From the Department of Safety Research on Biologics,
National Institute of Infectious Diseases, Tokyo 208-0011, Japan and
¶ Laboratory of Molecular Growth Regulation, NICHD, National
Institutes of Health, Bethesda, Maryland 20892
Received for publication, February 23, 2001
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ABSTRACT |
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Interferon regulatory factor-2 (IRF-2) is a
transcription factor of the IRF family that represses
interferon-mediated gene expression. In the present study, we show that
human monocytic U937 cells express truncated forms of IRF-2 containing
the DNA binding domain but lacking much of the C-terminal regulatory
domain. U937 cells are shown to respond to phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) to induce
expression of histone acetylases p300 and p300/CBP-associated factor
(PCAF). In addition, TPA treatment led to the appearance of
full-length IRF-2, along with a reduction of the truncated protein.
Interestingly, full-length IRF-2 in TPA-treated U937 cells occurred as
a complex with p300 as well as PCAF and was itself acetylated.
Consistent with these results, recombinant IRF-2 was acetylated by p300
and to a lesser degree by PCAF in vitro. Another IRF
member, IRF-1, an activator of interferon-mediated transcription, was
also acetylated in vitro by these acetylases. Finally, we
demonstrate that the addition of IRF-2 but not IRF-1 inhibits core
histone acetylation by p300 in vitro. The addition of IRF-2
also inhibited acetylation of nucleosomal histones in TPA-treated U937
cells. Acetylated IRF-2 may affect local chromatin structure in
vivo by inhibiting core histone acetylation and may serve as a
mechanism by which IRF-2 negatively regulates interferon-inducible transcription.
Nucleosomal histones are post-translationally modified by
various mechanisms including acetylation, deaceylation,
phosphorylation, and methylation (1, 2). High levels of histone
acetylation have been linked to the transcriptionally active region of
chromatin, while low histone acetylation is associated with the
transcriptionally repressed regions (3). Consistent with a link between
histone acetylation and transcriptional activation, several histone
acetylases are recruited to transcriptionally active promoters by
interacting with sequence-specific transcription factors (4, 5).
Histone acetylases are classified into several groups including the
conserved Gcn5-related N-acetyltransferase family and
the p300/CBP, Myst, TAF, and nuclear receptor co-activator families
(6). A number of transcription factors associate with
p300/CBP,1 originally known
as the global co-activators, and with PCAF and GCN5 belonging to the
Gcn5-related N-acetyltransferase family. Recruitment
of these histone acetylases is thought to alter chromatin structures,
required as an integral part of transcriptional activation. As a result
of interaction with histone acetylases, some transcription factors
become themselves acetylated, which often results in enhanced transcriptional activity (7-11). Some non-DNA-binding regulatory factors are also acetylated by histone acetylases (6, 12).
In addition to transcription, the status of histone acetylation is
thought to influence cell growth and differentiation (13, 14). In
support of a link between histone modification and cell growth, histone
acetylation is under control of growth and differentiation signals. For
example, it has been shown that treatment of human tissue culture cells
with epidermal growth factor increases phosphorylation and acetylation
of histone H3 (15). In addition, p300/CBP, by virtue of the interaction
with a number of cell cycle regulatory proteins, affects proliferation
and differentiation of some cells (16). Activity of p300/CBP is
also regulated during the cell cycle, peaking at G1/S
transition (17).
Proteins of the interferon regulatory factor (IRF) family
regulate type I interferon (IFN)-mediated transcription of many genes
(18). Some members regulate IFN gene expression as well. All IRF
proteins carry the conserved DNA binding domain (DBD) consisting of
~110 amino acids in the N-terminal region, through which they bind to
the IFN-stimulated response element (ISRE) present in IFN-inducible
genes. They have a less conserved C-terminal regulatory domain with
variable lengths. IRF-1 and IRF-2 are founding members of the family,
which have opposing activities. While IRF-1 activates transcription
from promoters carrying the ISRE, IRF-2 represses transcription of
these promoters. IRF-2, however, has a cryptic activation domain and
can activate transcription from some promoters (19, 20). Moreover,
IRF-1 can act as a tumor suppressor, while IRF-2 can act as an
oncogenic factor (21). Some IRF members are shown to interact with
histone acetylases. IRF-3, upon viral infection, interacts with
p300/CBP, and this interaction is a necessary step for its functional
activation (22-24). IRF-1 associates with p300/CBP and PCAF to form a
part of multiprotein complexes that assemble on the IFN- In the present study, we show that IRF-2 is truncated at the C-terminal
domain in untreated U937 cells, and TPA treatment induces expression of
full-length IRF-2. Coinciding with increased expression of p300/CBP and
PCAF following TPA treatment, full-length IRF-2 became associated with
these histone acetylases. As a result, IRF-2 was acetylated in
vivo in TPA-treated U937 cells. Last, we demonstrate that
acetylated IRF-2 inhibits p300-mediated acetylation of core histones.
This inhibition may be a part of mechanisms by which IRF-2 represses
transcription from some target genes and regulates cell growth.
Cell Culture--
Human monocytic U937 cells were maintained in
RPMI 1640 supplemented with 10% fetal bovine serum and gentamicin (25 µg/ml). Cells were treated with 10 nM TPA or 3 µM protein kinase C inhibitor GF109203X (Calbiochem) (31)
for the indicated periods of time. U937 cells stably expressing PCAF
were maintained in the above medium supplemented with 200 µg/ml
Geneticin (29). For in vivo labeling of
[14C]acetate in U937 cells, 20 µCi of
[14C]acetate (Amersham Pharmacia Biotech) were added to
U937 cell culture incubated at 37 °C in the CO2
incubator 1 h before isolation of cell lysate.
Semiquantitative PCR--
We used the methods by Colle et
al. (32) with a small modification. cDNAs were synthesized
using total RNA from TPA-treated U937 cells using Molony murine
leukemia virus reverse transcriptase (Life Technologies, Inc.) in a
reaction mixture containing 75 mM KCl, 3 mM
MgCl2, 50 mM Tris-HCl (pH 8.3), 0.25 mM dNTPs, 0.8 units of RNasin, and random hexamer primers
(Promega). Each PCR mixture contained 10 µl of cDNA, 50 mM KCl, 3 mM MgCl2, 10 mM Tris-HCl (pH 9.0), 250 µM dNTPs, 1 unit of
Taq DNA polymerase (Promega), and 1 µg of sense and
antisense primers in a total volume of 50 µl. A total of 30 cycles
were carried out for all samples. Southern blot hybridization was
performed as in Ref. 29.
GST Fusion Proteins--
A BamHI and SmaI
fragment of IRF-2 cDNA corresponding to the DNA binding domain
(amino acids 1-129) was subcloned into pGEX4T (Amersham Pharmacia
Biotech). GST fusion proteins were isolated by using
glutathione-Sepharose beads (Amersham Pharmacia Biotech) from bacteria
extracts. In vitro translated, full-length or truncated IRF-2 were produced from pcDNA3.1(+) (Invitrogen) harboring
appropriate inserts.
Immmunoblot Analysis--
Rabbit antibody against the C-terminal
domain of IRF-2 was described previously (29). Rabbit antibody against
the DBD of IRF-2 was produced by using a purified bacterial
protein produced in a pET-15b vector (Novagen) containing a PCR
fragment corresponding to residues 1-129 of IRF-2. The antibody
reacted with the DBD of IRF-2 but not with the DBD of IRF-1 or ICSBP.
Mouse antibody to p300 and rabbit antibody to histone deacetylase 1 were obtained from Upstate Biotechnology, Inc. Rabbit antibody to PCAF
(29) was a gift from Dr. Y. Nakatani (Dana-Farber Cancer Institute). Monoclonal M2 anti-FLAG antibody was purchased from Eastman Kodak Co.
For immunoblot analysis, the indicated amounts of nuclear extracts were
resolved on SDS 10% polyacrylamide gel electrophoresis (PAGE), gels
were transferred to a Immobilon P polyvinylidine fluoride membrane
(Millipore Corp.) and blocked with 1% skim milk in phosphate-buffered
saline containing 0.1% Tween 20. The membranes were incubated with an
appropriate dilution of primary antibodies and then with horseradish
peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Amersham
Pharmacia Biotech). The membranes were then developed using the ECL
detection kit according to the instructions provided by the
manufacturer (Amersham Pharmacia Biotech).
FLAG Pull-down Assay and Immunoprecipitation--
Extracts were
lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride and 0.1 mg/ml aprotinin
and centrifuged at 15,000 rpm for 20 min. Supernatants were incubated
with M2 anti-FLAG antibody (Sigma) or anti-IRF-2 antibody conjugated to
agarose beads for 2 h and washed three times in lysis buffer.
Bound materials were eluted in SDS-sample buffer and resolved on
SDS-10% PAGE followed by immunoblot analysis using M2 anti-FLAG
antibody. One µg of FLAG-tagged recombinant p300 or recombinant PCAF
produced in baculovirus vector (29, 33) was conjugated to 40 µl of M2
anti-FLAG antibody agarose beads and then incubated with in
vitro translated 35S-labeled IRFs in buffer containing
100 mM NaCl, 20 mM HEPES (pH 7.9), 10%
glycerol, 0.5 mM EDTA, and 0.1% Nonidet P-40 for 30 min at
40 °C, washed three times, eluted in SDS sample buffer, resolved on
SDS-12% PAGE, and autoradiographed.
Analysis of Histone Acetylase Activity--
Nuclear suspensions
were prepared as described in Ref. 34. Core histones (500 ng; Roche
Molecular Biochemicals) or nuclear preparations were incubated with 100 ng of baculovirus recombinant p300 or PCAF in the presence of
[14C]acetyl-CoA and the indicated amounts of baculovirus
recombinant IRF-2, IRF-1, or ICSBP for 30 min at 30 °C in histone
acetyltransferase reaction buffer as previously described (33). Reacted
materials were mixed with SDS-sample buffer and electrophoresed on
SDS-12.5% PAGE and subjected to PhosphorImager analysis using Fuji BAS
2000 or stained with Coomassie Brilliant Blue.
Affinity DNA Binding Assay and Electrophoretic Mobility Shift
Assay (EMSA)--
The DNA affinity binding assay was performed
essentially as described (29). Briefly, the indicated amounts of
baculovirus recombinant IRF-2 were incubated with recombinant p300 and
PCAF in the presence of 100 µM acetyl-CoA for 30 min at
30 °C, and reaction mixtures were incubated with magnetic beads
conjugated to biotinylated ISRE DNA from the ISG15
gene. Bound materials were immunoblotted with anti-IRF-2 antibody.
Nuclear extracts were prepared by the method of Dignam with
modifications that included the addition of protease inhibitors to the
buffers. Oligonucleotide probes were labeled with
[ TPA Treatment Increases Histone Acetylation in U937
Cells--
Phorbol ester, TPA, mediates various biological effects
through the activation of protein kinase C (30). Among other effects, TPA stimulates U937 human monocytic cells to differentiate into macrophage-like cells (31). Consistent with the role of protein kinase
C, TPA-induced differentiation is inhibited by protein kinase C
inhibitor. Previously, we reported that TPA treatment induced
expression of histone acetylases PCAF and p300 in U937 cells (29). We
were interested in determining whether TPA induction of histone
acetylase expression was affected by a protein kinase C inhibitor and
whether TPA treatment changed the status of histone acetylation in U937
cells. Results in Fig. 1A show
that while TPA treatment strongly induced expression of p300 and PCAF,
the co-addition of a specific inhibitor, GF109203X, greatly inhibited expression of both histone acetylases. To address the functional consequence of the induction of acetylases, we examined histone acetylation in U937 cells by incubating nuclear preparations with 14C-labeled acetyl-CoA. As shown in Fig. 1B, TPA
treatment led to a marked increase in the acetylation of histone H3 and
histone H2A and H2B, although acetylation of histone H4 did not appear to change significantly. The co-addition of inhibitor GF109203X strongly reduced histone acetylation levels. Coomassie Blue staining of
nuclear preparations confirmed that changes in histone acetylation levels were not due to differential sample loading. These results indicate that TPA induction of p300 and PCAF is dependent on protein kinase C activation and that this induction leads to increased acetylation of core histone in U937 cells.
Truncated IRF-2 and Expression of Full-length IRF-2 after TPA
Treatment--
During the course of these studies, we observed that
the molecular size of IRF-2 changes in U937 cells upon TPA treatment. This observation was made by the use of two anti-IRF-2 antibodies, one
reacting with the DBD and another reacting with the C-terminal end of
IRF-2. As shown in Fig. 1A, antibody to the DBD revealed two
IRF-2 bands in untreated U937 cells, corresponding to ~41-46 kDa in
size. These bands were smaller than a band expected of full-length
IRF-2, which should run near 49 kDa. In TPA-treated U937 cells, this
DBD antibody revealed a band of ~49 kDa along with weak smaller
bands. The addition of the protein kinase C inhibitor inhibited the
expression of the 49-kDa band (Fig. 1A). The smaller bands
were not a product of fortuitous cross-reactivity, since they were not
detected with sera absorbed with recombinant IRF-2 DBD (not shown).
These results suggested that IRF-2 proteins in untreated U937 cells
was truncated, and that TPA treatment restored expression of
full-length IRF-2 protein. In light of the previous study documenting
that IRF-2 undergoes C-terminal cleavage upon viral infection (35), we
further investigated whether the smaller IRF-2 bands present in
untreated U937 cells represent cleavage products of IRF-2. In Fig.
2A, reactivity of the two
anti-IRF-2 antibodies was compared side by side. The antibody against
the C-terminal region of IRF-2 did not reveal a discernible band in
untreated cells, while it recognized a 49-kDa band in TPA-treated
cells. This anti-C terminus IRF-2 antibody also reacted with a 49-kDa
band in Namalwa B cell extracts run as a control. In contrast, the
antibody against IRF-2 DBD (labeled N-terminal in Fig. 2A)
revealed several bands ranging from 41 to 46 kDa in size in untreated
U937 cells, but after TPA treatment the intensity of these bands was
decreased, while the intensity of the 49-kDa band was markedly
increased. Time course experiments using anti-IRF-2DBD antibody showed
that the increase in full-length IRF-2 coincided with the decrease
in truncated IRF-2 during 3 days of TPA treatment (Fig. 2B).
Namalwa B cells expressed predominantly a full-length band of 49 kDa
and a smaller 46-kDa band. These results indicate that untreated U937
cells express cleaved forms of IRF-2 and that TPA treatment causes
expression of full-length IRF-2. It is unlikely that IRF-2 is degraded
during extract preparations, because the sizes and levels of other
proteins such as histone deacetylase 1 (HDAC1; Fig.
2B), ICSBP, and PU.1 (not shown) remained unchanged before
and after TPA treatment, although this possibility cannot be formally
excluded. As shown in Fig. 2C, TPA treatment increased IRF-2
mRNA expression beginning at around 16 h, and the levels remained high for the subsequent 48-h period. The increase in IRF-2
transcripts correlated with the expression of full-length IRF-2 protein
(Fig. 2B), indicating that the TPA-induced change in the
size of IRF-2 involves increased gene expression. In Fig. 2D, we examined whether TPA treatment of U937 cells changed
factor binding activity for the ISRE, a target DNA element for the IRF family proteins. Extracts from untreated U937 cells produced several fast migrating bands (lane 2). On the other hand,
extracts from TPA-treated cells did not reveal fast migrating bands.
Instead, they produced three more slowly migrating bands
(lane 4). The upper two bands contained IRF-2 and
may represent a monomer and dimer of IRF-2, since these bands were
supershifted by anti-C terminus IRF-2 antibody (Fig. 2D).
The antibody did not react with bands from untreated cells. These
results are consistent with the view that untreated U937 cells express
truncated IRF-2 proteins that contain the DBD and that TPA treatment
increases expression of full-length IRF-2, reducing the truncated
species.
Full-length IRF-2 Is Complexed with p300 and PCAF in
TPA-treated U937 Cells--
We then investigated the interaction of
IRF-2 with histone acetylase PCAF or p300 in vivo.
Co-immunoprecipitation analysis performed with extracts of TPA-treated
U937 cells using anti-C terminus IRF-2 antibody showed that full-length
IRF-2 was co-precipitated with PCAF and p300 (Fig.
3A). However, neither IRF-2
nor the histone acetylases were precipitated from extracts of untreated
cells. Control IgG did not precipitate these proteins (Fig.
3A). It should be noted here that it was not possible to
test whether truncated forms of IRF-2 were also complexed with the
histone acetylases, because the antibody reacting with the IRF-2 DBD
did not work in immunoprecipitation experiments, although it did work
for immunoblot experiments. It is possible that epitopes recognized by
the antibody are sequestered in the native protein. However, based on
the low levels of histone acetylase expression in untreated U937 cells (Fig. 1) and data presented below, it seems unlikely that the truncated
forms of IRF-2 are complexed with histone acetylases.
To further verify the formation of IRF-2-histone acetylase
complex in U937 cells, we tested whether PCAF, known to interact with
p300, also forms a complex with IRF-2. For this purpose, immunoprecipitation analysis was performed using U937 cells stably expressing a FLAG-tagged PCAF (29). Anti-FLAG antibody precipitated full-length IRF-2 but not the truncated form of IRF-2 (Fig.
3B, middle panel). These cells
expressed significant levels of truncated IRF-2 as judged by immunoblot
analysis (see Input, bottom panel). Interestingly, a mutant PCAF lacking the catalytic domain of PCAF, on
the other hand, did not precipitate IRF-2. These results indicate that
full-length IRF-2, but not the truncated counterpart, interacts with
intact PCAF in vivo and forms a complex.
Given a number of reports that some transcription factors are
acetylated in vivo (7, 8, 10-12, 36), we sought to test the
possibility that IRF-2 is acetylated in vivo. U937 cells
were pulse-labeled with [14C]acetate for 60 min, and
extracts were immunoprecipitated with anti-C terminus IRF-2 antibody.
As shown in Fig. 3C, IRF-2 in TPA-treated U937 cells was
labeled with [14C]acetate, indicating that it is
acetylated. IRF-2 was not precipitated from untreated cells, and no
radioactive band was seen in the precipitates. Consistent with the
specificity of immunoprecipitation, control IgG did not reveal
acetylated IRF-2 from either extracts. Thus, IRF-2 is modified by
acetylation following TPA treatment of U937 cells.
Acetylation of IRF-2 by p300 and PCAF in Vitro--
We next tested
whether IRF-2 is acetylated in vitro by p300 and PCAF. Two
hundred ng of recombinant, full-length IRF-2 was incubated with
recombinant PCAF or p300 in the presence of
[14C]acetyl-CoA, and radiolabeled IRF-2 was detected
after SDS-PAGE separation. For comparison, we also tested recombinant
IRF-1 and ICSBP for in vitro acetylation. Both proteins are
members of the IRF family, and the former (but not the latter) is shown
to interact with PCAF (29). As shown in Fig.
4A, both p300 and PCAF were labeled with 14C-acetate, although acetylation was more
extensive for p300 than PCAF, indicating autoacetylation. In addition,
IRF-2 was acetylated by both p300 and PCAF. Interestingly, p300 was
more potent in acetylating IRF-2 than PCAF. IRF-1 was also acetylated
by p300, although to a much lesser degree than IRF-2. On the other
hand, PCAF did not acetylate IRF-1 to an appreciable degree. In
contrast, a detectable level of acetylation was not seen with ICSBP by
either p300 or PCAF. Coomassie Blue staining of reaction mixtures shown in Fig. 4B confirmed that each reaction contained equivalent
amounts of proteins.
Since a number of transcription factors are shown to gain increased DNA
binding activity upon acetylation (6, 8, 10, 37), it was of interest to
test whether acetylation of IRF-2 led to higher ISRE binding activity.
To this end, we carried out DNA affinity binding assay in which
increasing amounts of recombinant IRF-2 and p300 or PCAF were incubated
with the ISRE conjugated to magnetic beads in the presence or absence
of acetyl-CoA, and bound IRF-2 was measured by immunoblot analysis. As
shown in Fig. 4C, binding of IRF-2 to the immobilized ISRE
DNA was not significantly affected by acetylation, since the addition
of acetyl-CoA did not change the amount of bound IRF-2. We favored the
above DNA affinity binding assay over EMSA, since in the latter assays
binding of IRF-2-p300 complex or IRF-2-PCAF to the ISRE was not
detected, although binding of IRF-2 itself to the ISRE was seen.
Nevertheless, IRF-2 DNA binding activity tested in EMSA was not
affected by the histone acetylases in the presence of acetyl-CoA either
(not shown).
We previously noted that IRF-2 interacted with PCAF through the DNA
binding domain (29). Thus, it was of interest to determine whether
IRF-2 DBD is acetylated in vitro by p300 and PCAF. In Fig.
5A, a GST-IRF-2 DBD fusion
protein was incubated with recombinant p300 or PCAF in the presence of
[14C]acetyl-CoA. The IRF-2 DBD peptide but not the
control GST peptide was strongly acetylated by p300 and weakly by PCAF.
Acetylation of IRF-2 DBD was also demonstrated by the reactivity with
antiacetyl lysine antibody (Fig. 5C). IRF-2 DBD peptides
were co-incubated with p300 or PCAF and reacted with the antibody in
the presence of acetyl-CoA. IRF-2 DBD reacted with the acetyl lysine
antibody when incubated with p300 or PCAF only in the presence of
acetyl-CoA and not in the absence. IRF-2 DBD showed stronger reactivity
when incubated with p300 than with PCAF, consistent with more efficient acetylation of IRF-2 by p300 than by PCAF. As shown in EMSA analysis in
Fig. 5D, binding of IRF-2 DBD to DNA was not significantly affected by acetylation, similar to the results with full-length IRF-2.
We noted that the addition of p300 and PCAF increased the apparent
binding of IRF-2 DBD to DNA in the presence and absence of acetyl-CoA.
The basis of this increase is not clear at present.
IRF-2 DBD Binds to p300--
Previous studies have shown that some
IRF family proteins interact with p300/CBP, including IRF-1, IRF-2, and
v-IRF (23-27, 38). Given the observation that p300 can
acetylate IRF-2, it was anticipated that p300 directly interacted with
IRF-2 DBD. In experiments shown in Fig.
6, we tested binding of in
vitro translated, full-length IRF-2, IRF-2 DBD, or IRF-2 without
DBD ( IRF-2 Inhibits Core Histone Acetylation--
Since v-IRF is shown
to inhibit histone acetylase activity of p300 in vivo, it
was of interest to examine whether IRF-2 could affect acetylation of
core histones. We first tested the effect of recombinant IRF-2 on
p300-mediated core histone acetylation in vitro. As shown in
Fig. 7A (left
panel), the addition of IRF-2 inhibited p300-mediated
acetylation of all core histones in a dose-dependent
manner. PCAF-mediated histone acetylation was modest and was not
significantly affected by IRF-2 under these conditions. On the other
hand, comparable amounts of IRF-1 did not influence p300-mediated
histone acetylation (Fig. 7A, right
panel). In addition, ICSBP had no effect on histone
acetylation by p300 and PCAF. We then investigated the effect of IRF-2
on acetylation of nucleosomal histones derived from U937 cells. As
shown in Fig. 7B, untreated U937 cells showed only modest
acetylation noticeable primarily for histone H4. In agreement with data
in Fig. 1B, TPA treatment markedly increased acetylation of
other histones, namely histone H3, H2A, and H2B, while acetylation of
H4 remained largely unchanged. The addition of IRF-2 substantially
reduced levels of acetylation of histones H2A and H2B in a
dose-dependent fashion. ICSBP and IRF-1 (not shown), on the
other hand, had no effect, similar to the results in Fig.
7A. These effects were not due to differential loading of
proteins, as verified by equivalent Coomassie Blue staining of samples.
We used suspension nuclei for this assay, but permeabilized nuclei also
gave the same results (data not shown). These results imply that IRF-2,
upon interaction with p300 becomes acetylated and inhibits acetylation
of nucleosomal histones in TPA-treated U937 cells.
We show here that TPA treatment of U937 cells induces expression
of p300 and PCAF, leading to increased acetylation of histones in U937
cells. It has been shown that TPA activates protein kinase C and
stimulates differentiation of U937 cells toward macrophage-like cells
(31). Supporting the importance of protein kinase C activation in
histone acetylation, the inhibitor GF109203X greatly inhibited histone
acetylase induction and blocked increased histone acetylation in
TPA-treated U937 cells. These results imply that TPA (and perhaps other
differentiation signals) influence the overall levels of core histone
acetylation in the cells by regulating histone acetylase expression.
Consistent with the idea that histone acetylation is subject to growth
and differentiation signals, Cheung et al. (15) showed that
epidermal growth factor triggers phosphorylation and acetylation of
histone H3 in tissue culture cells. Several lines of evidence support a
link between increased histone acetylation and differentiation. First,
various histone deacetylase inhibitors are shown to cause
differentiation in many types of cancer cells (13). Second,
quinidine-induced differentiation of breast cancer cells is reported to
coincide with histone H4 hyperacetylation (14).
Along with changes in histone acetylase expression, truncated forms of
IRF-2 predominantly expressed in untreated U937 cells were largely
replaced by full-length IRF-2 after TPA treatment. Since there was no
evidence for alternative splicing of IRF-2 mRNA, it is likely that
in untreated U937 cells IRF-2 is cleaved by an endogenous protease(s).
Although the protease(s) that specifically cleaves IRF-2 has not been
identified, a previous report demonstrated that full-length IRF-2 was
cleaved in some cells upon viral infection (35). Interestingly, like
the truncated IRF-2 seen in U937 cells, the IRF-2 cleaved after viral
infection is shown to lack the C-terminal domain, while retaining the
intact DBD. The truncated IRF-2 in virus-infected cells acts as a
negative regulator of intact IRF-2. The emergence of full-length IRF-2
after TPA treatment is likely to be accounted for by newly synthesized
IRF-2, since there was a significant increase in the levels of IRF-2
mRNA expression after TPA treatment. In addition, it is possible
that increased stability of IRF-2 may have also contributed to the
appearance of full-length IRF-2.
In the present work, we have demonstrated that IRF-2 in
TPA-treated U937 cells was acetylated and occurred as a complex with p300 and PCAF (See Fig. 8 for a diagram).
Since PCAF is shown to directly interact with p300 (39), IRF-2 may be
part of a large PCAF-p300 complex. Recently, PCAF and GCN5 have been
shown to occur as a large stable complex containing many other factors (40, 41). However, neither IRF-2 nor p300 is shown to be a component of
a stable PCAF complex. It is likely that the IRF-2-containing complex
we detected by co-immunoprecipitation is a subset of heterogeneous PCAF
complexes that may be less stable than other complexes analyzed in
the previous studies. Although we do not have conclusive data, it seems
likely that only full-length IRF-2 is acetylated by p300 and PCAF
in vivo, based on the results that FLAG-tagged PCAF was not
co-precipitated with truncated IRF-2 in untreated U937 cells. Furthermore, the paucity of histone acetylases expressed in untreated cells would also make it less likely to cause acetylation of truncated IRF-2, although in vitro acetylation experiments showed that
the IRF-2 DBD can be acetylated by p300. Interestingly, although both IRF-1 and IRF-2 were acetylated in vitro, the level of
acetylation was significantly higher for IRF-2 than IRF-1. Senger
et al. (42) recently reported that IRF-2 inhibits the
recruitment of CBP to IFN-
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
promoter (25). Recently, it has been shown that v-IRF of Kaposi's
sarcoma-associated herpesvirus that acts as a repressor of interferon
inducible promoters also interacts with p300 (26-28). Previously, we
have demonstrated that IRF-1 and IRF-2 both interact with PCAF in
vivo and in vitro and that this interaction plays an
important role in transcription from relevant promoters (29). We also
observed that expression of histone acetylases PCAF and p300/CBP was
induced in response to phorbol ester,
12-O-tetradecanoylphorbol-13-acetate (TPA) in human
monocytic U937 cells (29). TPA has been shown to activate protein
kinase C and stimulate U937 cells to differentiate into macrophage-like
cells (30).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (Amersham Pharmacia Biotech) by T4
polymerase kinase (Promega). EMSA and supershift analysis were
performed as described (29).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of histone acetylase expression and
increased histone acetylation following TPA treatment of U937
cells. A, 30 µg of nuclear extracts from untreated
U937 cells (lane 1), cells treated with 3 µM protein kinase C inhibitor GF109203X (lane
2), cells treated with 10 nM of TPA
(lane 3), or cells treated with both for 3 days
(lane 4; GF109203X was added 30 min before TPA
addition) were separated on SDS-10% PAGE and analyzed for immunoblot
using antibody to p300, PCAF, IRF-2, and histone deacetylase 1 (HDAC1). B, nuclear preparations (3 × 104 nuclei) from U937 cells treated as above were reacted
with [14C]acetyl-CoA for 30 min at 30 °C. Reactions
were separated on SDS-12.5% PAGE and visualized by autoradiography
(upper panel). The gels were stained with
Coomassie Brilliant Blue for loading (CBB; lower
panel).
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Fig. 2.
IRF-2 is truncated in untreated U937
cells. A, 30 µg of nuclear extracts from untreated
U937 cells (lane 1) or cells treated with 10 nM TPA for 72 h (lane 2) and
untreated Namalwa B cells (lane 3) were analyzed
by immunoblot assay. IRF-2 was detected by anti-C terminus IRF-2
antibody (left) and anti-N terminus IRF-2 antibody
(right). Approximate molecular masses are indicated on the
left. B, time course of TPA effects. Immunoblot
analysis was performed with nuclear extracts from untreated U937 cells
(lane 1) and cells treated with TPA for 1 day
(lane 2), 2 days (lane 3),
or 3 days (lane 4) using anti-DBD IRF-2 antibody
(upper panel) or anti-histone deacetylase 1 (HDAC1) antibody (lower panel).
C, IRF-2mRNA expression following TPA treatment of U937
cells. Semiquantitative RT-PCR was performed with RNA from U937 cells
treated with TPA for the indicated periods. PCR products were run on
1% agarose gel and Southern blotted with probes for IRF-2 or HPRT.
D, alterations of ISRE binding activity following TPA
treatment of U937 cells. EMSA analysis was performed with 10 µg of
nuclear extracts from untreated U937 cells (lanes
2 and 3) and cells (lanes 4 and 5) treated with TPA for 72 h, incubated with
control IgG (lanes 2 and 4) or C
terminus IRF-2 antibody (lane 3 and 5)
using a 32P-labeled ISRE probe of the
ISG15 gene. Bars on the right
indicate IRF-2 complexes. An asterisk indicates an unknown
band that TPA treatment induced in U937 cells.
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Fig. 3.
Co-precipitation of PCAF and p300 with IRF-2
in TPA-treated U937 cells. A, extracts from untreated
(lanes 1 and 3) and TPA-treated (3 days) U937 (lanes 2 and 4) were
incubated with control rabbit IgG (lanes 1 and
2) or anti-IRF-2 IgG (lanes 3 and
4) for 1 h followed by incubation with protein
G-agarose for another 1 h. Bound materials were separated on
SDS-10% PAGE and immunoblotted using antibody to p300, PCAF, or IRF-2.
B, extracts from U937 cells stably expressing FLAG-tagged
PCAF, FLAG-tagged mutant PCAF without the catalytic domain ( HAT), or
control U937 cells with empty vector (PCNX) were incubated with
anti-FLAG M2 antibody conjugated to agarose beads for 2 h and
analyzed by immunoblot using anti-FLAG M2 (top
panel) or anti-IRF2 DBD (middle panel)
antibody. 30 µg of extracts from each of the U937 transfectants were
blotted with anti-IRF-2DBD antibody (Input;
bottom panel). C, untreated and
TPA-treated U937 cells were incubated with [14C]acetate
(20 µCi) for 1 h before isolation of cell lysate. Radiolabeled
cell lysates were immunoprecipitated (IP) with rabbit IgG or
anti-C terminus IRF-2 IgG and separated on SDS-10% PAGE followed by
PhosphorImager analysis.
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Fig. 4.
Acetylation of IRF-2 by p300 and PCAF
in vitro. A, 200 ng of recombinant
IRF-2 (lanes 1, 4, and 7),
ICSBP (lanes 2, 5, and 8),
or IRF-1 (lanes 3, 6, and
9) was incubated with [14C]acetyl-CoA in the
presence of 100 ng of BSA (lanes 1-3), p300
(lanes 4-6), or PCAF (lanes
7-9) for 30 min at 30 °C. Reaction mixtures were
separated on SDS 10%-PAGE and autoradiographed. B, gels in
A were stained with Coomassie Brilliant Blue. C,
affinity DNA binding assay for acetylated IRF-2. Indicated amounts of
recombinant IRF-2 (rIRF-2) were mixed with 100 ng of p300 or
PCAF for 30 min in the absence (lanes 1-4) or
presence (lanes 5-8) of acetyl-CoA and incubated
with magnetic beads conjugated to biotinylated ISRE. Bound IRF-2 was
detected by immunoblot assay.
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Fig. 5.
Acetylation of IRF-2 DBD by p300 and PCAF
in vitro. A, 200 ng of GST-IRF-2DBD
peptides (lanes 1, 3, and
5) or GST peptides (lanes 2,
4, and 6) were incubated with 100 ng of
recombinant p300 (lanes 1 and 2) or
PCAF (lanes 3 and 4) in the presence
of 14C acetyl-CoA for 30 min at 30 °C and analyzed as in
Fig. 4A. B, gels in A were stained
with Coomassie Brilliant Blue. C, 200 ng of GST-IRF-2DBD
peptides (lanes 1, 3, and
5) or GST peptides (lanes 2,
4, and 6) were incubated with 100 ng of
recombinant p300, PCAF, and BSA in the presence or absence of 100 µM acetyl-CoA for 30 min at 30 °C. Reaction mixtures
were immunoblotted using antiacetyl lysine antibody. D, DNA
binding activity of IRF-2 DBD. 200 ng of GST-IRF-2DBD peptides or GST
peptide were incubated with 100 ng of p300, PCAF, or BSA in the
presence or absence of acetyl-CoA and 32P-labeled ISRE
probe.
DBD) to FLAG-tagged p300 immobilized to M2 antibody beads.
Full-length IRF-2 and IRF-2 DBD bound to p300 but not to control M2
beads. In contrast,
DBD and in vitro translated
luciferase did not bind to p300-conjugated beads. These results show
that IRF-2 interacts with p300 through the DBD. Our results are
consistent with the recent report that v-IRF interacts with p300
through the N-terminal region corresponding to the DBD (28). These
results are analogous with our previous observation that interaction of
IRF-1 and IRF-2 with PCAF is dependent on the DBD (29).
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Fig. 6.
Interaction of IRF-2 DBD with p300 in
vitro. Recombinant FLAG-tagged p300 (lanes
3, 6, 9, and 12) or control
extracts with FLAG alone (lanes 2, 5,
8, and 11) were incubated with anti-FLAG
M2 antibody conjugated to agarose beads and incubated with
35S-labeled in vitro translated full-length
IRF-2 (lanes 1-3), IRF-2 DBD (lanes
4-6), IRF-2 lacking DBD (lanes 7-9),
or control luciferase product (lanes 10-12).
Bound materials were analyzed by autoradiography.
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Fig. 7.
IRF-2 inhibits histone acetylation by
p300. A, core histones (500 ng) were incubated with 100 ng of recombinant p300 or with PCAF and [14C]acetyl-CoA
in the presence of the indicated amounts of BSA, recombinant IRF-2,
ICSBP, or IRF-1 for 30 min at 30 °C, resolved on 12.5% SDS-PAGE,
and analyzed by a PhosphorImager (upper panel).
Reaction mixtures were stained with Coomassie Brilliant Blue
(CBB) for protein loading. B, nuclear
preparations (3 × 104 nuclei) from untreated U937
(lanes 1-4) or cells treated with TPA for 3 days
(lanes 5-8) were incubated with
[14C]acetyl-CoA in the presence of BSA (400 ng;
lanes 1 and 5), IRF-2 (200 and 400 ng;
lanes 2, 3, 5, and
6), or ICSBP (400 ng; lanes 4 and
8) in the presence of [14C]acetyl-CoA and
analyzed as in A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gene. Although our results are seemingly
contradictory with this report, it is important to stress that in the
present study we examined IRF-2 that was not bound to a specific
element, while Senger et al. focused on the IFN-
gene,
suggesting that action of IRF-2 may be dependent on the context of the
promoter. We note that among nine mammalian IRF members, IRF-2
possesses the highest number of lysine residues. Furthermore, the DBD
contains more lysines than the C-terminal domains in both IRF-1 and
IRF-2. It is reasonable to assume that the greater number of
acetylatable lysines present in IRF-2 accounts for greater levels of
acetylation for this protein. Our study places IRF-2 among the growing
list of DNA-binding transcription factors that are acetylated by
p300/CBP or PCAF/GCN5. The current list of acetylated proteins includes P53, Myb, GATA-1, Sp1, MyoD, and others (6-11, 43-45). Most of these
factors are reported to gain increased DNA binding activity and
enhanced transcriptional activity upon acetylation. For IRF-2, we did
not observe increased DNA binding activity after acetylation by 300 or
PCAF. Nevertheless, considering that the DNA binding domain is
acetylated, it may be reasonable to anticipate that the mode with which
IRF-2 interacts with a target DNA element is modified by acetylation in
the native, chromatinized promoters.
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Fig. 8.
Diagram for post-translational modification
of IRF-2 in U937 cells. In untreated U937 cells IRF-2 occurs as a
truncated form. TPA treatment leads to expression of full-length IRF-2
along with the induction of p300 and PCAF and increased histone
acetylation. Full-length IRF-2 interacts with p300 and PCAF and becomes
acetylated. Concomitantly, IRF-2 inhibits core histone acetylation,
resulting in an alteration of local chromatin structure. These changes
may provide a mechanism by which IRF-2 acts as a repressor and affects
transcription and cell growth.
Acetylation of IRF-2 may have a significant functional consequence in
transcription. We showed that the addition of recombinant IRF-2 but not
IRF-1 inhibited core histone acetylation by p300 in vitro as
well as nucleosomal acetylation of U937 cells after TPA treatment. We
noted that inhibition was more prominent for histone H-2A and H-2B than
histones H3 and H4, suggesting that IRF-2 inhibited histone acetylase
activity of p300 more strongly than PCAF, since p300 is capable of
acetylating all four histones, while PCAF/GCN5 acetylates predominantly
H3 and H4 (46). A simple interpretation of the observed inhibition
would be that IRF-2, by acting as a substrate for histone acetylases,
competitively inhibits histone acetylation. Consistent with this
interpretation, appreciable inhibition of histone acetylation was not
observed by IRF-1 and ICSBP, a weaker substrate for the acetylases.
Together, it is attractive to assume that IRF-2 inhibition of histone
acetylation is relevant to transcriptional repression of IFN-responsive
genes by IRF-2 (47, 48). It is of note that although for many factors acetylation leads to enhanced transcriptional activity, it can have a
negative regulatory effect, as reported for T-cell factor in
Drosophila (49). Interestingly, another IRF member, v-IRF, derived from Kaposi's sarcoma-associated herpesvirus, also interacts with p300 and inhibits core histone acetylation, resulting in repressed
transcription of interferon-responsive genes (28, 50). It would be of
interest to determine whether v-IRF inhibits core histone acetylation
by acting as a competitor. Among several transcriptional activators
involved in IFN- enhancer element, IRF-2 acts on CBP recruitment
negatively. In contrast, IRF-2 binds to p300 directly and inhibits its
histone acetylation in our study. It is important to note that since
IRF-2 can act as an activator for certain genes (19, 20, 51), the
effect of reduced local histone acetylation is likely to be dependent
on the promoter context.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Y. Nakatani for plasmids, Dr. K. Komuro for support, members of Dr. T. Taniguchi's laboratory for useful discussions, and Dr. M. Nishijima and Dr. H. Fukazawa for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by the Ministry of Health Science and Welfare of Japan.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.
§ To whom correspondence should be addressed: Safety Research on Biologics, 4-7-1, Gakuen, Musashimurayama-shi, National Institute of Infectious Diseases, Tokyo, Japan. Tel.: 81-425-61-0771; Fax: 81-425-65-3315; E-mail: amasumi@nih.go.jp.
Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M101707200
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ABBREVIATIONS |
---|
The abbreviations used are: CBP, CREB-binding protein; IRF, interferon regulatory factor; v-IRF, viral IRF; TPA, 12-O-tetradecanoylphorbol-13-acetate; ISRE, interferon-stimulated response element; DBD, DNA binding domain; PCR, polymerase chain reaction; ICSBP, interferon consensus sequence-binding protein; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; IFN, interferon; BSA, bovine serum albumin; PCAF, p300/CBP-associated factor.
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