo, FLAG-p65 was immunoprecipitated from transfected
cells that had been labeled with [3H]NaAc or
[35S]Met/Cys. Acetylation of p65 was enhanced in cells
cotransfected with p65 and PCAF or p300 (Fig. 2C) confirming
that p65 is a substrate for p300 and PCAF in vivo. In
contrast, cotransfection of HDAC3 with p65 significantly reduced p65
acetylation. TSA treatment inhibited HDAC3-mediated deacetylation of
p65 (lane 5). No effect of HDAC2 on p65 acetylation was
observed (lanes 6 and 7).
[35S]Met/Cys labeling shows the level of expression of
the different plasmids used. Taken together, the results show that
acetylation of p65 is regulated in vivo by p300, PCAF, and HDAC3.
Acetylation of p65 Does Not Affect Its Interaction with
I Acetylation of p65 Reduces Its Binding to p65 Is Acetylated in the Nucleus and Accumulates in the
Cytoplasm--
Because acetylation plays a role in destabilizing the
p65/
To further examine the site of p65 acetylation in cells, p65 was
blocked in the cytoplasm by its inhibitor, I
To further characterize the effect of I
Immunofluorescence analysis was performed to analyze the subcellular
localization of acetylation-competent (wild type) and acetylation-incompetent (KK-RR or KK-AA) forms of p65. HeLa cells were
transfected with either FLAG-p65 wild type, KK-RR, or KK-AA expression
vectors, and cells were stained with anti-FLAG to detect p65 and
anti-I Acetylation of p65 Represses Its Transcriptional Activity and Is
Involved in the Attenuation of p65-mediated Transcription--
It has
been shown previously that p300/CBP and PCAF are transcriptional
coactivators for NF-
We next analyzed the combined effects HAT and FAT activities of p300
and PCAF, HDAC3, and their respective enzymatic activity mutants on
p65-mediated transcriptional activation of HIV-1 LTR-luciferase reporter (Fig. 6C). As previously reported, p300 and PCAF
enhanced p65 transcriptional activity. Interestingly, p300 and PCAF
coactivated p65KK-RR transcriptional activity to a significantly higher
level than p65 wild type. Thus, elimination of acetyl-acceptors for FAT
activity leads to higher coactivation between p65 and HATs. On the
other hand, HDAC3 repressed p65KK-RR transcriptional activity more than
that of p65 wild type. These results, taken together with our other
experiments, strongly suggest that HAT activity of p300 and PCAF
potentiates p65-mediated transcription, whereas their FAT activity is repressive.
The opposing effects of HAT and FAT activities of p300 and PCAF in
NF-
I In this report, we examined the functions of p300 and PCAF
coactivators in NF-B represents a family of eukaryotic
transcription factors participating in the regulation of various
cellular genes involved in the immediate early processes of immune,
acute-phase, and inflammatory responses. Cellular localization and
consequently the transcriptional activity of NF-
B is tightly
regulated by its partner I
B
. Here, we show that the p65 subunit
of NF-
B is acetylated by both p300 and PCAF on lysines 122 and 123. Both HDAC2 and HDAC3 interact with p65, although only HDAC3 was able to
deacetylate p65. Acetylation of p65 reduces its ability to bind
B-DNA. Finally, acetylation of p65 facilitated its removal from
DNA and consequently its I
B
-mediated export from the nucleus.
We propose that acetylation of p65 plays a key role in
I
B
-mediated attenuation of NF-
B transcriptional activity
which is an important process that restores the latent state in
post-induced cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B/Rel family of inducible transcription factors is
involved in the expression of numerous genes involved in processes such
as growth, development, apoptosis, and inflammatory and immune responses (1, 2). The Rel family includes p65 (RelA), p105/p50, p100/p52, RelB, and c-Rel, which homo- or heterodimerize to form transcriptionally competent or repressive complexes referred to as
NF-
B (3). The most abundant form of NF-
B is a p50/p65 heterodimer
in which p65 contains the transcriptional activation domain. The
activity of NF-
B is regulated by its subcellular localization. In
unstimulated cells, NF-
B exists in an inactive form sequestered in
the cytoplasm by its inhibitor, I
B. The I
B family includes
several members of which the best characterized is I
B
(2). Cell
activation by a multitude of extracellular signals (4) converges on
phosphorylation of I
B by I
B kinase, which triggers its rapid
ubiquitination and degradation by the proteasome (5). Degradation of
I
B unmasks the nuclear localization signal
(NLS)1 present in NF-
B,
which then enters the nucleus to activate target gene expression.
B activity is the regulation of NF-
B
subcellular localization through its interaction with I
B in both
pre-induced and post-induced cellular states. I
B
contains both a
nuclear import sequence (6, 7), and a strong nuclear export sequence
that utilizes the exportin/CRM1 pathway (8-12). One of the target
genes of NF-
B is I
B
, resulting in rapid induction of newly
synthesized I
B
protein, which enters the nucleus and dissociates
NF-
B from
B-DNA to repress NF-
B function (13, 14).
NF-
B·I
B complexes are exported to the cytoplasm where
they may serve for additional rounds of activation or restore the
original latent state (6, 8).
B is regulated by transcriptional coactivators
that may function by bridging sequence-specific activators to the basal
transcriptional machinery and also play a role in chromatin remodeling
via their intrinsic histone acetyltransferase (HAT) or deacetylase
(HDAC) activity (15). p65 binds to CBP (CREB-binding protein) and its
homologue p300 as well as PCAF (p300/CBP-associated factor), whereas
p50 fails to recruit transcriptional coactivators (16-21). p65
phosphorylation by protein kinase A stimulates NF-
B-dependent gene expression by enhancing its
interaction with CBP (21). Enhancement of NF-
B transcriptional
activity requires the acetyltransferase activity of CBP/p300 (20) and
PCAF (19).
B was recently shown to be acetylated (22). It
was proposed by Chen et al. that reversible acetylation regulates the interaction between p65 and I
B
and, therefore, controls the duration of the NF-
B response. Here, we show that p65
is acetylated on dual lysine residues K122/123 by p300 and PCAF and
deacetylated by HDAC3. Contrary to Chen et al., we could not
demonstrate any significant effect of acetylation on the interaction between p65 and I
B
. Rather, we show that acetylation reduces binding of p65 to
B-containing DNA, facilitating its removal by
I
B
and subsequent export to the cytoplasm. We propose that acetylation of p65 contributes to the mechanism of post-induction turn-off of NF-
B-mediated transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
, LTRluc-wt, PCAF, p300, HDAC1,
HDAC2, HDAC3, and HDAC3mut have been described previously (23-25).
FLAG-p65 (wt) was generated by PCR using the oligonucleotides (the FLAG sequence is highlighted in boldface): (forward)
5'-TCCAAGCTTCACCATGGACTACAAAGACGATGACGACAAGGACGAACTGTTCCCCCTCATCTTCCCG-3' (reverse) 5'-CGCGGATCCCCCCTTAGGAGCTGATCTGACTCAG-3'. PCR fragments were cloned into pTarget (Promega), and the clones were fully sequenced. FLAG-p65 was used to generate p65 mutants. Mutagenesis was
performed by QuikChange site-directed mutagenesis (Stratagene). Mutations were generated with the following pairs of mutagenic oligonucleotide primers (mutations are highlighted in boldface): K122A/K123A: (forward)
5'-CTGGGAATCCAGTGTGTGGCGGCGCGGGACCTGGAGCAGG-3' and
(reverse)
5'-C CTGCTCCAGGTCCCGCGCCGCCACACACTGGATTCCCAG-3'. K122R/K123R: (forward)
5'-CTGGGAATCCAGTGTGTGAGGAGGCGGGACCTGGAGCAGG-3' (reverse) and
5'-CCTGCTCCAGGTCCCGCCTCCTCACACACTGGATTCCCAG-3'. Mutated clones were fully resequenced. The mutated plasmids were designated as p65KK-AA and p65KK-RR. To produce GST fusion proteins, p65 WT and p65KK-AA were cloned into pGEX-4T (Amersham Biosciences). GST-p65 and GST-p65KK-AA fusion proteins were expressed and purified as
previously described (26). Antibodies used were anti-p65 (C-20),
anti-p50 (E-10), anti-I
B
(FL), anti-HDAC1 (H-11), and anti-HDAC2
(C-19) (Santa Cruz Biotechnology), anti-FLAG M2 (Sigma), and anti-HDAC3
(23).
B
followed by incubation with Cy5-conjugated anti-mouse and anti-rabbit fluorescein isothiocyanate for detection of FLAG-p65 and I
B
, respectively.
70 °C.
-32P]ATP-labeled HIV-1
B probe. Reactions were
performed in binding buffer (25 mM Hepes, pH 7.9, 100 mM KCl, 20% glycerol, 0.01% Nonidet P-40, 1 µM ZnSO4, 1 mM DTT) for 15 min at
room temperature, resolved on 4% acrylamide gels, dried, and exposed
to x-ray film.
-32P]dCTP. PCR products were analyzed
by electrophoresis on 4% 1× TBE polyacrylamide gels and
autoradiography. The IL-8 promoter intensities obtained from
immunoprecipitates were first normalized to the starting chromatin
extracts (input). The -fold enrichment is defined as the ratio of the
normalized intensities for the transfected samples to the
mock-transfected sample. PCR primer sets for the human IL-8 promoter
region
121/+61 and the human IL-8 upstream region
1042/
826 have
been described previously (28).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and regulate NF-
B transcriptional activity. To test
whether p65 is modified by acetylation, we performed in vivo
acetylation assays. Jurkat cells were untreated or treated with PMA to
activate NF-
B, or the HDAC inhibitor TSA or a combination of both,
biosynthetically labeled for 1 h with [3H] sodium
acetate (NaAc) or [35S]methionine plus cysteine
(Cys/Met), and lysed, and protein was immunoprecipitated using either
p65-specific or p50-specific antisera. Acetylated p65 was observed only
in PMA-treated cells (Fig.
1A). TSA treatment alone did
not result in p65 acetylation. However, treatment with both PMA and TSA
enhanced p65 acetylation when compared with PMA treatment alone. In
contrast, no acetylation of p50 was observed (lanes 5-8).
This is consistent with a recent report showing that p50 acetylation
can be detected only in the presence of HIV-1 Tat (29). This experiment
shows that p65 is acetylated in vivo, and the acetylation is
dependent on NF-
B activation.
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Fig. 1.
p65 is acetylated at lysines 122 and 123 in vivo and in vitro.
A, p65 acetylation in vivo requires NF- B
activation. Jurkat cells were mock-treated (lanes 1 and
5), treated overnight with PMA 10 ng/ml (lanes 2 and 6), TSA 200 nM (lanes 3 and
7), or PMA plus TSA (lanes 4 and 8).
p65 and p50 were immunoprecipitated from cells that had been
pulse-labeled with either [3H]NaAc or
[35S]Met/Cys. Immunoprecipitates were analyzed by
SDS-PAGE and autoradiography. Autoradiographs corresponding to
[3H]p65 and [3H]p50 were exposed to film
for 5 days and 30 days, respectively. B, p65 is acetylated
in vitro by both p300 and PCAF. Histone H3, 100 ng
(lanes 1-8) or varying amounts of GST-p65 as indicated were
incubated in acetylation buffer with either recombinant p300
(lanes 1-4) or recombinant PCAF (lanes 5-8).
Reaction products were separated by 4-20% SDS-PAGE, and the gels were
Coomassie Blue-stained (bottom panel), dried, and visualized
by autoradiography (top panel). C and
D, p300 and PCAF acetylate lysines 122 and 123 in
vitro. C, synthetic peptides (1 µg) corresponding to
amino acids 21-60 (p1), 61-99 (p2), 111-130
(p3), 210-230 (p4), and 290-320 (p5)
of p65 were incubated with PCAF and [14C]acetyl-CoA for
1 h at 37 °C. Reaction products were resolved in 16.5%
Tris-Tricine acrylamide gels followed by autoradiography. Shown is the
quantification of the radiolabeled peptides. D, GST-p65 wild
type, GST-p65KK-AA (AA), and GST-p50 were incubated in
acetylation buffer with either recombinant p300 (lanes 1-3)
or recombinant PCAF (lanes 4-6). Reaction products were
analyzed by SDS-PAGE and autoradiography. E, lysines 122 and
123 of p65 are acetyl-acceptors in vivo. FLAG-tagged p65
wild type, KK-RR, or KK-AA were immunoprecipitated from transfected 293 cell extracts that had been pulse-labeled with either
[3H]NaAc or [35S]Met/Cys.
Immunoprecipitates were analyzed by SDS-PAGE and autoradiography.
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Fig. 2.
HDAC3 deacetylates p65 in vitro
and in vivo. A, p65 interacts
with HDAC2 and HDAC3 but not HDAC1. HeLa extracts were
immunoprecipitated using anti-p65, anti-HDAC1, anti-HDAC2, and
anti-HDAC3 antibodies. Two different pre-immune sera (PI)
were used as controls. The presence of p65 in immunoprecipitates was
analyzed by Western blotting using anti-p65 antibody. The presence of
HDAC3, HDAC2, and HDAC1 in the respective immunoprecipitates is shown
(right panel, lanes 7-9). The arrows
indicate the HDAC proteins. The upper band in lane
7 corresponds to IgG. B, HDAC3 but not HDAC2
deacetylates p65 in vitro. HDAC2, HDAC3, and
3H-labeled FLAG-p65 ([3H]p65) were
immunopurified separately from 293 cells transfected with the
respective expression plasmids. [3H]p65 was incubated in
deacetylation buffer alone (lane 1) or with HDAC2 or HDAC3
in the presence or absence of TSA (lanes 2-6). Acetylated
histone H3 was incubated in deacetylation buffer (lanes
7-9) either alone or in the presence of HDAC2 or HDAC3 as a
control. Reactions were analyzed by SDS-PAGE and autoradiography
(top panels) or Western blotting using anti-FLAG antibody
(lower panel). C, HDAC3 deacetylates p65 in
vivo. HeLa cells transfected with FLAG-p65 expression vector
either alone or with the indicated plasmids were metabolically labeled
with [3H]NaAc in the presence or absence of TSA
(lanes 1-7) or [35S]Met/Cys (lanes
8-12). FLAG-p65 was immunoprecipitated from cell extracts, and
bound complexes were resolved by SDS-PAGE and detected by
autoradiography.
B
--
It was recently reported that acetylation of p65
prevents its interaction with I
B
(22). We therefore examined
whether mutation of acetyl-acceptor lysines 122 and 123 of p65 affected its interaction with I
B
. As shown in Fig.
3A, the affinities for
I
B
of p65 wild type, KK-RR, and KK-AA were equivalent. We next
examined directly whether or not the acetylated form of p65 interacted
with I
B
(Fig. 3B). FLAG-p65 and T7-I
B
were
either transfected alone or cotransfected under conditions in which
I
B
was overexpressed relative to p65. Extracts of transfected
cells that had been pulse-labeled with either
[3H]NaAc or [35S]Met/Cys were subjected
to immunoprecipitation using anti-FLAG (lane 1) or anti-T7
(lane 2). The extracts from I
B
-p65-cotransfected cells
were subjected to two sequential rounds of immunoprecipitation with
anti-T7 (lanes 3 and 4) followed by a third round
with anti-FLAG (lane 5). Most of 35S-p65 was
found in association with I
B
, because immunodepletion of
T7-I
B
resulted in immunodepletion of p65 from the cells (compare lane 3 to lane 5). Analysis of acetylated p65
showed that all of [3H]p65 was also detected in complexes
with I
B
(compare lanes 3 to 5, lower
panel). Moreover, using GST-I
B
and in vitro
acetylated p65 (14C-p65) or in vitro translated
35S-labeled p65 (35S-p65) we show that
GST-I
B
is able to interact equally with acetylated and
non-acetylated p65 (Fig. 3C, compare lanes 3 to 1). Taken together, the data in Fig. 3 strongly suggests
that acetylation of p65 does not abolish its binding to I
B
.
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Fig. 3.
Acetylation of p65 does not affect its
interaction with
I B
.
A, immunoprecipitated FLAG-p65 wt, KK-RR, and KK-AA were
incubated with 2-fold decreasing amounts of 35S-labeled
I
B
. The complexes were washed extensively and retention of
I
B
was analyzed by SDS-PAGE and autoradiography. p65 wt and
mutants were also analyzed directly by Western blotting
(input, lanes 1 to 3). B,
extracts of HeLa cells transfected with FLAG-p65 (lane 2) or
T7-I
B
(lane 5) alone were immunoprecipitated with
the indicated antibodies. Extracts of HeLa cells, cotransfected with
FLAG-p65 and T7-I
B
, were subjected to two sequential
immunoprecipitations using anti-T7 antibody overnight at 4 °C
(lanes 3-4) followed by a third immunoprecipitation using
anti-FLAG antibody overnight at 4 °C (lane 5).
Immunoprecipitates were washed extensively and analyzed by 10%
SDS-PAGE and autoradiography. C, in vitro
acetylated p65 (top panel) or in vitro translated
35S-labeled p65 (lower panel) was incubated with
GST-I
B
or GST for 2 h at 4 °C. The beads were washed five
times in buffer containing 250 mM KCl and resuspended in
loading buffer. The presence of p65 was analyzed by SDS-PAGE followed
by autoradiography. Flow-through (ft) corresponding to
GST-I
B
is shown in lane 2.
B-DNA--
The
crystal structure of the p50/p65 heterodimer bound to DNA revealed
that, among the p65 residues involved in DNA binding, acetyl-acceptor
lysines 122 and 123 identified in this report are the only residues
that contact the DNA in the minor groove (31). Because acetylation
would be expected to neutralize the positive charge on Lys-122 and
Lys-123
-amino groups, we sought to determine how acetylation of p65
might affect its interaction with
B-DNA. To investigate this,
FLAG-p65 immunopurified from transfected 293 cells (Fig.
4A, Coomassie Blue staining)
was acetylated or mock-acetylated in vitro by PCAF (Fig.
4A, autoradiography). EMSA analysis of the acetylated and
mock-acetylated products showed that acetylation of FLAG-p65 reduces
its ability to bind
B-DNA (Fig. 4B). Similar results were
observed when p300-acetylated p65 or PCAF-acetylated GST-p65 were used
(data not shown). Nuclear extracts of HeLa cells transfected with p65
wild type, KK-RR, and KK-AA were next analyzed for binding to
B-DNA
(Fig. 4C). After normalization for the expression level of
the transfected vectors (Fig. 4D), wild type p65 and KK-RR
mutant bound
B-DNA with similar affinities while the KK-AA mutant
was incompetent in binding DNA (Fig. 4C). Thus, consistent
with predictions from crystal structure analysis (31), the
acetyl-acceptor lysines 122 and 123 identified in this report
participate in high affinity binding of p65 to
B-DNA.
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Fig. 4.
p65 acetylation by PCAF affects its DNA
binding activity. A, immunopurified FLAG-p65 and PCAF
were incubated in acetylation buffer in the presence or absence of
[14C]acetyl-CoA. 14C-labeled p65 protein was
analyzed by SDS-PAGE and autoradiography. B, varying amounts
of acetylated FLAG-p65 (p65-Ac, lanes 1-3) and
non-acetylated FLAG-p65 (p65, lanes 4-6) were incubated
with 32P-labeled B probe and analyzed by EMSA.
Reactions were resolved by 4% acrylamide gel electrophoresis and
analyzed by autoradiography. C, various amounts of nuclear
extracts (NE) of HeLa cells transfected with either FLAG-p65
(wt), FLAG-p65KK-RR (RR), or FLAG-p65KK-AA
(AA) were analyzed by EMSA as described in b.
D, expression of p65 wild type (wt), KK-RR
(RR), and KK-AA (AA) in nuclear extracts of
transfected HeLa cells detected by Western blotting using
anti-FLAG.
B-DNA interaction, we next investigated whether p65 acetylation occurs in the cytoplasm or nucleus. HeLa cells transfected with FLAG-p65 expression vector were untreated or treated with leptomycin B
(LMB), which blocks the exportin/CRM1 pathway and inhibits
I
B
-mediated export of NF-
B resulting in the accumulation of
p65 in the nucleus (9-11). Cellular protein synthesis was blocked
24 h post-transfection by incubation in Met/Cys-deficient media.
Cells were maintained in the presence of LMB throughout the experiment.
Immediately prior to lysis, cells were pulse-labeled with
[3H]NaAc. FLAG-p65, immunoprecipitated separately from
cytoplasmic and nuclear extracts, was analyzed by Western blot (Fig.
5A, lanes 1-4). In
the absence of LMB, p65 was found in both cytoplasmic and nuclear
compartments (Fig. 5A, lanes 1-2). LMB treatment
led to accumulation of p65 in the nucleus (Fig. 5A,
lanes 3-4). Acetylated p65 in immunoprecipitates was
analyzed by SDS-PAGE and autoradiography (Fig. 5A,
lanes 5-8). In the absence of LMB, acetylated p65
accumulated in the cytoplasm (Fig. 5A, compare lane
1 to 2 and lane 5 to 6). In
contrast, LMB treatment resulted in localization of acetylated p65 in
the nucleus (Fig. 5A, compare lane 3 to
4 and lane 7 to 8). These results
suggest that p65 is acetylated in the nucleus and accumulates in the
cytoplasm in the absence of LMB.
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Fig. 5.
p65 is acetylated in the nucleus and
accumulates in the cytoplasm. A, HeLa cells transfected
with FLAG-p65 were mock-treated or treated with LMB overnight as
indicated. Cells were incubated in Met/Cys-deficient media for 3 h
at 37 °C and labeled in the same media supplemented with
[3H]NaAc for 1 h at 37 °C. p65 was
immunoprecipitated from cytoplasmic (C) and nuclear
(N) extracts. Immunoprecipitates were resolved by SDS-PAGE,
and the presence of p65 was analyzed by Western blot (lanes
1-4) and autoradiography (lanes 5-8). B,
the experiment was performed as described in A except
that HeLa cells were transfected with FLAG-p65 expression
vector alone or cotransfected with I B
expression vector as
indicated. C, acetylation of p65 facilitates its nuclear
export by I
B
. The experiment was performed as described in
B except that protein synthesis in the transfected cells was
not inhibited. Cells were labeled with either
[35S]Met/Cys or [3H]NaAc as indicated.
D, mutation of acetyl-acceptor lysines affects induction of
I
B
expression and its subsequent I
B
-mediated export
from the nucleus. HeLa cells transfected with the indicated expression
plasmids were stained with Cy5-conjugated anti-mouse and anti-rabbit
fluorescein isothiocyanate for detection of FLAG-p65 and I
B
,
respectively. The arrow highlights a p65KK-RR-expressing
cell in which I
B
is induced. Average percentages of cells
displaying nuclear localization of p65 are shown in the lower
right corner of each panel. Results are derived from
examination of at least 200 transfected cells from two independent
experiments. Bar represents 25 µm.
B
. I
B
causes retention of p65 in the cytoplasm by masking the NF-
B NLS
(2). The experiment was performed under conditions where protein
synthesis was inhibited as described for Fig. 5A. In the
absence of I
B
, p65 was found in both cytoplasmic and nuclear
fractions, whereas in the presence of I
B
, p65 localized
exclusively to the cytoplasmic fraction (Fig. 5B).
Autoradiography analysis showed that, in the absence of I
B
,
acetylated p65 was detected in both cytoplasmic and nuclear fractions
at levels approximately corresponding to the amount of p65 detected in
each compartment (Fig. 5B, lane 5 and
7). However, when p65 was blocked in the cytoplasm by
coexpression with I
B
, no acetylated p65 was detected (Fig.
5B, lanes 6 and 8). This experiment
shows that cytoplasmic p65 is not a substrate for acetylation. Taken
together, these results demonstrate that p65 is acetylated in the
nucleus and accumulates in the cytoplasm.
B
on p65 acetylation
in vivo, we performed the same experiment as in Fig.
5B except under conditions in which protein synthesis was
not inhibited. Under these conditions, NF-
B and I
B
shuttle
between the cytoplasm and nucleus. 35S-p65 was found in
both cytoplasmic and nuclear extracts of p65-transfected cells (Fig.
5C, lanes 1 and 3). When p65 and
I
B
were cotransfected, 35S-p65 was found mainly in
the cytoplasm but was also present in the nucleus, due to expression of
newly synthesized p65 (Fig. 5C, lanes 2 and
4). Analysis of acetylated p65 ([3H]p65)
showed that in the absence of I
B
, [3H]p65 was
found in both cytoplasmic and nuclear fractions (Fig. 5C,
compare lane 5 to 7). However, when p65 and
I
B
were coexpressed under conditions of ongoing protein
synthesis, acetylated p65 accumulated in the cytoplasm (Fig.
5C, compare lane 6 to 8).
Interestingly, I
B
was found to enhance p65 acetylation under
these conditions (Fig. 5C, compare lanes 5 to
6). Thus, acetylation of p65 may facilitate its nuclear
export by I
B
.
B
antibodies to detect the induction of endogenous I
B
. Wild type p65 was found in the nucleus only in cells in which
I
B
was not induced (Fig. 5D, panels 1 and
2). Induction of endogenous I
B
by p65 led to its
export to the cytoplasm (data not shown). Cotransfection of p65 and
I
B
led to the export of p65 to the cytoplasm in the majority of
transfected cells (Fig. 5D, panels 5 and
6). The p65KK-AA mutant was localized almost exclusively in
the nucleus (Fig. 5D, panels 7 and 8).
This is likely due to its weak transcriptional activity (Fig.
6A) and, consequently, lack of
I
B
induction by p65KK-AA (data not shown). In agreement with
this, cotransfection of p65KK-AA and I
B
expression vectors lead
to accumulation of p65KK-AA in the cytoplasm (Fig. 5D,
panels 9 and 10). The p65KK-RR mutant induced
high level expression of endogenous I
B
(data not shown), likely
due to its high transcriptional activity (Fig. 6A). However,
in a significant number of cells, this mutant localized in the nucleus
even in the presence of strong induction of I
B
expression (Fig.
5D, panels 11 and 12, see cell indicated by the arrow) suggesting that it binds tightly to DNA, which prevents its cytoplasmic export by I
B
.
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Fig. 6.
Reversible acetylation of p65 regulates its
transcriptional activity. A, mutation of
acetyl-acceptor residues affects p65-mediated transcription. HeLa cells
were cotransfected with pLTR-luc (1 µg), and either 1 or 2 µg of
p65 wt or p65 mutant as indicated and pRL-TK (100 ng). The relative
luciferase activity was calculated following normalization for
Renilla luciferase activity expressed from the TK promoter
present in the pRL-TK internal control plasmid. The -fold activation
was calculated relative to transfections in the absence of p65
expression plasmids. B, HeLa cells were transfected with 50 ng of GAL4 DNA-binding domain (GAL4-BD), GAL4-p65wt,
GAL4-p65KK-RR, or GAL4-p65KK-AA together with 100 ng of
5XGAL4-luciferase reporter plasmid and 20 ng of pRL-TK using
LipofectAMINE. The -fold activation was calculated relative to
transfections performed in the absence of activator expression
plasmids. C, HeLa cells were transfected using LipofectAMINE
with LTR-luc (500 ng), p65 wild type (100 ng) or p65KK-RR (100 ng),
pCMV-Renilla (20 ng), and the indicated plasmids, and
assayed for luciferase activity. DNA concentrations indicated on the
figure are in nanograms. Results are expressed as described in
A.
B (16-21). Recruitment of these coactivators by
p65 may induce localized chromatin remodeling via their intrinsic
histone-directed acetylation activities resulting in enhancement of
p65-mediated transcription. To investigate the role of factor
acetyltransferase (FAT) activity of p300 and PCAF toward p65 on its
transcriptional activity in vivo, HeLa cells were
transfected with plasmids expressing wild type p65 or mutants in which
the two acetyl-acceptor residues (Lys-122 and Lys-123) were substituted
with arginines (KK-RR) or alanines (KK-AA). The KK-RR mutation, which
conserves the positive charge, enhanced p65 transcriptional activity,
whereas alanine substitutions that neutralize the positive charge
reduced p65-mediated transactivation of the HIV-1 LTR-luciferase
reporter (Fig. 6A). Because the KK-RR mutant and wild type
p65 bound to
B-DNA equivalently after normalization to the
expression level of the transfected vectors (Fig. 3C), the
enhanced transcriptional activity of p65KK-RR is not due to increased
DNA binding. These results suggest that acetylation of p65
down-regulates its transcriptional activity. Furthermore, when fused to
the GAL4 DNA-binding domain, the transcriptional activities of wild
type p65, KK-RR, and KK-AA mutants were equivalent toward
5XGAL4-luciferase reporter plasmid (Fig. 6B).
B-dependent gene expression led us to the hypothesis that the FAT activity of p300 and PCAF may be involved in turning off
NF-
B-dependent gene expression.
NF-
B-dependent gene expression is in part regulated by
induction of its inhibitor, I
B
. I
B
binds to and dissociates
NF-
B from
B-DNA thus contributing to attenuation of NF-
B
transcriptional activity (6, 8). The data presented suggest a model in
which acetylation of p65 may contribute to I
B
-mediated export of
p65 to the cytoplasm resulting in accumulation of acetylated p65 in the
cytoplasm. To examine this hypothesis, we analyzed the effect of
I
B
on the transcriptional activity of wild type p65 and KK-RR
mutant. I
B
was more efficient in inhibiting wild type
p65-mediated transcriptional activity than the KK-RR mutant (Fig.
7A) suggesting that
acetylation of p65 negatively regulates its transcriptional activity
and may contribute to the attenuation of p65 activity to restore the
original latent state.
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Fig. 7.
Acetylation of p65 facilitates attenuation of
p65 transcriptional activity and its removal from
B-DNA by
I
B
.
A, acetylation of p65 affects I
B
-induced inhibition
of p65-mediated transcription. HeLa cells were transfected with
pLTR-luc-wt (1 µg), p65 wt or KK-RR mutant (1 µg), increasing
concentrations of I
B
(0.5, 1, and 2 µg), and pRL-TK (100 ng).
Results are shown as percent transcriptional activity relative to that
of p65 wild type or KK-RR mutant in the absence of I
B
.
B, acetylation of p65 facilitates its removal from
B-DNA
by I
B
in vivo. 293 cells were mock-transfected or
transfected with 5 µg of expression vector for either wild type
FLAG-p65 or FLAG-p65KK-RR mutant, alone or together with 5 µg or 2.5 µg of I
B
expression vector as indicated in the figure. ChIP
assays were performed with anti-FLAG antibody. Chromatin
immunoprecipitates were analyzed for the presence of IL-8 promoter
region (
121/+61) or IL-8 promoter 5'-region (
1042/
826). The
left panel (input) shows the starting chromatin
extracts and the right panel (IP) shows the
enrichment of the IL-8 promoter region that contains the NF-
B
response element. Signals obtained from immunoprecipitates were first
normalized to the starting chromatin extract (input). The
-fold enrichment was determined by comparing normalized signals from
transfected samples to the mock-transfected sample. Lanes 1 and 9 correspond to PCR reactions performed in the absence
of DNA. C, Western blotting analysis showing the expression
of FLAG-p65 wild type, FLAG-p65KK-RR, and I
B
in transfected 293 cells used for ChIP analysis. Cells were transfected with FLAG-p65 wild
type (lane 2) or FLAG-p65KK-RR (lane 5) alone, or
cotransfected with FLAG-p65 and I
B
(lanes 3,
4, 8, and 9) or FLAG-p65KK-RR and
I
B
(lanes 6, 7, 10, and
11). Concentrations of DNA transfected are as described in
B.
B
-mediated removal of wild type and non-acetylated forms
of p65 from the NF-
B elements associated with the IL-8 promoter in vivo was assessed by ChIP analysis (32, 33). ChIP assay using anti-FLAG antibody was performed on cells transfected with FLAG-p65 or FLAG-p65KK-RR in the presence and absence of I
B
. The
IL-8 promoter intensities obtained from immunoprecipitates (Fig.
7B, IP, right panel) were first
normalized to the starting chromatin extracts (Fig. 7B,
input, left panel). The -fold enrichment was
determined by dividing the normalized intensities of the transfected samples by that of the mock-transfected sample. ChIP analysis of cells
transfected with FLAG-p65 (lane 13, upper panel)
or FLAG-p65KK-RR (lane 16, upper panel) alone
resulted in ~6- and 9-fold enrichment of sequences containing the
IL-8 promoter, respectively. When I
B
was cotransfected with wild
type p65, 4- and 1.6-fold enrichment was observed, depending on the
concentration of I
B
transfected. However, 7.6- and 7-fold
enrichment was observed when the same concentrations of I
B
were
cotransfected with p65KK-RR mutant. The expression levels of p65,
p65KK-RR, and I
B
are shown in Fig. 7C. Therefore,
p65KK-RR is more resistant than wild type p65 to I
B
-mediated
removal from NF-
B elements of the IL-8 promoter in
vivo.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-mediated transcription. Both p300 and PCAF acetylate p65 and, moreover, target the same residues within p65: lysines 122 and 123 that are important for high affinity binding of p65
to
B-DNA. We identified HDAC3 as the histone deacetylase responsible
for p65 deacetylation. Fig. 8 shows a
schematic representation of the proposed role of p65 acetylation in
NF-
B-mediated transcription. Following immune stimulation of cells,
which results in degradation of I
B
, NF-
B is translocated to
the nucleus via the newly exposed NLS in p65 and binds tightly to
B-DNA elements. Recruitment of p300 and PCAF to the promoter region
results in activation of NF-
B-mediated transcription presumably
through their associated histone-directed acetylase activity, which
induces localized chromatin remodeling. Subsequently, acetylation of
p65 by p300 or PCAF at two DNA-binding residues, lysines 122 and 123, lowers its affinity for
B-DNA. This facilitates removal of NF-
B
from enhancer elements by newly synthesized I
B
whose expression
is induced by NF-
B, and subsequent export of NF-
B·I
B
from
the nucleus to the cytoplasm. The NF-
B·I
B
cytoplasmic
complex can subsequently either establish a post-induced latent state
or serve for additional rounds of activation following deacetylation of
p65 by HDAC3. Thus, acetylation of p65 is essential for turning off
NF-
B-mediated gene expression.
View larger version (23K):
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Fig. 8.
Schematic model for the proposed role of
p300- and PCAF-induced acetylation of p65 in regulating
NF- B-mediated transcription. See text for
details.
Recently, the reversible acetylation of p65 was reported by Chen
et al. (22). Although both studies show that reversible acetylation of p65 by p300 and HDAC3 plays a critical role in NF-B-transcriptional activity, the function attributed to p65 acetylation contrasts sharply between the two studies. Chen et al. proposed a model in which deacetylation of p65 promotes
binding between NF-
B and I
B
, which mediates export of the
complex to the cytoplasm to establish the latent state. In contrast, we
have shown that acetylation of p65 reduces its interaction with
B DNA and thereby promotes I
B
-mediated nuclear export. We were unable to demonstrate any significant effect of p65 acetylation on its
interaction with I
B
(Fig. 3). Because Chen et al. did not identify the specific acetyl acceptor residues present in p65,
their conclusions were based on experiments using the broadly acting
HDAC inhibitor, TSA, to enhance acetylation. However, TSA induces
global effects on acetylation in the cell and would influence the
function of many acetylated substrates, which include coactivators, components of the basal transcription machinery, and proteins involved
in nuclear import (34, 35). Chen et al. showed that cotreatment of cells with tumor necrosis factor
and TSA enhances p65 DNA-binding activity. In contrast, a direct comparison of
B-DNA
binding by acetylated and non-acetylated forms of p65 showed that
acetylation of p65 lowered its affinity for
B-DNA (Fig. 4), which
consequently promotes its I
B
-mediated export to the cytoplasm
(Figs. 5, 7, and 8). Using a GST-I
B
pull-down assay, Chen
et al. showed that cotransfection of p300 and p65 reduces the ability of GST-I
B
to interact with p65 and concluded that acetylation of p65 prevents its interaction with I
B
. We directly analyzed binding of I
B
to acetylated p65 by coimmunoprecipitation analysis using extracts from either [3H]NaAc or
[35S]Met/Cys-labeled cells and found that most of the
acetylated p65 in cells can be found in association with I
B
.
Indeed, given that acetylation of p65 occurs in the nucleus (Fig. 5),
the finding that acetylated p65 accumulates in the cytoplasm in an
I
B
-dependent manner indicates that the interaction
between p65 and I
B
is not abolished, because studies using
knock-out mice have shown that nuclear export of NF-
B depends on its
interaction with I
B
. The discrepancy between the conclusions
reached by the two studies may be due to differences in the
experimental approaches used.
The importance of IB-mediated NF-
B export from the nucleus and
consequently the termination of NF-
B-dependent
transcription has been provided from studies using I
B
knockout
mice (36, 37). However, the mechanism by which I
B
removes NF-
B
from DNA is unknown. The p50/p65 heterodimer has a particularly high affinity for
B-DNA, between 10
10 and
10
13 (38, 39), whereas the affinity of I
B
for the
p50/p65 heterodimer is 10
9 (40). Thus, the
NF-
B/
B-DNA interaction would need to be destabilized so that
I
B can compete for the removal of NF-
B from DNA. In agreement
with this, Munshi et al. (41) found that I
B
was not
able to remove NF-
B from DNA in the context of the INF-
enhanceosome. The crystal structure of the p50/p65 heterodimer bound to
B-DNA revealed that, among p65 residues involved in DNA
binding, lysines 122 and 123 are the only residues that contact the DNA
in the minor groove (31). Our results show that acetylation of lysines
122 and 123 within p65 by p300 and PCAF reduces its affinity to
B-DNA (Fig. 3). Thus, we propose a model in which acetylation of p65
plays a role in I
B-mediated removal of NF-
B from DNA and
termination of NF-
B-mediated transcription. In agreement with this
model, the acetylated form of p65 accumulated in the cytoplasm in an
I
B
-dependent manner, and the p65KK-RR mutant was less
sensitive to I
B
-mediated termination of transcription and removal
from
B-DNA than wild type p65 (Fig. 7). Analogous to the function
attributed to p65 acetylation in this report, acetylation of high
mobility group I(Y) by CBP has been shown to destabilize its
interaction with DNA, which disrupts the enhanceosome and turns off
INF-
gene expression (41).
The activity of NF-B has been shown to be regulated by
transcriptional coactivators that function by bridging the
sequence-specific activators to the basal transcriptional machinery and
play a role in chromatin remodeling via their intrinsic HAT or HDAC
activity (15). p65 binds to p300/CBP and PCAF (16-21). Protein kinase A phosphorylation of p65 enhances the p65/CBP interaction leading to
increased NF-
B-dependent gene expression (21).
Furthermore, acetyltransferase activity of CBP/p300 and PCAF enhance
NF-
B transcriptional activity (19, 20). Here, we show that p300 and
PCAF HAT activities potentiate p65 transcriptional activity, whereas
their FAT activities are repressive. Factor acetylation of p65
negatively regulates its transcriptional activity by lowering binding
to
B-DNA and facilitating its removal and export by I
B
. Additionally, HDAC3 was more repressive toward p65KK-RR than p65 wt,
suggesting that factor deacetylase activity of HDAC3 is involved in
activating p65 transcriptional activity, perhaps by enhancing p65
binding to
B-DNA. Thus, p300 and PCAF acetyltransferase activities play a role in both transcriptional activation and post-induction turn-off of p65-mediated transcription. A similar functional duality has been previously demonstrated for high mobility group I(Y), which, together with other transcriptional activators, recruits HAT
activity to the enhanceosome to stimulate transcription and serves
itself as a target for acetylation leading to enhanceosome disassembly
and turn-off of transcription (41).
The demonstration that p300 and PCAF are involved in both
transcriptional activation and post-induction turn-off implies that their acetylation activity would need to be regulated in a temporal and
substrate-dependent manner. What prevents p300 and PCAF
from acetylating p65 immediately after their recruitment to the
promoter? One possibility is that other substrates such as histones
provide better targets for acetylation. In support of this, we have
observed that acetylation of p65 by p300 and PCAF in vitro
is ~100-fold less efficient than that of nucleosomes (data not
shown). Alternatively, the substrate specificity of p300 and PCAF
acetylation activity may be regulated by post-translational
modifications. Therefore, recruitment of p300 and/or PCAF by
B-DNA-bound p65 may lead first to chromatin derepression and
transcriptional activation. Subsequently, p300 and PCAF may acetylate
p65 to facilitate its removal from DNA by I
B
and its export to
the cytoplasm to terminate NF-
B-mediated transcription.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank F. Arenzana-Seisdedos, R. Weil, and K.-T. Jeang for helpful discussions; P. Travo, Head of IFR24 Integrated Imaging Facility (www.crbm.cnrs-mop.fr/Imagcell.html) for his constant interest and support; Dr. Yoshida for the gift of LMB; D. Ballard, E.Verdin, and W. Fischle for the gift of plasmids; and J. Demaille for his support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Agence Nationale de Recherche sur le SIDA, Human Frontier Science Program, Minister de la Recherche (ACI) (to M. B.), ARC (to S. E.), Human Frontier Science Program (to C. S.), and Grants HK-RGC and HKU (to D.-Y. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an ANRS fellowship.
** Supported by a Ligue National Contre le Cancer fellowship.
A Leukemia and Lymphoma Society Scholar.
¶¶ To whom correspondence should be addressed: Laboratoire de Virologie Moléculaire, Institut de Génétique Humaine, 141 rue de la Cardonille, Montpellier 34396, France. Tel.: 33-4-99-61-99-32; Fax: 33-4-99-61-99-01; E-mail: bmonsef@igh.cnrs.fr.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209572200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NLS, nuclear localization signal; HAT, histone acetyltransferase; HDAC, histone deacetylase; CBP, CREB-binding protein; PCAF, p300/CBP-associated factor; FBS, fetal bovine serum; DTT, dithiothreitol; TSA, trichostatin A; EMSA, electrophoretic mobility shift assay; HIV-1, human immunodeficiency virus, type 1; ChIP, chromosomal immunoprecipitation; IL-8, interleukin-8; PMA, phorbol 12-myristate 13-acetate; GST, glutathione S-transferase; LMB, leptomycin B; CMV, cytomegalovirus; FAT, factor acetyltransferase; LTR, long terminal repeat; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TK, thymidine kinase.
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REFERENCES |
---|
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---|
1. | Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735[CrossRef][Medline] [Order article via Infotrieve] |
2. | Ghosh, S., May, M. J., and Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225-260[CrossRef][Medline] [Order article via Infotrieve] |
3. | Chen, F. E., and Ghosh, G. (1999) Oncogene 18, 6845-6852[CrossRef][Medline] [Order article via Infotrieve] |
4. | Pahl, H. L. (1999) Oncogene 18, 6853-6866[CrossRef][Medline] [Order article via Infotrieve] |
5. | Karin, M. (1999) Oncogene 18, 6867-6874[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Sachdev, S.,
Hoffmann, A.,
and Hannink, M.
(1998)
Mol. Cell. Biol.
18,
2524-2534 |
7. |
Tam, W. F.,
Wang, W.,
and Sen, R.
(2001)
Mol. Cell. Biol.
21,
4837-4846 |
8. |
Arenzana-Seisdedos, F.,
Turpin, P.,
Rodriguez, M.,
Thomas, D.,
Hay, R. T.,
Virelizier, J. L.,
and Dargemont, C.
(1997)
J. Cell Sci.
110,
369-378 |
9. |
Huang, T. T.,
Kudo, N.,
Yoshida, M.,
and Miyamoto, S.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
1014-1019 |
10. |
Johnson, C.,
Van Antwerp, D.,
and Hope, T. J.
(1999)
EMBO J.
18,
6682-6693 |
11. |
Rodriguez, M. S.,
Thompson, J.,
Hay, R. T.,
and Dargemont, C.
(1999)
J. Biol. Chem.
274,
9108-9115 |
12. |
Tam, W. F.,
Lee, L. H.,
Davis, L.,
and Sen, R.
(2000)
Mol. Cell. Biol.
20,
2269-2284 |
13. | Le Bail, O., Schmidt-Ullrich, R., and Israel, A. (1993) EMBO J. 12, 5043-5049[Abstract] |
14. | Sun, S. C., Ganchi, P. A., Ballard, D. W., and Greene, W. C. (1993) Science 259, 1912-1915[Medline] [Order article via Infotrieve] |
15. | Roth, S. Y., Denu, J. M., and Allis, C. D. (2001) Ann. Rev. Biochem. 70, 81-120[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Gerritsen, M. E.,
Williams, A. J.,
Neish, A. S.,
Moore, S.,
Shi, Y.,
and Collins, T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2927-2932 |
17. |
Hottiger, M. O.,
Felzien, L. K.,
and Nabel, G. J.
(1998)
EMBO J.
17,
3124-3134 |
18. |
Perkins, N. D.,
Felzien, L. K.,
Betts, J. C.,
Leung, K.,
Beach, D. H.,
and Nabel, G. J.
(1997)
Science
275,
523-527 |
19. |
Sheppard, K. A.,
Rose, D. W.,
Haque, Z. K.,
Kurokawa, R.,
McInerney, E.,
Westin, S.,
Thanos, D.,
Rosenfeld, M. G.,
Glass, C. K.,
and Collins, T.
(1999)
Mol. Cell. Biol.
19,
6367-6378 |
20. |
Vanden Berghe, W., De,
Bosscher, K.,
Boone, E.,
Plaisance, S.,
and Haegeman, G.
(1999)
J. Biol. Chem.
274,
32091-32098 |
21. | Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661-671[Medline] [Order article via Infotrieve] |
22. |
Chen, L.,
Fischle, W.,
Verdin, E.,
and Greene, W. C.
(2001)
Science
293,
1653-1657 |
23. |
Emiliani, S.,
Fischle, W.,
Van Lint, C., Al-,
Abed, Y.,
and Verdin, E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2795-2800 |
24. |
Kiernan, R. E.,
Vanhulle, C.,
Schiltz, L.,
Adam, E.,
Xiao, H.,
Maudoux, F.,
Calomme, C.,
Burny, A.,
Nakatani, Y.,
Jeang, K. T.,
Benkirane, M.,
and Van Lint, C.
(1999)
EMBO J.
18,
6106-6118 |
25. | Scherer, D. C., Brockman, J. A., Chen, Z., Maniatis, T., and Ballard, D. W. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11259-11263[Abstract] |
26. |
Benkirane, M.,
Chun, R. F.,
Xiao, H.,
Ogryzko, V. V.,
Howard, B. H.,
Nakatani, Y.,
and Jeang, K. T.
(1998)
J. Biol. Chem.
273,
24898-24905 |
27. |
Fajas, L.,
Paul, C.,
Vie, A.,
Estrach, S.,
Medema, R.,
Blanchard, J. M.,
Sardet, C.,
and Vignais, M. L.
(2001)
Mol. Cell. Biol.
21,
2956-2966 |
28. |
Nissen, R. M.,
and Yamamoto, K. R.
(2000)
Genes Dev.
14,
2314-2329 |
29. |
Furia, B.,
Deng, L., Wu, K.,
Baylor, S.,
Kehn, K., Li, H.,
Donnelly, R.,
Coleman, T.,
and Kashanchi, F.
(2002)
J. Biol. Chem.
277,
4973-4980 |
30. |
Ashburner, B. P.,
Westerheide, S. D.,
and Baldwin, A. S., Jr.
(2001)
Mol. Cell. Biol.
21,
7065-7077 |
31. | Chen, F. E., Huang, D. B., Chen, Y. Q., and Ghosh, G. (1998) Nature 391, 410-413[CrossRef][Medline] [Order article via Infotrieve] |
32. | Braunstein, M., Sobel, R. E., Allis, C. D., Turner, B. M., and Broach, J. R. (1996) Mol. Cell. Biol. 16, 4349-4356[Abstract] |
33. | Orlando, V., Strutt, H., and Paro, R. (1997) Methods 11, 205-214[CrossRef][Medline] [Order article via Infotrieve] |
34. | Bannister, A. J., Miska, E. A., Gorlich, D., and Kouzarides, T. (2000) Curr. Biol. 10, 467-470[CrossRef][Medline] [Order article via Infotrieve] |
35. | Imhof, A., Yang, X. J., Ogryzko, V. V., Nakatani, Y., Wolffe, A. P., and Ge, H. (1997) Curr. Biol. 7, 689-692[Medline] [Order article via Infotrieve] |
36. | Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S., and Baltimore, D. (1995) Nature 376, 167-170[CrossRef][Medline] [Order article via Infotrieve] |
37. | Klement, J. F., Rice, N. R., Car, B. D., Abbondanzo, S. J., Powers, G. D., Bhatt, P. H., Chen, C. H., Rosen, C. A., and Stewart, C. L. (1996) Mol. Cell. Biol. 16, 2341-2349[Abstract] |
38. | Thanos, D., and Maniatis, T. (1992) Cell 71, 777-789[Medline] [Order article via Infotrieve] |
39. | Urban, M. B., and Baeuerle, P. A. (1990) Genes Dev. 4, 1975-1984[Abstract] |
40. |
Phelps, C. B.,
Sengchanthalangsy, L. L.,
Huxford, T.,
and Ghosh, G.
(2000)
J. Biol. Chem.
275,
29840-29846 |
41. | Munshi, N., Merika, M., Yie, J., Senger, K., Chen, G., and Thanos, D. (1998) Mol. Cell 2, 457-467[Medline] [Order article via Infotrieve] |