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INTRODUCTION |
Control of immune responses and inflammatory reaction is
mediated by intercellular communication through direct cell-to-cell interactions and soluble factors such as cytokines. Cytokine-mediated intercellular communication is often orchestrated through cross-talk between different classes of cytokines and extracellular stimuli. Interferon-
(IFN-
)1
promotes the development of cell-mediated immunity and functions cooperatively with other extracellular stimulus such as tumor necrosis factor
(TNF
) or lipopolysaccharide to induce the
expression of a number of proinflammatory genes including major
histocompatibility complex class I (1), inducible nitric-oxide synthase
(2, 3), intercellular adhesion molecule 1 (4), and interferon-inducible chemokine CXC ligand 10 (CXCL10)/IFN-inducible protein of 10 kDa (5).
Cytokine-mediated transcriptional activations of inflammatory genes has
been studied extensively. NF-
B plays critical roles in
transcriptional regulation of numerous genes involved in host-defense mechanisms (6). Prototypically, the NF-
B1 (p50)/RelA (p65) heterodimer is sequestered in the cytoplasm by inhibitor protein I
B.
Upon stimulation with extracellular signals such as proinflammatory cytokines or bacterial or viral components, I
B is phosphorylated by
I
B kinases, ubiquitinated, and degraded by 26 S proteasomes. After
degradation of I
B, NF-
B is translocated to the nucleus and binds
to
B elements found in many inflammatory genes (7). Signal
transducers and activators of transcription (STATs) are latent
cytoplasmic transcription factors that are phosphorylated at a single
tyrosine residue via members of the Jak kinase family following
stimulation with cytokines, hormones, or growth factors; are assembled
in dimeric form; are translocated to the nucleus; and become bound to
specific DNA sequence motifs (8-10). IFN-
activates the STAT1
homodimer that binds to IFN-
activation sequences (11) found in the
promoter region of a number of IFN-
-inducible genes including
interferon regulatory factor 1 (IRF-1) (12) and chemokine CXCL9, which
is a monokine induced by IFN-
(MIG) (13, 14). The NF-
B-
and STAT-dependent signaling pathways are integral to the
transcriptional regulation of many inflammatory genes, and these
transcriptional factors often cooperatively regulate the
transcriptional activation of many genes (4, 5, 15, 16). Previous
studies have demonstrated that IFN-
-induced STAT1
and
TNF
-induced NF-
B synergistically regulate the transcription of
the intercellular adhesion molecule-1 and IRF-1 genes (4, 15, 16),
although the molecular mechanisms involved in the STAT1
/NF-
B-mediated transcriptional cooperation remain to be elucidated.
Transcriptional coactivator CREB-binding protein (CBP) and closely
related p300 play a critical role in various aspects of transcriptional
regulation (17-19). One of the major functions of coactivator CBP/p300
is to function as a bridging factor between sequence-specific
transcriptional activator and basal transcriptional machinery and to
assemble them to form a stable multiprotein complex. CBP/p300 also
possesses an intrinsic histone acetyltransferase (HAT) activity, which
modifies the histone tail to destabilize the chromatin structure and
thus increase the accessibility of the basal transcriptional machinery
to the DNA template (20-22). Furthermore, HAT alters the activities of
a number of nonhistone transcription factors such as p53 by acetylating
them and thereby stimulates their DNA binding and transcriptional
activities (23).
Because CBP has been shown to function as a coactivator for STAT1 and
NF-
B (24-28), we hypothesized that CBP might play an essential role
in the transcriptional synergy between STAT1 and NF-
B in
inflammatory gene expression. In this study we explored the mechanisms
through which CBP integrates the cross-talk between IFN-
/STAT1 and
TNF
/NF-
B signaling pathways to cooperatively induce the
transcriptional activation of the gene for CXCL9, an IFN-
-inducible
chemokine (13, 29). The results presented in this study demonstrate
that CBP mediates the transcriptional synergy between IFN-
/STAT1 and
TNF
/NF-
B in the CXCL9 gene. Whereas the HAT activity of CBP was
dispensable for the synergy, the N-terminal 450 residues and C-terminal
region between amino acids 1779 and 2027 were required to mediate the
transcriptional synergy. Consistent with this and extending prior
studies, STAT1 and NF-
B were shown to simultaneously interact with
the N- and C-terminal regions of CBP. Furthermore, the results of a
chromatin immunoprecipitation (ChIP) assay demonstrated that IFN-
and TNF
cooperatively recruited STAT1 and CBP-RNA polymerase II to
the promoter region of the CXCL9 gene. These results indicate that CBP
mediates the IFN-
/STAT1 and TNF
/NF-
B-induced transcriptional synergy by recruiting the RNA polymerase II complex to the CXCL9 promoter via simultaneous interaction with STAT1 and NF-
B.
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EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Recombinant IFN-
and TNF
were
obtained from R & D Systems (Minneapolis, MN). Antibodies against
STAT1, CBP, NF-
B p50, RelA (p65), and RNA polymerase II were
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and
anti-acetylated histone H3 and H4 were from Upstate Biotechnology, Inc.
(Lake Placid, NY). Anti-V5 epitope tag antibody came from Invitrogen.
Goat anti-mouse IgG labeled with Alexa (488 nm) and SYBR Gold staining
reagent were purchased from Molecular Probes, Inc. (Eugene, OR). Other
reagents used in this study were described previously (15).
Reporter Plasmid and Expression Constructs--
The luciferase
reporter construct containing the mouse CXCL9/MIG gene promoter (
328
bp) (14, 30) and a mutant construct of the 5'-half site of the
RE
motif in the CXCL9 promoter were described previously (31). The 3'-half
site of the
RE motif and the
B1,
B2, and
B3 sites were
mutated by site-directed mutagenesis with a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA). The mutant sequences (sense
strand) utilized were the following: 3'-
RE,
CTCCCCGTTTgTGTctAATGGAAGTAGAAC;
B1,
GGGAAGGAAAAGcGATTTggTAAATAAATATGATCC;
B2,
CTGAGAGTAGccTTTTCgCCAGGACGATC;
B3,
GTAGAACATGCAcAAATTCgCTGGGATCTG. Lowercase letters
represent the mutant nucleotides, and the underlined sequences are the
consensus sequences for
RE and
B motifs. pCMV-CBP expression
plasmid was kindly provided by Dr. C. K. Glass (University of
California, San Diego) (32). Deletion mutants of CBP were generated as
a PCR fragment by PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA) and subcloned into pcDNA3 (Invitrogen). The deletion construct CBP 775-1779NLS contained the nuclear localization signal from SV40 T antigen at the C-terminal region of the truncated protein.
Mutation in the HAT domain of CBP was generated by introducing 2 amino
acid substitutions at amino acid residues 1690 (Leu
Lys) and 1691 (Cys
Leu) by using the QuikChange site-directed mutagenesis kit.
This mutation was previously shown to abolish the HAT activity of CBP
(33) and has been used in different systems (34, 35). pRC-STAT1 was
kindly provided by Dr. G. Stark (Cleveland Clinic Foundation).
pCMV-RelA was described previously (36).
Cell Culture and Transient Transfection--
Mouse NIH3T3 cells
and human embryonic kidney 293 (HEK293) cells were cultured in
Dulbecco's modified Eagle's medium containing L-glutamine, penicillin/streptomycin, and 10% fetal bovine
serum as described previously (5, 37). Cells were transiently
transfected with luciferase reporter plasmids, pRL-TK reference
Renilla luciferase plasmid (Promega, Madison, WI), and
expression plasmids by using Polyfect transfection reagents (Qiagen,
Valencia, CA) according to the manufacturer's instructions. For
standardization of the transfection efficiencies for the luciferase
reporter assay, the transfected cells were harvested, pooled, and
seeded in 24-well culture plates. After 24 h, the cells were
treated with IFN-
and/or TNF
for 8 h. Firefly and
Renilla luciferase activities were assayed by using reagents
provided by Promega according to the manufacturer's instructions.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared as described previously (5) by use of a
modification of the method of Dignam et al. (38). The
following oligonucleotides were used in the EMSA:
RE,
5'-GATCCCTTACTATAAACTCCCCGTTTATGTGAAATGGA-3';
B1,
5'-tcgaAAAAGGGATTTCCTAAAT-3';
B2, 5'-tcgaAGTAGGGTTTTCCCCAGGA-3';
B3, 5'-tcgaATGCAGAAATTCCCTGGG-3'.
Binding reactions and antibody supershift assays were described
previously (5, 15).
Immunoprecipitation and Western Blot Analysis--
Cells were
washed with ice-cold phosphate-buffered saline, harvested, resuspended
in lysis buffer (50 mM Hepes (pH 7.9), 150 mM
NaCl, 1 mM EDTA, 2.5 mM, 0.1% Nonidet P-40,
10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin, antipain, aprotinin, and pepstatin), and
kept on ice for 10 min. After preclearing of the lysate, the whole cell
lysate (~500 µg of protein) was incubated with anti-V5 (1 µg)
antibody or normal mouse IgG and protein G-Sepharose (50% slurry) for
16 h at 4 °C. The immunoprecipitates were washed four times
with 1 ml of lysis buffer, eluted with SDS-PAGE sample buffer, resolved
on 7.5% SDS-PAGE, and analyzed by Western blotting.
Immunocytochemistry--
Cells grown on Lab-tek chamber slides
(Nunc, Rochester, NY) were fixed at room temperature in 4%
paraformaldehyde and 0.5% Triton X-100. They were then reacted with
mouse anti-V5 antibody at room temperature for 1 h, and unbound
antibody was subsequently removed by washing with phosphate-buffered
saline. Bound antibody was detected with goat anti-mouse IgG conjugated
with Alexa (488 nm) (Molecular Probes). Immunofluorescence was detected
by confocal laser-scanning microscopy (LSM 510; Carl Zeiss, Goettingen, Germany).
ChIP--
ChIP was performed as described previously (39, 40)
with some modification. Briefly, confluent monolayers of NIH3T3 cells were fixed with formaldehyde (1% v/v) overnight at 4 °C. Following cross-linking, the cells were resuspended in ChIP lysis buffer (5 mM PIPES (pH 8.0), 85 mM KCl, 0.5% Nonidet
P-40, and 10 µg/ml of proteinase inhibitors) and sonicated with a
Bioruptor sonication apparatus (Toso Electronics, Tokyo). Soluble
chromatin was collected by centrifugation, precleared with Protein
G-agarose, and immunoprecipitated with the desired antibodies overnight
at 4 °C. The immunoprecipitates were sequentially washed once with
sonication buffer (50 mM Hepes (pH 7.9), 140 mM
NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.1%
sodium deoxycholate), twice with high salt buffer (50 mM Hepes (pH 7.9), 500 mM NaCl, 1 mM EDTA, 1%
Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate), twice with low
salt buffer (20 mM Tris-Cl (pH 8.0), 250 mM
LiCl, 0.5% Nonidet P-40, and 0.1% sodium deoxycholate), and twice
with TE buffer before elution with elution buffer (50 mM
Tris-Cl (pH 8.0), 1 mM EDTA, and 1% SDS). The eluted
samples were reverse cross-linked at 65 °C for 5 h and treated
with RNase A and proteinase K for 1 h. The recovered DNA was
purified with a DNA clean up kit (Qiagen), and samples of input DNA
were also prepared in the same way. The purified DNA was subjected to
PCR with a set primer and analyzed on a 2% agarose gel with SYBR Gold
(Molecular Probes) staining. The stained bands were analyzed by using a
Molecular Imager (Bio-Rad). Primers for the promoter region of the
CXCL9 gene were TTCCACATCCAGGTAGCAACTTTG (5' primer) and
TGTTGGAGTGAAGTCCGAGAATGT (3' primer).
Preparation of RNA and Northern Hybridization
Analysis--
Total cellular RNA was extracted by the guanidine
isothiocyanate-cesium chloride method (41). Northern hybridization
analysis and cDNA probes for CXCL9/monokine induced by IFN-
and
glyceraldehyde-3-phosphate dehydrogenase were described previously (31,
42).
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RESULTS |
CBP Potentiates IFN-
- and TNF
-induced CXCL9 Promoter
Activity--
Previous studies demonstrated that IFN-
-induced STAT1
and TNF
-induced NF-
B acted synergistically in the transcription
of many inflammatory genes (4, 5, 15, 16). Since CBP has been shown to
function as a transcription coactivator for various transcription
factors including STAT1 and NF-
B (17-19, 24-28), we wished to
examine whether CBP mediated this STAT1- and
NF-
B-dependent transcriptional synergy. For this
purpose, we analyzed the transcriptional regulation of the CXCL9/MIG
gene, an IFN-
-inducible chemokine for activated T cells (13, 14, 29)
known to be cooperatively regulated by IFN-
and TNF
, although the
mechanisms involved in this cooperation remain to be determined. HEK293
cells were cotransfected with a luciferase reporter construct
containing 328 bp of a 5'-flanking sequence of the CXCL9 gene and an
expression vector encoding CBP or empty vector. After transfection, the
cells were stimulated with IFN-
and/or TNF
or left untreated
before analysis of the luciferase reporter gene activity. As shown in Fig. 1, although IFN-
and TNF
alone
had only a minimum effect on the CXCL9 promoter activity in the absence
of CBP, costimulation with IFN-
and TNF
synergistically induced
the promoter activity. When the cells were cotransfected with the
expression vector encoding CBP, the cooperative response to IFN-
and
TNF
was further potentiated. This result indicates that CBP
functioned to mediate the synergy between IFN-
and TNF
for the
transcription of the CXCL9 gene.

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Fig. 1.
Coactivator CBP potentiates
IFN- and TNF -induced
cooperative promoter activity of the CXCL9 gene. A,
schematic representation of a luciferase reporter construct containing
the 5'-flanking sequence of the CXCL9/MIG gene (14, 30, 31). The
numbers above the promoter region refer to the
nucleotide position relative to the transcription start site.
B, HEK293 cells were transiently co-transfected with either
empty vector or the CBP expression plasmid (2 µg) and the CXCL9
luciferase reporter construct (Mig-328, 1 µg). 24 h after
transfection, the cells were either left untreated or treated with
IFN- and/or TNF (10 ng/ml each) for 8 h prior to measurement
of luciferase activity. The relative luciferase activity is shown as
-fold induction compared with the activity of unstimulated samples.
Each column and bar represents the mean ± S.E. of three independent experiments.
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RE and
B2 Sites Are Required for IFN-
- and TNF
-induced
Transcriptional Synergy--
In previous studies, we and others showed
that both a STAT1 binding element and NF-
B binding site were
required for the IFN-
- and TNF
-induced transcriptional synergy
(4, 5, 15, 16). There are several potential regulatory elements in the
promoter region of the CXCL9 gene (Fig.
2A). Although
RE has been
identified as an IFN-
-responsive site that is recognized by the
STAT1 tetramer (14, 31), the functional significance of several
putative
B sites within
200 bp of the promoter has not been
analyzed. To determine the regulatory elements responsible for the
CBP-mediated transcriptional synergy, we mutated the
RE and these
putative
B sites and analyzed the mutant ones for their possible
role in IFN-
- and TNF
-induced promoter activity. As shown in Fig. 2B, mutation of 5'-
RE or 3'-
RE abolished the
cooperative response to IFN-
and TNF
without affecting the
TNF
-induced luciferase activity (lanes 3 and
4). Whereas mutation of the
B1 site had little effect on
the promoter activity (lane 5), mutation of the
B3 one diminished the cooperative response to IFN-
and TNF
(lane 7). Furthermore, mutation of the
B2 site
abolished the response to TNF
and markedly reduced the cooperativity
for IFN-
and TNF
(lane 6). Mutation of both
the 3'-
RE and
B2 sites almost completely eliminated the
sensitivity to both stimuli (lane 9). The
requirement of the
RE and
B2 sites for mediating the IFN-
- and
TNF
- induced transcriptional synergy was also observed in NIH3T3
cells (Fig. 2C). Taken together, these results indicate that
both the
RE and
B2 sites are required to mediate the
transcriptional synergy of the CXCL9 gene.

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Fig. 2.
The RE and
B sites in the promoter region of the CXCL9 gene
are required for IFN- - and
TNF -induced transcriptional synergy.
A, schematic representation of wild-type and mutant Mig-328
luciferase reporter constructs. B, HEK293 cells were
transiently co-transfected with either empty vector or the CBP
expression plasmid (2 µg) and the indicated wild-type or mutant
Mig-328 luciferase reporter construct (1 µg). 24 h after
transfection, the cells were either left untreated or treated with
IFN- and/or TNF (10 ng/ml each) for 8 h prior to measurement
of luciferase activity. The relative luciferase activity is shown as
-fold induction compared with the activity of the unstimulated samples.
Each column and bar represents the mean ± S.E. of three independent experiments. C, individual NIH3T3
cell cultures were transiently transfected with the indicated wild-type
or one of the mutant Mig-328 luciferase reporter constructs (3 µg).
Luciferase activity was measured as described above. Each
column and bar represents the mean ± S.E.
of three independent experiments. D, the nucleotide
sequences of the RE and B sites are illustrated.
Underlined sequences represent the RE and B
motifs.
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RE and
B DNA Binding Activities in Nuclear Extracts from
IFN-
- and TNF
-stimulated Cells--
An EMSA study was carried
out to examine the
RE and
B DNA binding activities in nuclear
extracts from IFN-
- and/or TNF
-stimulated cells. As seen in Fig.
3A, IFN-
induced formation
of complex I and modestly increased the binding activity of complex II
(lane 2), but co-stimulation with IFN-
and
TNF
did not enhance these DNA binding activities (lane
4). The antibody supershift assay demonstrated that
complexes I and II contained STAT1 (Fig. 3B, lane 8), which correspond to the previously
identified
RF-1 and
RF-2, respectively (14, 31). Antibody to
NF-
B1 (p50) and RelA (p65) also reduced the binding activity of
complexes II and III (lanes 9-11), indicating
that complexes II and III contained NF-
B1 (p50)/RelA (p65) and
NF-
B1 (p50) homodimer, respectively. In this regard, NF-
B was
previously reported to bind to the
RE of the CXCL9 gene as well as
to the IFN-
activation sequence of the IRF-1 gene (15, 16, 31). In
our EMSA study using the
B motifs from the promoter region of the
CXCL9 gene as probes, a marked DNA binding activity was observed at the
B2 motif (Fig. 3C, lanes 5-8),
consistent with the result of the promoter analysis showing that
mutation of the
B2 site significantly reduced the promoter activity
(Fig. 2). When the cells were costimulated with IFN-
and TNF
, the
binding activity toward these
B motifs was unchanged (Fig.
3C, lanes 4, 8, and
12). The antibody supershift assay showed that NF-
B1
(p50)/RelA (p65) and NF-
B1 (p50) homodimer bound to the
B2 motif
(Fig. 3D). Thus, these results indicate that STAT1 and
NF-
B had the ability to bind to these sites independently.

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Fig. 3.
RE and
B DNA binding activities in nuclear extracts from
IFN- - and
TNF -stimulated cells. NIH3T3 were either
left untreated or treated with IFN- and TNF (10 ng/ml each) for
30 min before the preparation of nuclear extracts. 10 µg of each
nuclear extract was analyzed for RE (A and B)
or B (C and D) binding activity by EMSA using
radiolabeled oligonucleotides as described under "Experimental
Procedures." In some experiments (B and D),
nuclear extracts were incubated with the indicated antibodies (1 µg)
before analysis of the binding activities. Supershifted complexes
(s.s.) and nonspecific binding (ns) are
indicated. Similar results were obtained from three separate
experiments.
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STAT1 and RelA Simultaneously Interact with the N- and C-terminal
Regions of CBP in Vivo--
Because STAT1 and NF-
B have been shown
to interact with CBP independently (24-28), synergistic
transcriptional activity of the CXCL9 promoter by IFN-
/STAT1 and
TNF
/NF-
B could result from the simultaneous physical interaction
of CBP with STAT1 and NF-
B. To determine whether CBP simultaneously
associates with STAT1 and NF-
B in vivo, we performed
coimmunoprecipitation experiments (Fig.
4). HEK293 cells were co-transfected with
an expression plasmid encoding the N-terminal (amino acids 1-777) or
the C-terminal region of CBP (amino acids 1758-2441) together with
STAT1 and RelA expression vectors. These transfected mutants resided in the nucleus, as shown by immunostaining (Fig. 4B). After
stimulation with IFN-
and/or TNF
, whole cell lysates were
prepared, immunoprecipitated with antibody against the V5 epitope tag,
and assessed by Western blotting with antibody against STAT1. After
detection of STAT1, the blots were stripped and reprobed with antibody
against RelA. As shown in Fig. 4C (lanes
5-8), whereas both STAT1 and RelA were constitutively detected in lysates immunoprecipitated with the N-terminal region of CBP 1-777, the association of CBP with RelA was
enhanced by TNF
stimulation (lanes 7 and
8). Immunoprecipitates of lysates from the C-terminal region
(CBP 1758-2441)-transfected cells also constitutively contained STAT1
and RelA (p65) (lane 13), although the
interaction with STAT1 was enhanced by IFN-
stimulation
(lanes 14 and 16). We also generated a
deletion mutant CBP 775-1779NLS lacking both the N-terminal and
C-terminal regions of CBP (Fig. 4A). This construct
contained a nuclear localization signal from the SV40 T antigen, since
the original construct did not translocate to the nucleus. Although the
CBP 775-1779NLS resided in both the cytoplasm and the nucleus (Fig.
4B), no associations with STAT1 and RelA were observed under
unstimulated or stimulated conditions (Fig. 4E), indicating
that the N-terminal and C-terminal regions of CBP specifically
interacted with STAT1 and RelA. Thus, these results indicate that CBP
is capable of interacting with STAT1 and NF-
B simultaneously.
Furthermore, the nature of the interaction between CBP and STAT1 or
NF-
B appeared to be distinct; i.e. IFN-
-activated
STAT1 preferentially interacted with the C-terminal region of CBP, and
TNF
-stimulated NF-
B RelA interacted with the N-terminal region of
CBP.

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Fig. 4.
STAT1 and RelA simultaneously interact with
the N- and C-terminal regions of CBP in vivo.
A, the diagram shows the structure of wild-type CBP
including its functional domains and those structures of N-terminal and
C-terminal truncated mutant constructs. Proteins known to interact with
CBP are indicated at the top of the diagram. Numbers denote
amino acid positions. RID, receptor-interacting domain;
CH, cysteine-histidine-rich region; KIX,
kinase-induced interaction domain; Bromo,
bromodomain; IBiD, IRF-3 binding domain. CBP 775-1779
contained an NLS from SV40 in front of the V5 epitope tag.
B, immunofluorescence microscopy of HEK293 cells transfected
with CBP mutants. HEK293 cells transfected with V5 epitope-tagged
deletion mutants were fixed and labeled with anti-V5 antibody.
Immunofluorescence staining was detected by confocal laser-scanning
microscopy. C and E, HEK293 cells were
transiently co-transfected with V5-epitope-tagged expression plasmid
together with STAT1 and RelA expression vectors. 24 h after
transfection, the cells were either left untreated or treated with
IFN- and/or TNF (10 ng/ml each) for 1 h. Whole cell extracts
were prepared, immunoprecipitated (IP) with anti-V5 antibody
or normal mouse IgG as indicated, and analyzed by Western blotting
using anti-STAT1 antibody. The blots were then stripped and analyzed
with anti-RelA and again stripped and treated with anti-V5 antibodies.
D, whole cell extracts prepared as described above were
assessed for STAT1 and RelA by Western blotting using ant-STAT1 and
anti-RelA antibodies.
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|
The N-terminal and C-terminal Regions of CBP Are Required to
Mediate the STAT1 and NF-
B-dependent Transcriptional
Synergy--
To determine the functional significance of these
interactions for mediating the transcriptional synergy, we assessed the N- and C-terminal deletion mutants of CBP for their transactivating function with respect to the CXCL9 promoter (Fig.
5). Although progressive C-terminal
deletions to amino acid residue 2027 had little effect on the
IFN-
/STAT1 and TNF
/NF-
B-mediated transcriptional synergy,
deletion to amino acid 1779 abolished the synergistic transcription,
indicating that the region between residues 2027 and 1779 was required
to mediate the transcriptional synergy. This region contains
cysteine-histidine-rich domain 3, which is known to interact with STAT1
as well as with RNA helicase A and RNA polymerase II (24, 25, 43).
Transfection with the N-terminal deletion mutant (CBP 450-2441), which
retains the CREB-binding domain KIX, also reduced the IFN-
/STAT1 and
TNF
/NF-
B-mediated transcriptional synergy. These results
demonstrate that both the N-terminal 450 amino acids and
cysteine-histidine-rich domain 3 of CBP are required to mediate the
IFN-
/STAT1 and TNF
/NF-
B-induced transcriptional synergy.

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Fig. 5.
The N-terminal and C-terminal regions of CBP
are required for the STAT1 and
NF- B-dependent-transcriptional
synergy. A, the diagram shows wild-type CBP and
N-terminal and C-terminal deletion mutants. Proteins known to interact
with CBP are indicated, as defined in the legend to Fig. 4.
B, HEK293 cells were transiently co-transfected with either
empty vector or the wild-type or the mutant CBP expression plasmid (2 µg) and Mig-328 luciferase reporter construct (1 µg). 24 h
after transfection, the cells were either left untreated or treated
with IFN- and/or TNF (10 ng/ml each) for 8 h prior to
measurement of luciferase activity. The relative luciferase activity is
shown as -fold induction compared with the activity of unstimulated
samples. Each column and bar represents the mean ± S.E. of three
independent experiments.
|
|
Previous studies have shown that STAT1 and
RelA/p65-dependent transcription require coactivator NcoA-3
(p/CIP) and NcoA-1 (SRC-1), respectively, which interact with the
region between residues 2058 and 2163 of CBP (44, 45), and this region
has been recently identified as the IRF-3 binding domain (IbiD) (46). Interestingly, whereas the deletion mutant CBP 1-2027, which lacks the
IBiD, was capable of mediating the synergistic response, the TNF
-induced promoter activity was significantly reduced. This result
is consistent with the previous finding that
RelA/p65-dependent transcription requires the NcoA-1
(SRC-1) interacting domain of CBP (45). Together, these results suggest
that although IBiD is required for individual RelA/p65- or
STAT1-dependent transcription, the IFN-
/STAT1- and
TNF
/NF-
B-mediated transcriptional synergy does not require this region.
The IFN-
/STAT1 and TNF
/NF-
B-induced
Transcriptional Synergy Does Not Require the HAT Activity of
CBP--
CBP possesses an intrinsic HAT domain that regulates the
transcriptional activities of various transcription factors (20-22). In order to determine whether the HAT activity of CBP was required for
the IFN-
/STAT1 and TNF
/NF-
B-induced transcriptional synergy, we tested an expression construct containing a mutant HAT domain for
its transactivating function. The mutation of the HAT domain used here
was previously demonstrated to abolish the HAT activity (33). As shown
in Fig. 6, the mutant construct (CBP
mHAT) was able to enhance the promoter activity in response to IFN-
and TNF
, indicating that the HAT activity of CBP is dispensable for the IFN-
/STAT1- and TNF
/NF-
B-induced transcriptional synergy of the CXCL9 promoter.

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Fig. 6.
CBP HAT activity is dispensable for the
IFN- /STAT1- and
TNF /NF- B-induced
transcriptional synergy. HEK293 cells were transiently
co-transfected with either empty vector or either of the CBP expression
plasmids (2 µg) as indicated and the Mig-328 luciferase reporter
construct (1 µg). 24 h after transfection, the cells were either
left untreated or treated with IFN- and/or TNF (10 ng/ml each)
for 8 h prior to measurement of luciferase activity. The relative
luciferase activity is shown as -fold induction compared with the
activity of unstimulated samples. Each column and
bar represents the mean ± S.E. of three independent
experiments.
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|
Costimulation with IFN-
and TNF
Does Not Induce Histone
Hyperacetylation at the CXCL9 Promoter--
It was earlier
demonstrated that highly acetylated histone correlates with
transcriptionally active chromatin, which facilitates recruitment of
the basal transcriptional machinery (22). Although the intrinsic HAT
activity of CBP is dispensable for the IFN-
/STAT1- and
TNF
/NF-
B-induced transcriptional synergy, CBP interacts with
other coactivators such as p/CAF, NcoA1 (SRC-1), and NcoA3 (p/CIP/ACTR), which also possess HAT activity (47-49). To determine whether histone hyperacetylation could be one of the mechanisms for the
transcriptional synergy, we assessed the acetylation status of the
promoter region of the CXCL9 gene in NIH3T3 cells by using a ChIP assay
(Fig. 7). Initially, we monitored the
acetylation status of the CXCL9 promoter in the presence of
trichostatin A (TSA), a histone deacetylase inhibitor (50). Whereas a
relatively low basal level of histone H4 acetylation was observed at
the promoter in untreated cells (Fig. 7B, lane
6), treatment of cells with TSA led to a
time-dependent increase in the amount of acetylated histone
H4 (lanes 7-10), indicating that the promoter
region of the CXCL9 gene is deacetylated by TSA-sensitive histone
deacetylase with the cells in the quiescent state. To determine whether
costimulation with IFN-
and TNF
led to hyperacetylation at the
CXCL9 promoter, we performed a ChIP assay with antibodies against
anti-acetylated histones H3 and H4 (Fig. 7C). Treatment of
cells with IFN-
or TNF
for 4 h induced a significant
increase in acetylated histone H3 (Fig. 7C, lanes
7 and 8). However, there was no further increase in the level of acetylated histone in IFN-
- and TNF
-treated cells
(lane 9) despite the fact that a marked synergism
between IFN-
and TNF
was observed in the expression of the
endogenous CXCL9 gene (Fig. 7D, lane
4). Interestingly, whereas the level of acetylated histone
H4 was also enhanced in IFN-
-stimulated cells (Fig. 7C,
lane 7), TNF
only modestly stimulated the
acetylation (lane 8), and no cooperative effect
on the histone acetylation was observed in IFN-
- and TNF
-treated
cells (lane 9). Thus, these results indicate that
IFN-
/STAT1 and TNF
/NF-
B-induced transcriptional synergy with
respect to the CXCL9 gene does not correlate with histone
hyperacetylation at the promoter.

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Fig. 7.
Histone acetylation at the promoter region of
the CXCL9 gene. A, schematic representation of the
promoter region of the CXCL9 gene. The region amplified by the primer
pairs used in the PCR step of the ChIP assay is illustrated. The
numbers refer to the nucleotide position relative to the
transcription start site. B, time-dependent
increase in the levels of histone H4 acetylation at the CXCL9 promoter
region by TSA treatment. NIH3T3 cells were treated with TSA (100 nM) for the indicated periods of time. Cross-linked
chromatin fragments were prepared and immunoprecipitated with
anti-acetylated histone H4. The recovered DNA was amplified by PCR with
the specific primers for the promoter region of the CXCL9 gene. The
amplified products were analyzed on a 2% agarose gel. DNA isolated
from sonicated cross-linked chromatin fragments were used as inputs.
C, costimulation with IFN- and TNF does not
cooperatively induce histone hyperacetylation at the CXCL9 promoter.
NIH3T3 cells were treated with IFN- and/or TNF (10 ng/ml each) or
TSA (100 nM) for 4 h, and cross-linked chromatin was
then prepared. Soluble chromatin was immunoprecipitated with
anti-acetylated histone H3 or H4. The recovered DNA was amplified and
analyzed as described above. D, Northern blots analysis of
the CXCL9 mRNA expression. NIH3T3 cells were treated with IFN-
and/or TNF (10 ng/ml each) for 4 h as described above prior to
preparation of total RNA and analysis of specific mRNA levels by
Northern hybridization.
|
|
Costimulation with IFN-
and TNF
Cooperatively Recruits STAT1,
CBP, and RNA Polymerase II to the CXCL9 Promoter--
Next, by using
the ChIP assay, we examined whether costimulation with IFN-
and
TNF
could induce a cooperative binding of STAT1 and/or NF-
B to
the CXCL9 promoter (Fig. 8A).
Although IFN-
alone modestly recruited STAT1 to the promoter
(lane 6), costimulation with IFN-
and TNF
led to an increase in occupancy of STAT1 (lane 8). We were, however, unable to detect significant occupancy
of NF-
B at the promoter region. It is possible that antigen
determinants of RelA might be masked by the multiple protein complex
bound to the promoter.

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Fig. 8.
Enhanced recruitment of STAT1, CBP, and RNA
polymerase II to the CXCL9 promoter region by costimulation with
IFN- and TNF . NIH3T3 cells
were treated with IFN- and/or TNF (10 ng/ml each) for 4 h,
and cross-linked chromatin was then prepared. Soluble chromatin was
immunoprecipitated with anti-STAT1 (A) or anti-CBP or
anti-RNA polymerase II (B) antibody. The recovered DNA was
amplified by PCR and analyzed on a 2% agarose gel as described in the
legend to Fig. 7.
|
|
Because STAT1 interacts with CBP, the cooperative binding of STAT1 may
lead to an increased recruitment of CBP at the promoter. Furthermore,
since cysteine-histidine-rich domain 3 (1805-1890 amino acids) of CBP
was required for the transcriptional synergy (Fig. 5) and this domain
has been demonstrated to interact with RNA polymerase II (RNA Pol II)
via RNA helicase A (43), it is conceivable that CBP recruits RNA Pol II
to the CXCL9 promoter in response to IFN-
/STAT1 and TNF
/NF-
B.
To test these possibilities, we next assessed the recruitment of CBP
and RNA Pol II to the CXCL9 promoter by using the ChIP assay. As shown
in Fig. 8B, whereas IFN-
and TNF
alone had only
minimum effect on the recruitment of CBP (lanes 6 and 7), costimulation with IFN-
and TNF
cooperatively recruited CBP to the promoter (lane 8).
Consistent with this, a marked increase in the recruitment of RNA Pol
II to it was observed in IFN-
- and TNF
-treated cells. Thus, taken
together, these results indicate that the IFN-
/STAT1- and
TNF
/NF-
B-induced transcriptional synergy is, at least partially,
mediated by recruiting RNA Pol II to the promoter region of the CXCL9
gene via CBP.
 |
DISCUSSION |
Transcription of the genes that contain STAT1-binding elements and
NF-
B-binding sites in their promoter regions are often cooperatively
regulated by extracellular stimuli that induce STAT1 and NF-
B, such
as IFN-
and TNF
or bacterial lipopolysaccharide (2-5, 15, 16).
We and others previously reported that IFN-
-induced STAT1
and
TNF
-induced NF-
B synergistically regulated the transcription of
many inflammatory genes (4, 5, 15, 16). Although independent
interaction of STAT1 and NF-
B with their cognate binding sites was
shown to be sufficient for mediating the transcriptional synergy (15),
the molecular mechanisms involved in the STAT1- and NF-
B-mediated
transcriptional synergy remained to be elucidated. In the present
study, we evaluated the potential role of coactivator CBP in the
control of the transcriptional synergy between IFN-
/STAT1 and
TNF
/NF-
B. Our results demonstrate that simultaneous interactions of CBP with IFN-
-induced STAT1 and TNF
-activated NF
B RelA
(p65) were required to mediate the transcriptional synergy.
Furthermore, the IFN-
/STAT1- and TNF
/NF-
B-induced
transcriptional synergy appears to be mediated by increased recruitment
of RNA polymerase II to the promoter region of the CXCL9 gene via CBP.
These conclusions are based on the following observations. 1)
Overexpression of CBP potentiated IFN-
/STAT1- and
TNF
/NF-
B-induced cooperative transcriptional activation of the
CXCL9 gene. 2) The CBP-mediated synergistic transcriptional activity of
the CXCL9 promoter was abolished by mutation of the
RE and the
B
sites. 3) The N-terminal 450 residues and the C-terminal region between
amino acids 2027 and 1779 were required for the CBP-mediated
transcriptional synergy. 4) IFN-
-induced STAT1 and TNF
-activated
NF-
B RelA (p65) simultaneously interacted with distinct regions of
CBP. 5) Costimulation with IFN-
and TNF
cooperatively recruited
STAT1, CBP, and RNA Pol II to the promoter region of the CXCL9 gene.
One of the mechanisms involved in the IFN-
/STAT1 and
TNF
/NF-
B-induced transcriptional synergy appears to be the
enhanced recruitment of RNA Pol II to the promoter region of the CXCL9 gene. Since CBP has been shown to associate with RNA Pol II via RNA
helicase A (43), the simultaneous interaction of CBP with STAT1 and
NF-
B might stabilize the binding of CBP to the promoter, and the
stabilized CBP could provide a stable scaffold for the RNA polymerase
II complex. Indeed, our data from the ChIP assay (Fig. 8) demonstrated
that co-treatment with IFN-
and TNF
led to an increase in the
recruitment of CBP and RNA Pol II to the CXCL9 promoter. The data from
the ChIP assay also demonstrated that the costimulation with IFN-
and TNF
induced a cooperative occupancy of STAT1 to the promoter.
Although we were unable to obtain data for the ChIP assay of NF-
B,
STAT1 and NF-
B bound to the CXCL9 promoter may create an
enhanceosome-like structure that leads to the cooperative
recruitment of CBP/Pol II to the promoter (51). Although STAT1 and
NF-
B have been shown to interact with other components of the
transcriptional machinery (52, 53), the role of these factors in the
IFN-
/STAT1- and TNF
/NF-
B-induced transcriptional synergy
remains to be determined.
STAT1 and NF-
B (RelA/p65) were previously shown to bind to the
N-terminal and the C-terminal regions of CBP (24-28). We confirmed these physical interactions in vivo and extended our
observations to show that STAT1 and NF-
B were capable of interacting
with CBP simultaneously to form a trimeric complex. Although both the N- and C-terminal regions of CBP have the capacity to interact with
STAT1 and NF-
B, the nature of the physical association appears to be
different. The interaction of STAT1 with the C-terminal region of CBP
was enhanced by IFN-
treatment (Fig. 3), suggesting that
tyrosine-phosphorylated STAT1 preferentially interacted with the
C-terminal region of CBP. In this regard, the C-terminal activation domain of STAT1 has been shown to bind to cysteine-histidine-rich domain 3 of CBP (24). In contrast, the association of STAT1 with the
N-terminal region of CBP was constitutive and much weaker than the
C-terminal interaction. Thus, it is likely that the C-terminal interaction of CBP with STAT1 may participate in mediating the transcriptional synergy in response to IFN-
. We did not, however, detect stimulus-dependent interaction of STAT1 with the
N-terminal region of CBP, as was previously reported (24, 25). Although the reason for this difference is currently unclear, the difference in
expression systems might be a possible explanation. In addition to the
interaction of STAT1 with the C-terminal part of CBP, interaction of
RelA with the N-terminal region (amino acids 1-450) of CBP appeared to
be required for mediating the transcriptional synergy. As was shown in
Figs. 4 and 5, although RelA had the ability to interact with both the
N-terminal and C-terminal region of CBP, deletion of
the N-terminal 450 amino acid residues but not the C-terminal deletion
to 2027 residues abolished the transcriptional synergy. Furthermore,
the interaction of RelA with the N-terminal region of CBP was enhanced
by TNF
treatment (Fig. 4), suggesting that nuclear translocated RelA
associates with the N-terminal region of CBP. The N-terminal region
of CBP (amino acids 1-450) has been shown to interact with the
transactivating domain of RelA (amino acids 313-550) (27, 28). Thus,
simultaneous interaction of CBP with IFN-
-activated STAT1
through
the C-terminal region of CBP and with TNF
-activated RelA through its
N-terminal region is likely to mediate the transcriptional synergy of
the CXCL9 gene.
The requirement for the coactivator NcoA-3 (p/CIP) and NcoA-1 (SRC-1)
interacting region of CBP, which has been recently identified as the
IRF-3 binding domain (46), to mediate the STAT1/NF-
B-induced transcriptional synergy appears to be different from that for individual STAT1 or NF-
B RelA/p65-dependent
transcription. Although previous studies have shown that STAT1 and
NF-
B RelA/p65-dependent transcription require
coactivator NcoA-3 (p/CIP) and NcoA-1 (SRC-1), respectively (44, 45),
as shown in this study, the C-terminal deletion mutant (CBP 1-2027),
which lacked the IRF-3 binding domain, was able to mediate the
STAT1/NF-
B-induced transcriptional synergy shown for the CXCL9
promoter (Fig. 5). This suggests that some other region(s) of CBP may
compensate for the transactivating function or that the requirement for
NcoA-3 (p/CIP) and NcoA-1 (SRC-1) to mediate the transcriptional
activation may depend upon the promoter context.
Although HAT-dependent and -independent transcriptional
activations have been demonstrated for various transcriptional factors in different promoter contexts (22, 33, 35, 45, 54), the IFN-
/STAT1
and TNF
/NF-
B-induced transcriptional synergy of the CXCL9 gene
did not require the CBP HAT activity (Fig. 6). This conclusion was
further supported by the finding that costimulation with IFN-
and
TNF
did not increase the levels of histone acetylation at the CXCL9
promoter (Fig. 7). The level of acetylated histone at the promoter in
the cells treated with IFN-
and TNF
was comparable with that with
TSA treatment, a histone deacetylase inhibitor (50), suggesting that
the promoter region is highly acetylated. This result suggests that
although CBP has been shown to interact with other histone acetylases
such as p/CAF, NcoA-1 (SRC-1), and NcoA-3 (p/CIP/ACTR) (47-49), the
histone hyperacetylation at the promoter region may not be the primary
mechanism for the transcriptional synergy of the CXCL9 gene.
Furthermore, histone acetylation per se is not sufficient
for mediating transcriptional activation of the CXCL9 gene. As seen in
the ChIP assay (Fig. 7C), although treatment of cells with
IFN-
or TNF
alone markedly acetylated the promoter region, either
stimulus alone did not significantly induce the transcriptional
activation of the CXCL9 gene (Figs. 2C and 7D).
Taken together, these results indicate that the histone acetylation at
the CXCL9 promoter may be necessary for some step in the transcription
but is not sufficient for mediating the transcriptional activation of
the CXCL9 gene. The requirement of histone acetylation for
STAT1/NF-
B-dependent transcriptional synergy in other genes remains to be determined.
STAT1 and NF-
B are integral transcription factors functioning in the
regulation of genes involved in immune and inflammatory reactions.
Activations of STAT1 and NF-
B are normally induced by distinct
classes of extracellular signals present in the microenvironment. Type
I and type II IFNs activate STAT1, whereas members of the TNF family
and ligands for Toll-like receptors including lipopolysaccharide induce
activation of NF-
B. When the cells are exposed to stimuli that
activate both signaling pathways, this could ultimately promote type I
immune responses, which are associated with host-defense mechanisms
against viral and bacterial infections and excessive immune response
that could result in some type of autoimmune disease (55, 56). Our
study presented here provides an insight into the molecular mechanisms
involved in the interplay between STAT1 and NF-
B to control the
synergistic transcriptional activation of the inflammatory genes
associated with type I responses.