From the
Departments of Safety Research on
Biologics, ¶Cell Biology and Biochemistry, and
||Bioactive Molecules, National Institute of
Infectious Diseases, Tokyo, Japan and the **Laboratory
of Molecular Growth and Regulation, NICHD, National Institute of Health,
Bethesda, Maryland 20892
Received for publication, December 20, 2002 , and in revised form, May 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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The IRF (interferon regulatory factor) family of transcription factors has been extensively studied in the context of host defense and oncogenesis. Members of this family have extensive homology within their DNA binding domain. By binding to the common target DNA, ISRE, and IRF proteins regulate transcription of interferon and interferon-inducible genes. Some IRF proteins interact with other transcription factors such as TFIIB, coactivators p300 and PCAF (2933). IRF-2 has generally been described as a transcriptional repressor, and it is thought to function by competing with the transcriptional activator IRF-1. However, IRF-2 can act as a positive regulator for ISRE-like sequences including the H4 promoter (34), VCAM promoter (35), and gp91phox promoter (36). Hence, IRF-2 is similar to factors such as YY1, RAP-1, Dorsal, and Kruppel, which exhibit both transcriptional activating and repressing activities (37). Biologically, IRF-2 plays an important role in cell growth regulation, and has been shown to be a potential oncogene, as overexpression of IRF-2 causes anchorage-independent growth in NIH 3T3 cells and tumor formation in mice (38). We previously reported that IRF-2 in 12-O-tetradecanoylphorbol-13-acetate-treated U937 cells is complexed with p300, as well as PCAF, and is itself acetylated (39). Consistent with these results, recombinant IRF-2 was acetylated by p300, and to a lesser degree by PCAF, in vitro (39), providing the first example of acetylation in the family. Since then, additional IRF members, IRF-3 and IRF-7, were shown to be acetylated in vivo and in vitro (40, 41). In a previous paper (39), we documented that acetylation of IRF-2 does not alter its DNA binding activity, although acetylation increases DNA binding activity and transcription for some transcription factors. However, acetylation of IRF-2 led to inhibition of histone acetylation by p300, suggesting a possible mechanism for transcriptional repression by IRF-2.
In this paper, we identified two lysine residues located in the IRF-2 DNA binding domain that are acetylated. We show that one residue, Lys-75 is important for the interaction with p300 and the stimulation of histone H4 promoter activity. Histone H4 is transcribed during cell replication, where IRF-2 functions as an activator (42). Providing further evidence of the biological significance of IRF-2 acetylation, we show that IRF-2 is acetylated only in rapidly growing NIH 3T3 cells but not in growth-arrested cells, although IRF-2 is expressed at a similar level in both type of cells. Chromatin immunoprecipitation analysis revealed that acetylated IRF-2 forms a complex and binds to the endogenous H4 promoter. Together, IRF-2 is acetylated by p300 in NIH 3T3 cells in a cell growth-dependent manner, enabling it to participate in gene regulation crucial for cell growth control.
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MATERIALS AND METHODS |
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Immunoprecipitation/Western BlottingNuclear extracts were prepared by the methods of Dignam with modifications including the addition of a protease inhibitor mixture (Sigma) to buffers. Immunoprecipitation was performed using an anti-IRF-2 antibody as described previously (39). The precipitates or the whole cell lysates containing equal amounts of total proteins were suspended in the SDS sample buffer with boiling, separated on SDS-10% polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore) for 1 h. The membrane was blocked with 5% nonfat dry milk in a PBS-T buffer (containing PBS and 0.5% Tween 20) for 1 h, incubated with anti-IRF-2 (Santa Cruz), anti-p300 (Santa Cruz), anti-acetyllysine (New England Biology), anti-glutathione S-transferase (GST) (Santa Cruz Biotechnology Inc.), and anti-FLAG (Sigma), anti-histidine (Qiagen) antibodies for 1 h, and washed in PBS-T. The antigen-antibody interaction was visualized by incubation in a chemiluminescent reagent (Amersham Biosciences) and exposure to x-ray film.
Analysis of Acetylation SiteTen micrograms of GST-IRF-2 DBD
was incubated with 1 µg of rp300 or rPCAF at 30 °C for 2 h in 60 µl
of acetyltransferase reaction buffer
(39). The reaction mixture was
incubated with 10 units of thrombin (Amersham Biosciences) for 16 h to cleave
the GST region and IRF-2DBD was purified by reverse-phase high performance
liquid chromatography (phenyl-5WRP). Purified GST-IRF-2 (4 µg) was
incubated with 100 ng of endopeptidase Lys-C (Roche Diagnostics) in a buffer
containing 25 mM Tris-HCl, pH 8.5, 1 mM EDTA, and 8
M urea for 16 h at 37 °C. Acetylated peptides were subjected to
mass spectrometry (high performance liquid chromatography ESI-MS).
Plasmid Constructs and Transient TransfectionGST-IRF-2DBD
(amino acids 1129) and GST-IRF-2DBD (amino acids 86349)
were generated by cloning a PCR-amplified fragment into pGEX-4T (Amersham
Biosciences). To make the IRF-2DBD mutant, a mutated oligonucleotide was
produced by PCR and inserted into pGEX-4T (Amersham Biosciences) at the
BamHI and SmaI sites. GST-IRF-2DBD mutant proteins were
synthesized in Escherichia coli and purified as previously described
(39). To make full-length
IRF-2 mutants, wild type IRF-2 was point mutated by changing to the
oligonucleotide produced by PCR to produce an IRF-2KR pcDNA3.1 mutant.
Adenovirus E1A and E1A
N plasmids were kindly provided from Dr. Y.
Nakatani (Dana-Farber Cancer Institute).
For H4 reporter analysis, wild type IRF-2pcDNA3.1 or
IRF-2KRpcDNA3.1 mutants were co-transfected into NIH 3T3 cells with
H4 reporter (30) by
LipofectAMINE Plus (Invitrogen), and luciferase activity was analyzed 24 h
after transfection. For transient transfection of 293T cells, IRF-2pcDNA3.1,
E1A, and E1AN were cotransfected by FuGENE (Roche Diagnostics).
Synthesis and Purification of Recombinant ProteinsFLAG-tagged human PCAF was purified in Sf9 cells by using a baculovirus expression system and purified as previously described (30). His6-tagged human p300 baculovirus were kindly gifted from Dr. Kraus and Dr. Ito (22). In vitro translatable products from plasmids were made using a rabbit reticulocyte TNT assay kit (Promega) as described previously (30).
In Vitro Acetylation AssaysSubstrate proteins (GST-IRF-2DBD) (200 ng) were incubated with 100 ng of recombinant p300 or PCAF in 20 µl of acetylation buffer containing 50 mM Tris, pH 8.0, 5% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium butyrate with 10 µM acetyl-CoA (Sigma) or [14C]acetyl-CoA (Amersham Biosciences). Reaction mixtures were incubated at 30 °C for 30 min, and resolved by SDS-PAGE. The reactions were analyzed first by Western blotting with an anti-acetyllysine antibody to evaluate acetylation activity, and the same gel was subsequently subjected to immunoblot with an anti-GST antibody to verify the amounts of proteins used in each reaction. Radiolabeled reactions were analyzed by autoradiography after staining by Coomassie Brilliant Blue.
FLAG-Pull Down AssayCell lysates from 293T transfectants were incubated with M2-agarose (Sigma) in a buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.1% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml aprotinin and washed three times. Bound materials were eluted in an SDS sample buffer, resolved on an SDS-12.5% PAGE, detected by Western blotting.
Affinity DNA Binding Assay and Electromobility Shift AssayThe DNA affinity binding assay was performed as described (30, 39). Briefly, nuclear extracts (500 µg of protein) were incubated with magnetic beads conjugated to biotinylated oligonucleotide from the H4 gene (34). Bound materials were immunoblotted with anti-IRF-2 and anti-acetyllysine antibodies. Electrophoretic mobility shift assays were performed using a 32P-labeled H4 promoter oligonucleotide (34) made by T4 kinase (Promaga). In vitro translated products were incubated with a reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 1 µg of poly(dI-dC)·poly(dI-dC) and 1 µg of salmon sperm DNA) in the presence of a labeled probe. Protein-DNA complexes were separated from the free probe by gel electrophoresis on 5% polyacrylamide gels in a 0.5 x TBE buffer. The gel was dried and analyzed by autoradiography.
Chromatin ImmunoprecipitationThe chromatin immunoprecipitation analysis was performed essentially as described (16) with some modifications. A total 3 x 107 NIH 3T3 cells were cross-linked with 1% formaldehyde for 15 min at room temperature. Cells were washed with PBS and resuspended in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) plus a protein inhibitor mixture (Sigma), incubated on ice for 10 min, and sonicated to an average size of 500 bp by an ultrasonic cell disruptor (MISONIX, MICROSONTM). One hundred-µl aliquots of sonicated chromatin were diluted in 1 ml of buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8) and precleared with 2 µg of sheared salmon sperm DNA and protein G-Sepharose (Invitrogen) (50 µl of 50% slurry in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA) for 2 h at 4 °C. Immunoprecipitation was performed overnight at 4 °C with no antibody, anti-IRF-2 (C-19: Santa Cruz sc-498), or rabbit IgG (Sigma). A 50-µl aliquot of protein G-Sepharose, and 2 µg of salmon sperm DNA were added to each immunoprecipitation and incubated for 1 h. Precipitates were washed sequentially for 10 min in a 1x washing buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), 2x washing buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 500 mM NaCl), 1x washing buffer III (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and 2x TE (10 mM Tris-HCl, pH 8, 1 mM EDTA). Samples were extracted twice with 250 µl of elution buffer (1% SDS, 0.1 M NaHCO3), heated at 65 °C overnight to reverse cross-links, and DNA fragments were purified with a QIAEX II Gel Extraction Kit (Qiagen). A 5-µl aliquot from a total of 40 µl was used in the PCR. Primers used in PCR were the H4 promoter (5'-AAGAAAAACAGGAAGATGATGCAA; 3'-AGCTAATGTAATCTGAAACACCAG) and the glyceraldehyde-3-phosphate dehydrogenase (5'-AGAACATCATCCCTGCCTCTACTG; 3'-CATGTGGGCCATGAGGTCCACCAC). Input DNAs were diluted 1:100. The PCR products were amplified for 35 cycles, which was established as the linear range of PCR.
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RESULTS |
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We next sought to determine individual lysine residues within the IRF-2 DBD that are acetylated by p300 and PCAF. To this end, the GST-DBD peptides were acetylated by p300 and PCAF in vitro, digested by endopeptidase Lys-C, and the resultant peptides were subjected to mass spectrometry analysis to identify acetylated peptides. We found that peptide 1 and peptide 2 in Fig. 1B were clearly modified by acetylation among other peptides. Peptide 1 covers amino acids 2632, containing three lysines, Lys-29, Lys-31, Lys-32, whereas peptide 2 encompasses amino acids 6578 containing Lys-71, Lys-75, and Lys-78. To further assess major acetylation sites within these regions, mutant DBDs were constructed in which each of the Lys residues was changed to arginine. These mutants were acetylated in vitro by p300 and PCAF, and the levels of acetylation were assessed by immunoblot with an anti-acetyllysine antibody. Wild type IRF-2DBD was intensely acetylated both by p300 and PCAF (Fig. 1C). Mutants K29R, K31R, K32R, and K71R were also acetylated, although slightly less than the wild type DBD. However, levels of acetylation were markedly reduced for K75R, observed both by PCAF and p300. In addition, acetylation by PCAF was significantly reduced for K78R, although the reduction was less significant when tested with p300 (Fig. 1C). These results indicate that Lys-75 is the major site of acetylation by p300 and PCAF, whereas Lys-78 is also acetylated by PCAF, albeit to a lesser degree. The lysine residue at 75 is shared between IRF-1 and IRF-2, but not in other IRF family members. Lys-78 is a residue conserved in all IRF family proteins, and is critical for ISRE binding activity (41). Thus, although mass spectrometry found other Lys residues (Lys-29, Lys-31, and Lys-32) to be acetylated, the overall acetylation level of these residues appear much less extensive.
Having identified Lys-75 and Lys-78 as the main sites of IRF-2 acetylation in vitro, it was important to determine whether these Lys residues were acetylated in vivo. FLAG-tagged wild type IRF-2 as well as FLAG-tagged, full-length IRF-2 mutants K75R and K78R were transfected into 293T cells. Extracts were precipitated by an anti-FLAG antibody and analyzed by immunoblot with an anti-acetyllysine antibody. As shown in Fig. 1D, the anti-acetyllysine antibody gave much stronger signals for wild type IRF-2 than the K75R and K78R point mutants and K75R,K78R double mutants. Acetylation of K75R,K78R double mutants was decreased further compared with each point mutant. Immunoblot with the anti-FLAG antibody verified that the four IRF-2 were expressed at comparable levels. These results verify that Lys-75 and Lys-78 are acetylated in vivo.
The Acetylation Mutant K75R Fails to Stimulate H4 Promoter ActivityWe previously showed that acetylation of IRF-2 does not alter DNA binding activity (39). Here, we tested whether the substitution of Lys to Arg in amino acid positions 75 or 78 affected DNA binding activity. In vitro transcribedtranslated mutants K75R and K78R were tested in gel mobility shift assays using the ISRE of the histone H4 promoter as a probe (Fig. 2A). The K75R mutant bound to the ISRE as well as the wild type IRF-2. In contrast, the K78R mutant failed to bind to the DNA. Lysine at 78 is conserved throughout the IRF family and is directly involved in DNA binding (43), and the mutation of this residue likely impaired a structure/conformation required for DNA binding, because mutation of the homologous residue in several IRF family proteins abolishes DNA binding activity (41, 44).
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IRF-2 acts as an activator or repressor depending on the promoter context.
IRF-2 functions as an activator for the H4 gene promoter in NIH 3T3
cells (34). To examine whether
the mutation of Lys-75 or Lys-78 influences the ability of IRF-2 to activate
H4 promoter activity, transfection assays were performed using
vectors for K75R and K78R mutants and a H4-luciferase reporter.
Results obtained with NIH 3T3 cells are shown in
Fig. 2B. As expected,
transfection of wild type IRF-2 enhanced the H4 promoter activity in
a dose-dependent manner. However, the K75R mutant only modestly enhanced
promoter activity, reaching 50% of the enhancement seen by wild type
IRF-2. The K78R mutant that did not bind to the target DNA, did not enhance
the H4 promoter. Thus, mutation of the acetylatable residue at Lys-75
diminishes activator function of IRF-2, supporting the role for acetylation of
Lys-75 in the transcriptional activity of IRF-2.
Assuming that full activation of H4 promoter activity depends on
acetylation of IRF-2, we wished to evaluate the contribution of p300/CBP or
PCAF to H4 promoter activity. Because levels of PCAF expression are
very low in NIH 3T3
cells,2 p300/CBP is
the likely acetylase affecting IRF-2 in these cells. It has been shown that
p300/CBP binds to the adenovirus E1A, and that this binding inhibits
p300-dependent transcription activation
(45). However, the N-terminal
mutant E1A (E1AN) fails to bind to p300, and does not inhibit
transcription
(2428,
46). In experiments shown in
Fig. 3, H4 promoter
activity was examined following cotransfection of IRF-2 and E1A along with the
H4-luciferase reporter. Cotransfection of wild type E1A strongly
inhibited the H4 promoter activated by IRF-2. On the other hand,
E1A
N did not inhibit the H4 promoter activation, suggesting
that endogenous p300 takes part in IRF-2-dependent activation of H4
promoter activity.
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To ascertain whether IRF-2 acetylation is inhibited by E1A, immunoblot
analysis was performed for FLAG-tagged IRF-2 cotransfected with wild type E1A
or E1AN in 293T cells. As shown in
Fig. 3B, coexpression
of E1A, but not of the mutant E1A
N, strongly reduced acetylation of
IRF-2 in vivo. These results suggest that p300/CBP acetylates IRF-2,
thereby enhancing its transcriptional activity.
To examine whether E1A affects the modest promoter activity of the IRF-2 mutant, E1A was cotransfected with IRF-2K75R mutant into NIH 3T3 cells. E1A transfection inhibited the modest activation of H4 promoter activity by mutant IRF-2 K75R as well as that of wild type IRF-2 (Fig. 3C).
IRF-2 Is Acetylated in Growing, but Not in Growth-arrested NIH 3T3 CellsIRF-2 has an oncogenic property, as evidenced by anchorage-dependent cell growth when overexpressed in NIH 3T3 cells (38).
To assess whether acetylation of IRF-2 is relevant to cell growth control, we examined the status of IRF-2 acetylation in exponentially proliferating, or confluent, growth-arrested NIH 3T3 cells. Endogenous IRF-2 was precipitated by an anti-IRF2 antibody and tested for acetylation by immunoblot. As seen in Fig. 4, IRF-2 was expressed at a comparable level in growing and growth-arrested cells. However, acetylation of IRF-2 was seen only in growing cells, but not in growth-arrested cells. These results indicate that acetylation of IRF-2 depends on the status of cell growth.
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These results were interesting, because histone H4 is a cell cycle-regulated gene, expressed during the S phase, and is not expressed in growth-inhibited cells (42). Thus, we sought to determine whether acetylated IRF-2 binds to the H4 promoter in a cell growth-dependent manner. To this end, we first performed a DNA affinity binding assay with biotinylated H4 promoter oligonucleotides that had been conjugated to magnetic beads. Nuclear extracts from growing and growth-arrested cells were incubated with the bead-conjugated H4 promoter DNA, and bound IRF-2 was detected by immunoblot. Results are shown in Fig. 4B. Binding of IRF-2 to the H4 promoter was detected only with extracts from growing cells, but not from growth-arrested cells, despite that levels of total IRF-2 in the extracts being comparable, suggesting that only acetylated IRF-2 binds to the H4 promoter. We found that in addition to IRF-2, H4 promoter DNA bound to p300, again only from growing cells, but not growth-arrested cells. Like IRF-2, the total levels of p300 were similar between growing and arrested cells.
Finally, it was of importance to verify that IRF-2 binds to the promoter of
the endogenous H4 gene in a cell growth-dependent manner. Thus, we
performed chromatin immunoprecipitation assays for growing and growth-arrested
NIH 3T3 cells. Cells were first treated with formaldehyde, and chromatin was
sonicated to produce 500-bp DNA fragments. Materials were precipitated by
an anti-IRF-2 antibody, and precipitated DNA was subjected to PCR to amplify a
150-bp H4 promoter fragment. As shown in
Fig. 4D, the IRF-2
antibody precipitated the H4 promoter DNA from growing cells.
However, the same antibody failed to precipitate DNA from growth-arrested
cells. The anti-IRF-2 antibody did not precipitate glyceraldehyde-3-phosphate
dehydrogenase, run as a control. These results confirm that IRF-2 binds to the
H4 promoter in growing NIH 3T3 cells, but not in their
growth-arrested counterparts, supporting the role of IRF-2 acetylation in
regulating H4 transcription and cell growth.
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DISCUSSION |
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Our results indicate that acetylation of Lys-75 is required for
transcriptional activity of IRF-2, because a mutation of Lys-75 substantially
diminished H4 promoter activity, coinciding with reduced levels of
IRF-2 acetylation in vivo (Fig.
2). K75R mutant seems to be acetylated at the Lys-78 site; data in
Fig. 1 shows that this residue
is acetylated both in vivo and in vitro. The K78R mutant no
longer binds to the ISRE, indicating that this residue is important for DNA
binding as well as acetylation. Based on the in vitro binding assays
and a report by Caillaud (41),
DNA binding activity may not depend on the acetylation of this residue. In the
absence of DNA binding activity by the K78R mutation, the significance of
acetylation could be studied only with the K75R mutant. We infer that
p300/CBP, rather than PCAF, is the main acetylase for IRF-2 in NIH 3T3 cells,
because p300 is expressed at a high level in NIH 3T3 cells, whereas PCAF
expression is very low (not shown). Because Lys-75 is acetylatable by both
p300 and PCAF in vitro, PCAF may acetylate IRF-2 at this residue in
other cell types. In agreement with the role of p300/CBP, E1A that competes
with cellular factors for p300/CBP binding inhibited H4 promoter
activity. H4 promoter activity was not inhibited by E1AN,
which does not bind to p300/CBP. Correlating with the E1A inhibition of
promoter activity, IRF-2 acetylation in vivo was inhibited by E1A.
These data indicate that p300/CBP interacts with IRF-2 and acetylates Lys-75
in NIH 3T3 cells, and that this event, susceptible to disruption by E1A, is
critical for IRF-2 activation of the H4 promoter. Recently, Paulson
et al. (48)
demonstrated the ability of E1A to bind to GCN5 and inhibit its activity in
interferon-stimulated transcription system. We cannot exclude the involvement
of GCN5, which has histone acetyltransferase activity and binds to the
N-terminal region of E1A similar to p300, in an IRF-2-mediated transactivation
system. However, p300 acetylates IRF-2 much higher than that of PCAF in
vitro, and a considerable amount of p300 associates with IRF-2 in NIH 3T3
cells. These finding suggest that at least p300 acetylase is required for
IRF-2 transcriptional function in living cells, although acetylase such as
GCN5 other than p300/CBP has to be examined in our system in the future. In
line with these results, E1A has been reported to interfere with the
acetylation of DNA binding factors, and hence transcription in several
systems. Puri et al.
(49) showed that MyoD forms a
multimeric complex with PCAF and p300/CBP leading to the acetylation of MyoD,
which is critical for promotion of myogenic transcription. However, E1A
inhibits this process by disrupting MyoD acetylation and the formation of the
complex. In addition, Hung et al.
(50) showed that CBP
stimulates acetylation of GATA-1 in vivo in an E1A-sensitive manner,
which is linked to transcriptional activity of GATA-1. Although acetylation is
not required for GATA-1 to bind to DNA, the mutation that impairs acetylation
reduces the ability of GATA-1 to induce cellular differentiation
(50).
IRF-2 has a dual activity and can act as a repressor for many interferon-inducible genes (51). In this context, we previously reported that acetylation of IRF-2 reduces p300-mediated acetylation of core histones, suggesting that acetylated IRF-2 can negatively affect transcription in some cases (39). Whether acetylated IRF-2 can act as an activator or repressor may be dependent on specific promoters.
We have extended our study to address the role of acetylated IRF-2 in cell growth, and showed that IRF-2 is acetylated only in growing cells, but not growth-arrested cells. By DNA affinity binding, as well as a chromatin immunoprecipitation assay, we found that IRF-2 binds to the H4 promoter only in growing cells, and that H4-bound IRF-2 was acetylated. That IRF-2 was expressed in growth-arrested cells at levels comparable with rapidly growing cells, but was not acetylated or bound to the H4 promoter in growth-arrested cells, suggests that only acetylated IRF-2 binds to the H4 promoter and activates H4 promoter activity. Our data indicate that acetylated IRF-2 has an increased affinity for the H4 promoter in vivo, although acetylation did not alter the DNA binding activity of recombinant IRF-2 in electrophoretic mobility shift assays (39). Different from a recombinant protein-DNA complex, endogenous IRF-2 is likely to bind to the H4 promoter by associating with multiple factors. Acetylated IRF-2 could form a complex distinct from that of non-acetylated IRF-2, and bind to the promoter more efficiently than non-acetylated IRF-2 in vivo. Thus, the effect of acetylation may not have been observed with electrophoretic mobility shift assays where DNA binding is detected without complex formation. In this context, Barlev et al. (52) showed that acetylated p53 binds more tightly to the transcriptional cofactors TRRAP and CBP than nonacetylated p53, although acetylated and nonacetylated p53 bind to the p21 promoter in the same manner. Interestingly, Suhara et al. (40) reported that IRF-3 was acetylated when incorporated into the large holocomplex that includes p300, and that a p300 lacking histone acetylase activity failed to confer IRF-3 DNA binding activity, indicating that acetylation of IRF-3 is important for the DNA binding activity of the IRF-3 holocomplex. These results support the notion that acetylation influences the DNA binding activity of IRF family members, albeit under varying conditions.
Our results indicate that IRF-2 is acetylated by p300/CBP in a cell growth-regulated manner. The temporal control of IRF-2 acetylation and consequently transcriptional activity may provide a mechanistic basis for cell cycle-dependent transcription of the H4 gene (34). It has been shown that although p300/CBP is expressed throughout the cell cycle, levels of phosphorylation and coactivator function change during the cell cycle (53). Importantly, CBP is phosphorylated by cyclin E-cdk2 at the G1/S phase, increasing the histone acetylase activity during this phase of the cell cycle (54). p300/CBP is also phosphorylated by another kinase, causing a different functional outcome (55). Thus, acetylation of IRF-2 may be attributed to cell cycle-dependent phosphorylation/acetylase activity of p300/CBP. In addition, some nuclear cofactors may be preferentially recruited to acetylated IRF-2, rather than unacetylated IRF-2, resulting in more efficient transcription. IRF-2 may interact with other proteins, but not with p300 in confluent NIH 3T3 cells and it cannot bind to the H4 promoter efficiently. In contrast, in growing NIH 3T3 cells IRF-2 associates with p300 and is acetylated. IRF-2 binds to the H4 promoter by recruiting some factors that access cell growth. Adenovirus E1A inhibits acetylation of IRF-2 and IRF-2-induced H4 gene activation by dissociation of IRF-2 and p300. In growth-arrested cells, IRF-2 may act only for those promoters where it can bind in an unacetylated state.
In summary, we identified Lys-75 as a major site of IRF-2 acetylation. The acetylation of this residue is required for full activation of histone H4 promoter. Furthermore, being acetylated in a cell growth-dependent manner by p300, IRF-2 regulates cell growth-dependent transcription of the H4 gene.
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FOOTNOTES |
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To whom correspondence should be addressed: Safety Research on Biologics,
National Institute of Infectious Diseases, 4-7-1, Gakuen, Musashimurayama-shi,
Tokyo, Japan. Tel.: 81-425-61-0771; Fax: 81-425-65-3315; E-mail:
amasumi{at}nih.go.jp.
1 The abbreviations used are: PCAF, p300/CBP-associated factor; IRF,
interferon regulatory factor; DBD, DNA binding domain; GST, glutathione
S-transferase; CBP, CREB-binding protein; CREB, cAMP-response
element-binding protein; PBS, phosphate-buffered saline.
2 A. Masumi, unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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