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Originally published online as doi:10.1189/jlb.0205112 on May 13, 2005

Published online before print May 13, 2005
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(Journal of Leukocyte Biology. 2005;78:515-523.)
© 2005 by Society for Leukocyte Biology

Interferon-{gamma}-activated STAT-1{alpha} suppresses MMP-9 gene transcription by sequestration of the coactivators CBP/p300

Zhendong Ma, Mi Jung Chang, Reesha C. Shah and Etty N. Benveniste1

Department of Cell Biology, University of Alabama at Birmingham

1 Correspondence: Department of Cell Biology, MCLM 395, University of Alabama at Birmingham, Birmingham, AL 35294-0005. E-mail: tika{at}uab.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interferon-{gamma} (IFN-{gamma}) is a pleiotropic cytokine involved in aspects of immune regulation, cell proliferation, and host defense mechanisms directed toward various cancers. Some of the biological functions of IFN-{gamma} are achieved through inhibition of gene expression, although the mechanisms by which IFN-{gamma} suppresses gene transcription are poorly understood. Herein, we demonstrate the molecular basis by which IFN-{gamma} mediates suppression of the matrix metalloproteinase-9 (MMP-9) gene. IFN-{gamma}-activated signal transducer and activator of transcription-1{alpha} (STAT-1{alpha}) suppresses MMP-9 gene transcription, which is dependent on phosphorylation of tyrosine 701 but not phosphorylation of serine 727. The coactivator cyclic AMP response element-binding protein-binding protein (CBP) is an important component of induction of MMP-9 gene transcription. IFN-{gamma} induces the in vivo association of STAT-1{alpha} and CBP and decreases the association of CBP to the MMP-9 promoter. IFN-{gamma} does not influence the stability of CBP nor does IFN-{gamma} affect chromatin-remodeling events on the MMP-9 promoter. IFN-{gamma} inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation and inhibiting recruitment of Pol II to the MMP-9 promoter. These findings indicate that IFN-{gamma}/STAT-1{alpha} exert their inhibitory effects by affecting multiple aspects of MMP-9 gene transcription.

Key Words: cytokines • transcription factors • gene regulation • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interferons (IFNs) are multifunctional cytokines that have antiviral, antiproliferative, and immunomodulatory effects [1 ]. IFNs are used in the clinical management of malignant tumors, multiple sclerosis, and viral infections. Beneficial effects of IFNs in attenuating angiogenesis in various tumors have also been reported, although the mechanism by which this occurs is unknown [1 ]. Activated T cells and natural killer cells predominantly produce IFN-{gamma}, the type II IFN [1 ]. The receptor for IFN-{gamma} has two subunits, IFN-{gamma}R1 and IFN-{gamma}R2. Upon engagement of the IFN-{gamma}R by IFN-{gamma}, Janus tyrosine kinase (JAK)1 and JAK2 are activated and subsequently phosphorylate the signal transducer and activator of transcription-1{alpha} (STAT-1{alpha}) protein, which dimerizes, translocates to the nucleus, and induces target gene transcription by binding to {gamma}-activated sequences (GAS) in the promoter of IFN-{gamma}-responsive genes [2 ]. IFN-{gamma} induces phosphorylation of STAT-1{alpha} at tyrosine 701 and serine 727 residues [2 ]. Phosphorylation of STAT-1{alpha} at tyrosine 701 is critical for STAT-1{alpha} dimerization, nuclear translocation, and DNA binding [2 ]. Phosphorylation of STAT-1{alpha} at serine 727 is important for optimal transactivational activity of STAT-1{alpha} [3 ]. Other post-translational modifications of STAT-1{alpha} have been reported, such as methylation, ubiquitination, sumoylation, and ISGylation, which contribute to the biological functions of STAT-1{alpha} [2 ]. Besides forming a STAT-1{alpha} homodimer or heterodimer with STAT-2, STAT-1{alpha} has been shown to interact with a variety of transcription regulators including cyclic AMP response element-binding protein-binding protein (CBP), p300, histone deacetylase 1 (HDAC-1), p300/CBP cointegrator protein (pCIP), N/myc/interactor (Nmi), and BRACA1, and these interactions are believed to be critical to regulate the function of STAT-1{alpha} [4 , 5 ].

The majority of IFN-{gamma}-mediated biological functions depend on STAT-1{alpha}-induced transcriptional regulation and/or direct protein–protein interactions mediated by STAT-1{alpha}, although STAT-1{alpha}-independent pathways have also been described [6 , 7 ]. Recently, an IFN-{gamma} signaling pathway dependent on inhibitor of {kappa}B (I{kappa}B) kinase was described [8 ]. Genes that are negatively regulated by IFN-{gamma} are far fewer than those positively induced. Among the negatively regulated ones are some of the matrix metalloproteinases (MMPs) such as MMP-1, -9, and -13 and stromelysin, type II collagen, interleukin-4, neu/HER-2, cell-cycle genes (c-myc, cyclin D, cyclin A), and the scavenger receptor A (SR-A) gene [9 10 11 12 ]. The detailed mechanisms of transcriptional suppression by IFN-{gamma} are still unclear, but STAT-1{alpha}-dependent and -independent processes have been implicated [10 ]. For instance, it has been shown that competition for limited amounts of CBP/p300 between IFN-{gamma}-activated STAT-1{alpha} and the activated protein (AP)-1 transcription factor is the mechanism underlying IFN-{gamma}-mediated inhibition of SR-A transcription [13 ]. The class II transactivator (CIITA) protein, which is induced by IFN-{gamma}, plays an important role in IFN-{gamma}-mediated suppression of the collagen {alpha} (2)(I) promoter and the MMP-9 promoter via direct binding to CBP [14 , 15 ], and in the case of the type II collagen gene, STAT-1{alpha}-dependent suppression of the general transcription machinery is involved [16 ].

The MMPs are a family of structurally conserved, zinc-dependent endopeptidases, which are involved in proteolytic modeling of the extracellular matrix [17 ]. MMP-9, also known as 97 kDa type IV collagenase, has important roles in normal growth and development, stem cell differentiation, and pathological conditions such as tumor invasion, angiogenesis, and inflammatory and autoimmune diseases [17 ]. In addition, MMP-9 cleaves myelin basic protein, cytokines, and chemokines, which is a crucial step in regulating immune function through proteolysis [18 ]. Previous studies have shown that transcriptional regulation is the rate-limiting step in MMP-9 synthesis and that nuclear factor (NF)-{kappa}B, specificity protein 1 (Sp1), and AP-1 factors play a critical role in MMP-9 transcription [19 ]. We have previously shown that IFN-{gamma} and IFN-ß suppress MMP-9 gene transcription in a STAT-1{alpha}-dependent manner [9 ].

Transcriptional activation of genes depends on the ordered recruitment of transcription factors, coactivators, chromatin modifiers/remodelers, and general transcription factors (GTFs) to the promoter of target genes [20 ]. We have recently described the molecular basis of mitogen-induced MMP-9 gene transcription [21 ]. Our results indicate that coincident with phorbol 12-myristate 13-acetate (PMA)-induced activation of the MMP-9 gene, transcription factors such as c-Fos, p65, p50, and Sp1 are recruited to the MMP-9 promoter after PMA treatment. Chromatin remodeling induced by Brg-1 is required for MMP-9 transcription, which occurs in concert with initiation of transcription. Coactivators are recruited to the MMP-9 promoter in a PMA-inducible manner; these include p300, CBP, and coactivator-associated arginine methyltransferase 1 (CARM1). Furthermore, histone modifications shift from repressive to permissive modifications concurrent with PMA activation of the MMP-9 gene. Recruitment of Pol II and phosphorylation of Ser5 Pol II C-terminal domain (CTD) also correlate with PMA-induced transcriptional activation of the MMP-9 gene. Collectively, these findings indicate that coordination of chromatin remodeling, histone modifications, and recruitment of transcription regulators is critical to regulate MMP-9 gene expression [21 ].

In this study, we investigated the molecular mechanism(s) by which IFN-{gamma} suppresses MMP-9 gene expression. Our results demonstrate that IFN-{gamma}-activated STAT-1{alpha} suppresses human MMP-9 gene expression, which is dependent on STAT-1{alpha} tyrosine 701 phosphorylation but not the phosphorylation of serine 727 of STAT-1{alpha}. IFN-{gamma} treatment does not influence chromatin-remodeling events on the MMP-9 promoter; rather, IFN-{gamma} inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation, association of CBP/p300 to the MMP-9 promoter, and inhibiting recruitment of Pol II.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and reagents
PMA was purchased from Sigma Chemical Co. (St. Louis, MO), antibodies against AcH3 (06-599), AcH4 (06-866), and STAT-1{alpha} (06-501) were purchased from Upstate Biotechnology (Lake Placid, NY), and antibody against p65 (7970) was purchased from Abcam (Cambridge, UK). Antibodies against c-Fos (sc-52), JunD (sc-74), Sp1 (sc-59), Brg-1 (sc-8749), Pol II (sc-899), CBP (sc-583 and sc-369), and p300 (sc-585 and sc-584) were purchased from Santa Cruz Biotechnology (CA). The human MMP-9 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Amersham Biosciences (Piscataway, NJ).

Cell lines
HeLa cells and U3A cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum (FBS).

MMP-9 ELISA
Cells were incubated in the absence or presence of PMA, IFN-{gamma}, or both for 36 h, and then supernatants were collected as described previously [9 ]. ELISA was performed following the instructions provided by the manufacturer. Results were normalized to the total protein concentration of the cell lysate.

Total RNA isolation and RNase protection assay (RPA)
Experiments were performed and quantified as described previously [9 ]. Total RNA (20 µg) was hybridized with human MMP-9 (50x103 cpm) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 25x103 cpm) riboprobes at 42°C overnight. The hybridized mixture was then treated with RNase A/T1 (1:200) at room temperature for 1 h and analyzed by 5% denaturing (8 M urea) polyacrylamide gel electrophoresis (PAGE). MMP-9 mRNA expression was normalized to GAPDH mRNA levels for each experimental condition.

Preparation of nuclei
Isolation of nuclei from HeLa cells was carried out as described [22 ]. Briefly, cells were spun at 1200 rpm for 5 min at 4°C and washed twice with ice-cold phosphate-buffered saline (PBS). Pelleted cells were resuspended in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% (v/v) Nonidet P-40. After 5 min of incubation on ice, the lysate was spun at 1500 rpm for 10 min at 4°C, and then the nuclei were resuspended in MNase digestion buffer.

Coimmunoprecipitation and immunoblotting
To identify the endogenous STAT-1{alpha}-CBP complex in vivo, HeLa cells were treated with IFN-{gamma} for 45 min. Then, nuclei were purified as described above and lysed in buffer containing 25 mM Hepes, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, 50 mM NaF, 1% Triton X-100, and 1x proteinase inhibitor cocktail. Protein (600 µg) was incubated with 5 µg normal rabbit immunoglobulin G (IgG), anti-STAT-1{alpha}, or anti-CBP antibodies at 4°C for 12 h with gentle agitation. Protein A/G beads (40 µl) were added and incubated for 2 h. The beads were then pelleted and washed three times in lysis buffer. The immune complexes were eluted from the beads by boiling for 5 min in Laemmli’s sample buffer and electrophoresed in 8% sodium dodecyl sulfate (SDS)-PAGE gels. Proteins were transferred to nitrocellulose and immunoblotted as described [9 ].

Metabolic labeling and chasing CBP
HeLa cells were starved for 30 min in methionine/cysteine-free DMEM plus 10% dialyzed FBS at 37°C and then were metabolically labeled for 30 min with 150 µCi/ml [35S]-methionine/cysteine (Amersham-Pharmacia, Piscataway, NJ). Cells were washed twice with PBS and treated with PMA or PMA plus IFN-{gamma} for 45 min. The labeled cells were chased for up to 4 h. At each point, the cells were lysed, and extracted protein was subjected to immunoprecipitation with anti-CBP antibody as described above. The immune complexes eluted from the beads were fractionated in 8% SDS-PAGE gels. The dried gels were exposed to the PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantified.

Restriction enzyme hypersensitivity analysis
Nuclei were isolated from cells as described above. The purified nuclei were digested with 5 U EcoRI per µg DNA for 20 min at 25°C. Genomic DNA was then purified and subjected to Southern blotting as described previously [12 ].

Southern blotting
Control DNA or DNA isolated from MNase-digested nuclei (20 µg) was first digested by appropriate restriction enzymes and then separated in a 1.5% (v/v) agarose gel and transferred to Hybond N+ membrane (Amersham-Pharmacia). The ultraviolet cross-linked blot was hybridized with random primer-labeled probes as indicated [21 ].

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed as described [23 , 24 ]. Nuclei from cross-linked cells were resuspended in Tris-EDTA buffer and sonicated. The soluble chromatin was adjusted into radio immunoprecipitation assay buffer (0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate, and 140 mM NaCl) and precleared. Immunoprecipitation was performed with 2–5 µg appropriate antibodies, and the immune complexes were absorbed with protein A beads (Upstate Biotechnology) or protein A/G beads (Pierce, Rockford, IL), blocked with bovine serum albumin and salmon sperm DNA. Immunoprecipitated DNA was amplified by a primer pair corresponding to a 269-base pair (bp) fragment (–67 to –336) of the human MMP-9 promoter and subjected to semiquantitative polymerase chain reaction (PCR) [21 ], whose products were resolved in 1.5% agarose gels in 1 x Tris acetate-EDTA buffer, and the gels were stained with ethidium bromide. In some experiments, PCR was performed for 25–28 cycles in the presence of 2.5 µCi [{alpha}-32P] deoxy-cytidine 5'-triphosphate, and the products were fractionated in 4% polyacrylamide gels. The dried gels were exposed to the PhosphorImager (Molecular Dynamics). Densitometry was used to quantify the PCR results, and all results were normalized by respective input values.

Transient transfection and luciferase assays
A luciferase reporter plasmid containing 670 bp of the human MMP-9 promoter was obtained from Douglas D. Boyd (MD Anderson Cancer Center, Houston, TX) [25 ]. A tandem GAS luciferase reporter construct was purchased from Stratagene (La Jolla, CA). Transient transfection was performed as described previously [9 ] by using Lipofectamine (Life Technologies, Grand Island, NY). Cells were also transfected with a promoterless vector control (pGL3-basic) and pRL null vector (Promega, Madison, WI). Cell extracts were assayed in triplicate with the Dual-Luciferase reporter assay system (Promega). The luciferase activity from the vector control was arbitrarily set at one for calculation of fold induction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{gamma} suppresses MMP-9 gene transcription in HeLa cells
We have previously determined that IFNs suppress PMA-induced MMP-9 gene expression in human astroglioma and human fibrosarcoma cell lines and that the STAT-1{alpha} transcription factor is crucial for IFN-mediated MMP-9 suppression [9 ]. In addition, we have recently elucidated the transcription program of the human MMP-9 gene, which is how cell signaling, chromatin remodeling, histone modifications, and transcription regulator recruitment are coordinated in MMP-9 gene transcription in HeLa cells [21 ]. Therefore, to elucidate how IFNs suppress MMP-9 gene transcription, we characterized the inhibitory effect of IFN-{gamma} on MMP-9 gene transcription in HeLa cells. Cells were treated in the absence or presence of PMA or PMA plus IFN-{gamma} for 36 h, supernatants collected, and then MMP-9 protein levels were determined by ELISA. IFN-{gamma} inhibited PMA-induced MMP-9 protein expression by ~52% (Fig. 1A ). RPA was used to study the effects of IFN-{gamma} on PMA-induced MMP-9 mRNA expression. Serum-starved HeLa cells were incubated in the absence or presence of PMA or PMA plus IFN-{gamma} for 10 h, and then total RNA was subjected to RPA analysis. PMA-induced MMP-9 mRNA expression was inhibited by IFN-{gamma} (~55% inhibition; Fig. 1B ). Time-course experiments indicated that IFN-{gamma} suppressed PMA-induced MMP-9 mRNA expression as early as 4 h after PMA stimulation (data not shown), which is the time of initiation of MMP-9 mRNA expression [21 ]. To determine whether IFN-{gamma} inhibits MMP-9 promoter activity, the 670-bp human MMP-9 promoter construct was tested by transient transfection in HeLa cells. PMA induced MMP-9 promoter activity by ~20-fold, and IFN-{gamma} suppressed MMP-9 promoter activity by ~42% (data not shown). Taken together, these data demonstrate that IFN-{gamma} inhibits MMP-9 gene expression through transcriptional suppression in HeLa cells.



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  		Figure 1. IFN-{gamma} suppresses MMP-9 gene transcription in HeLa cells (A), which were treated in the absence or presence of PMA (50 ng/ml), IFN-{gamma} (400 U/ml), or both for 36 h. Concentrated supernatants were subjected to ELISA analysis for MMP-9 protein levels. Mean ± SD of three experiments. (B) Total RNA was isolated from HeLa cells treated in the absence or presence of PMA (50 ng/ml), IFN-{gamma} (400 U/ml), or both for 10 h and analyzed by RPA for MMP-9 and GAPDH expression. Fold induction is shown. Representative of five experiments.

 
Differential effects of STAT-1{alpha} mutations on IFN-{gamma}-mediated suppression of MMP-9 gene transcription
Our previous study determined that the inhibitory effect of IFN-{gamma} on MMP-9 gene expression requires STAT-1{alpha}, and introduction of STAT-1ß had only a modest ability to restore the inhibitory effect of IFN-{gamma} [9 ]. To further elucidate the mechanism by which IFN-{gamma}-activated STAT-1{alpha} suppresses MMP-9 gene expression, we used two STAT-1{alpha} mutants, STAT-1{alpha} Y701F and STAT-1{alpha} S727A (Fig. 2A ), to study the functional domains of STAT-1{alpha} that are required for suppression of MMP-9 gene transcription. U3A cells, which are deficient in STAT-1{alpha} [26 ], were used to test whether these mutants can restore the inhibitory effects of IFN-{gamma}. We have previously shown that IFN-{gamma} fails to suppress PMA-induced MMP-9 expression in STAT-1{alpha}-deficient U3A cells, but introduction of STAT-1{alpha} successfully restores the inhibitory effects [9 ] (Fig. 2B) . The STAT-1{alpha} Y701F mutant prevents STAT-1{alpha} dimerization and subsequent nuclear translocation [2 ]. Introduction of the STAT-1{alpha} Y701F mutant was incapable of restoring IFN-{gamma} inhibition (Fig. 2B) and was also incapable of activating the GAS-driven promoter construct, as shown in Figure 2C . The STAT-1{alpha} S727A mutant is able to translocate to the nucleus but has only 10–20% of transactivation function compared with wild-type STAT-1{alpha} [3 , 27 ]. Introduction of the STAT-1{alpha} S727A mutant restored the inhibitory effect of IFN-{gamma} in a manner comparable with wild-type STAT-1{alpha} (Fig. 2B) . This suggests that the transactivation function of STAT-1{alpha} is not crucial for the ability of IFN-{gamma} to inhibit MMP-9 promoter activity. However, in confirmation of previous findings, this mutant was defective in mediating IFN-{gamma}-induced activation of the tandem GAS promoter construct in U3A cells, as activation was only ~30% of that induced by STAT-1{alpha} [3 ] (Fig. 2C) . Therefore, these data suggest that the inhibitory effect of IFN-{gamma} on MMP-9 gene expression is dependent on phosphorylation of tyrosine 701 of STAT-1{alpha} and its ability to translocate to the nucleus, but not on the phosphorylation of serine 727 of STAT-1{alpha} and possibly its transactivation function.



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  		Figure 2. Differential effects of STAT-1{alpha} mutants on IFN-{gamma}-mediated suppression of MMP-9 gene transcription. (A) Schematic diagram of functional domains of STAT-1{alpha}, STAT-1{alpha} (Y701F), and STAT-1{alpha} (S727A). N, N-terminal domain; CC, coiled-coil domain; DNA, DNA-binding domain; L, linker domain; SH2, Src homology 2 domain; and TAD, transactivation domain. (B) The MMP-9 promoter construct (0.2 µg) was transiently transfected into U3A cells with pcDNA3, STAT-1{alpha}, STAT-1{alpha} (Y701F), or STAT-1{alpha} (S727A) expression vectors (0.1 µg). After 24 h of recovery, the cells were treated with serum-free medium (lanes C), PMA (P), IFN-{gamma} ({gamma}), or both (P{gamma}) for 12 h, and then luciferase activity was determined. Mean ± SD of four experiments. (C) A tandem GAS construct (0.2 µg) was transfected into U3A cells with pcDNA3, STAT-1{alpha}, STAT-1{alpha} (Y701F), or STAT-1{alpha} (S727A). Transfected cells were treated in the absence (lanes C) or presence of IFN-{gamma} ({gamma}) for 12 h, and then luciferase activity (Luc) was determined. Mean ± SD of three experiments.

 
IFN-{gamma} induces the in vivo association of CBP and STAT-1{alpha}, and overexpression of CBP relieves IFN-{gamma}-mediated MMP-9 gene suppression
Our data suggest that the nuclear translocation of STAT-1{alpha} is required for IFN-{gamma} to suppress MMP-9 gene transcription, yet STAT-1{alpha} does not bind directly or indirectly to the MMP-9 promoter (data not shown). This suggests that nuclear protein(s) associated with STAT-1{alpha} may play an important role in mediating MMP-9 gene inhibition. Previous studies have shown that STAT-1{alpha} can bind CBP/p300 in vitro through its N-terminal domain and CTD [28 ]. This suggested the possibility that the interaction between STAT-1{alpha} and CBP/p300 may lead to inhibition of MMP-9 gene expression. Coimmunoprecipitation was used to examine the in vivo association of endogenous CBP and STAT-1{alpha}. Serum-starved HeLa cells were incubated in the absence and presence of IFN-{gamma} and nuclear extracts from these cells subjected to immunoprecipitation with antibodies against CBP or STAT-1{alpha}. The precipitated immune complexes were then immunoblotted for the presence of STAT-1{alpha} or CBP. In unstimulated cells, anti-CBP antibody did not precipitate the STAT-1{alpha} protein (Fig. 3A , lane 2), and in IFN-{gamma}-treated cells, anti-CBP antibody immunoprecipitated STAT-1{alpha} (lane 5). As a negative control, IgG did not immunoprecipitate CBP or STAT-1{alpha} (lanes 1 and 4). It is interesting that in unstimulated cells, modest levels of STAT-1{alpha} protein were detected in the nuclear extracts (lane 3). Stimulation by IFN-{gamma} resulted in enhanced nuclear accumulation of STAT-1{alpha} (lane 6). The STAT-1{alpha} blot was reprobed with anti-STAT-1{alpha}-phosphotyrosine and anti-STAT-1{alpha}-phosphoserine antibodies, and the results indicate that IFN-{gamma} stimulation also induces phosphorylation of tyrosine 701 and serine 727 in total STAT-1{alpha} protein and CBP-associated STAT-1{alpha} proteins (Fig. 3A , lanes 5 and 6). The anti-STAT-1{alpha} antibody did not immunoprecipitate the CBP protein efficiently, as anti-STAT-1{alpha}-immunoprecipitated CBP could only be detected following a long exposure (Fig. 3A , lane 6). This may be a result of a partial blockage of STAT-1{alpha} epitopes by CBP. Similar results were obtained for p300; i.e., p300 also associates with STAT-1{alpha} in an IFN-{gamma}-dependent manner (data not shown). These results indicate that STAT-1{alpha} does not associate with CBP or p300 in unstimulated cells; however, IFN-{gamma} treatment can induce the in vivo association of STAT-1{alpha} and CBP or p300.



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  		Figure 3. IFN-{gamma} induces the in vivo association of CBP and STAT-1{alpha}, and overexpression of CBP relieves IFN-{gamma}-induced MMP-9 gene suppression. (A) HeLa cells were treated with serum-free medium (Control) or IFN-{gamma} for 45 min, and then nuclear extracts were obtained and subjected to coimmunoprecipitation, as described in Materials and Methods. For each condition, IgG, anti-CBP, and anti-STAT-1{alpha} were used to immunoprecipitate (IP) the STAT-1{alpha} and CBP complex. The immune complexes were fractionated and immunoblotted by anti-CBP antibody, anti-STAT-1{alpha}, anti-STAT-1{alpha}-phosphotyrosine, and anti-STAT-1{alpha}-phosphoserine antibodies. Representative of four experiments. (B) HeLa cells were transiently transfected with the MMP-9 promoter construct (0.2 µg), without and with increasing amounts (0.1–0.4 µg) of a cytomegalovirus-CBP construct, and the total amount of transfected DNA was normalized by pcDNA3. Transfected cells were treated with serum-free medium (lane C), PMA (P), IFN-{gamma} ({gamma}), or both (P{gamma}) for 12 h, and then luciferase activity was determined. Mean ± SD of five experiments. (C) Pulse-chase analysis of CBP levels in the presence of PMA or PMA plus IFN-{gamma} for 45 min. Graph shows the decay rate of CBP, and the levels of CBP at the 0-h time-point were set as 100%. Mean ± SD of three experiments.

 
CBP/p300 play critical roles in the PMA-induced program of MMP-9 gene transcription [21 ]. Therefore, it is possible that STAT-1{alpha} suppresses MMP-9 gene activation by interfering with the binding of CBP or p300 to the MMP-9 promoter. Transient transfection assays were first used to examine whether overexpression of CBP could reverse IFN-{gamma}-mediated MMP-9 suppression. Indeed, overexpression of CBP reversed IFN-{gamma}-induced MMP-9 gene suppression in a dose-dependent manner (Fig. 3B) . Overexpression of CBP alone had only a modest effect on MMP-9 promoter activity, as previously shown by our laboratory [15 ]. Similar reversion of IFN-{gamma}-mediated MMP-9 gene suppression was also observed upon overexpression of p300 (data not shown). Therefore, these data suggest that the IFN-{gamma}-induced interaction between STAT-1{alpha} and CBP/p300 is in part responsible for suppression of MMP-9 expression by IFN-{gamma}.

Transcriptional inhibition by affecting CBP stability was reported recently [29 ]. Therefore, we examined whether IFN-{gamma} affects the stability of the CBP protein. HeLa cells were incubated with PMA or PMA plus IFN-{gamma} for 45 min, and then pulse-chase analysis was used to determine the steady-state levels of CBP. The results indicate that IFN-{gamma} treatment does not change the stability of CBP, as the decay rate of CBP was comparable in the absence or presence of IFN-{gamma} (Fig. 3C) . Similar results were also obtained for p300 (data not shown). Thus, IFN-{gamma} does not interfere with the stability of the CBP or p300 proteins.

PMA-induced chromatin remodeling on the MMP-9 promoter is not affected by IFN-{gamma}
PMA-induced chromatin remodeling is critical for MMP-9 gene transcription and coincides with initiation of transcription [21 ]. To test whether IFN-{gamma} inhibits MMP-9 transcription through interfering with chromatin remodeling, restriction enzyme hypersensitivity analysis was used to examine chromatin remodeling upon IFN-{gamma} treatment. We focused on the N4 nucleosome, which covers the NF-{kappa}B, distal AP-1, and Sp1 elements, as the remodeling of this nucleosome is critical for MMP-9 gene transcription [21 ] (Fig. 4A ). Nuclei purified from HeLa cells treated in the absence or presence of PMA, IFN-{gamma}, or PMA plus IFN-{gamma} for 4 h were digested with EcoRI, and purified DNA was further digested with XmaI/BglII and then blotted with the probe at the EcoRI site. By comparing the percentage of accessibility, the data demonstrate that IFN-{gamma} does not inhibit chromatin remodeling on the MMP-9 promoter induced by PMA (Fig. 4B) . Therefore, IFN-{gamma} suppression of the MMP-9 gene transcription is not through interference with chromatin remodeling of the MMP-9 gene.



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  		Figure 4. PMA-induced chromatin remodeling on the MMP-9 promoter is not affected by IFN-{gamma}. (A) Schematic diagram of nucleosomes located in the –670 region of the human MMP-9 promoter. The positions of the four nucleosomes spanning the –670 promoter region are shown. (B) HeLa cells were treated in the absence or presence of PMA, IFN-{gamma}, or both for 4 h, nuclei purified, and then digested by EcoRI. The extracted genomic DNA was then subsequently digested by XmaI and BglII. DNA fragments were analyzed by Southern blotting with the probe downstream of the EcoRI site. Accessibility was defined as the ratios of in vivo digestion normalized against the sums of in vivo and in vitro digestion. Mean ± SD of three experiments.

 
IFN-{gamma} inhibits the assembly of the MMP-9 transcription complex by suppressing CBP/p300 association, H3/H4 acetylation, and inhibiting recruitment of Pol II
To examine whether IFN-{gamma} affects MMP-9 transcription complex assembly in vivo, ChIP assays were performed with antibodies recognizing transcription factors, histone acetylation, chromatin-remodeling complexes, Pol II, CBP, and p300, all of which are involved in regulation of MMP-9 gene transcription [21 ]. ChIP assays were performed using HeLa cells treated in the absence or presence of PMA, IFN-{gamma}, or PMA plus IFN-{gamma}. Our results indicate that IFN-{gamma} does not affect the binding of the transcription factors c-Fos, JunD, p65, or Sp1 to the MMP-9 promoter, nor does it interfere with the association of Brg-1, the major component of the chromatin-remodeling complex, to the MMP-9 promoter (Fig. 5 ). However, IFN-{gamma} treatment resulted in suppression of PMA-induced H3/H4 histone acetylation and also inhibited PMA-induced binding of CBP and p300 to the MMP-9 promoter (Fig. 5) . Although IFN-{gamma} did not affect the association of general transcription factors such as TATA box-binding protein (TBP)-transcription factor IIA (TFIIA), TFIIB, TBP, TFIIE, and TFIIH (data not shown), it suppressed the binding of Pol II to the MMP-9 promoter (Fig. 5) . Taken together, the data from the ChIP assays illustrate that IFN-{gamma} interferes with transcription of the MMP-9 gene by affecting the assembly of transcription complexes on the MMP-9 promoter.



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  		Figure 5. IFN-{gamma} inhibits the assembly of the MMP-9 transcription complex by suppressing CBP/p300 association and H3/H4 acetylation and inhibiting recruitment of Pol II. HeLa cells were treated in the absence (Con) or presence of PMA, IFN-{gamma}, or both for 4 h or in case of c-Fos, JunD, p65, Sp1, and p300, for 2 h. ChIP was performed using antibodies against c-Fos, JunD, p65, Sp1, AcH3, AcH4, Brg-1, Pol II, CBP, and p300. The basal level was set as 1.0, and fold induction upon PMA, IFN-{gamma}, or PMA plus IFN-{gamma} treatment was compared with that. Representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Eukaryotic gene transcription is controlled by complicated mechanisms that include regulatory events at multiple levels of gene transcription; for instance, activators, coactivators/corepressors, chromatin remodeling, general transcription machinery, and signal transduction are possible regulatory targets [20 ]. How the gene transcriptional machinery integrates signals from different pathways is a central question of modern biology. In exploring the suppressive effects of IFN-{gamma} on MMP-9 gene transcription, we found that STAT-1{alpha} suppression of human MMP-9 gene transcription depends on its ability to be phosphorylated on tyrosine 701, but not serine 727. IFN-{gamma} induces the in vivo association of STAT-1{alpha} and CBP or p300 and decreases the association of CBP or p300 to the MMP-9 promoter. In addition, IFN-{gamma} inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation and inhibiting recruitment of Pol II.

Among several genes that are inhibited by IFN-{gamma}, c-myc has been shown to require STAT-1{alpha}-dependent and STAT-1{alpha}-independent pathways, and notably, there is a GAS element in the c-myc promoter that is necessary, but not sufficient, to confer the inhibitory effects of IFN-{gamma} [10 ]. As for the SR-A and type II collagen genes, the inhibitory effects of IFN-{gamma} are absolutely dependent on STAT-1{alpha}; however, there are no GAS elements in these promoters [13 ]. We previously found that STAT-1{alpha} is necessary and sufficient to inhibit MMP-9 gene transcription by IFN-{gamma} [9 ]. The MMP-9 promoter lacks GAS elements, and ChIP assays indicate that IFN-{gamma}-activated STAT-1{alpha} does not associate with the MMP-9 promoter (data not shown); thus, suppression of MMP-9 gene expression by IFN-{gamma}-activated STAT-1{alpha} is not dependent on the direct or indirect binding of STAT-1{alpha} on the MMP-9 promoter. Our results indicate that tyrosine 701 phosphorylation of STAT-1{alpha} is indispensable for suppression of the MMP-9 gene, and this indicates that nuclear translocation and localization of STAT-1{alpha} are required to inhibit the MMP-9 gene. Based on these results, we speculate that suppression of the MMP-9 gene depends on the ability of activated STAT-1{alpha} to interact with other proteins inside the nuclei such as CBP and p300, sequester them from the MMP-9 promoter, and thus, suppress MMP-9 transcription.

Another interesting finding is that phosphorylation of serine 727 of STAT-1{alpha} is not essential to suppress MMP-9 gene transcription, as the STAT-1{alpha} serine 727 mutant has the same ability to suppress MMP-9 gene transcription as does wild-type STAT-1{alpha}. Thus, this suggests that IFN-{gamma} suppression of the MMP-9 gene may be independent of downstream proteins induced by STAT-1{alpha}. Sanceau et al. [30 ] reported that IFNs inhibit tumor necrosis factor {alpha}-mediated MMP-9 activation by a mechanism involving IFN regulatory factor 1 (IRF-1)-competitive inhibition of NF-{kappa}B binding to the MMP-9 promoter. In addition, they demonstrated that IRF-1 binds to the MMP-9 promoter 16 h after IFN stimulation at an IFN responsive-like element overlapping the NF-{kappa}B site [30 ]. However, we found that IFN-{gamma} suppresses MMP-9 expression as early as 4–6 h after stimulation and that there was no association of IRF-1 on the MMP-9 promoter at that time-point (data not shown). In addition, we did not detect changes in PMA-induced p65 binding upon IFN-{gamma} treatment. Thus, the involvement and importance of IRF-1 in IFN-mediated suppression of MMP-9 gene transcription are not clear at this time. These results, coupled with the findings presented herein, indicate that IFN-{gamma} has evolved several mechanisms to inhibit gene expression. First, activated STAT-1{alpha} can interact directly with coactivators such as CBP and/or p300, leading to suppression of MMP-9. In addition, IFN-{alpha}, via the activated STAT-1{alpha} protein, induces the CIITA protein, which inhibits MMP-9 expression by sequestration of CBP at later time-points [15 ]. The direct inhibitory effect of STAT-1{alpha} may be operative early in the course of MMP-9 gene expression, and the inhibitory influence of STAT-1{alpha}-induced proteins, such as CIITA or IRF-1, would occur at a later time, as induction of these proteins by IFN-{gamma} requires a longer time course [30 , 31 ].

In addition to functioning as histone acetyltransferases, CBP/p300 serve as scaffold proteins in transcription complex formation [32 ]. Given the fact that the total amount of CBP/p300 is limited compared with the amount of other transcription regulators, it has been proposed that cross-talk or competition of different signaling pathways may be mediated by CBP/p300 [13 , 32 ]. It has been shown that competition for limited amounts of CBP/p300 between the IFN-{gamma}-activated JAK/STAT pathway and the Ras/AP-1 signaling pathway is the mechanism underlying IFN-{gamma}-mediated inhibition of SR-A gene transcription [13 ]. Moreover, CBP has been shown to have critical functions in the cell fate determination of neural stem cells. Neurogenin-dependent neuronal differentiation requires sequestration of CBP from the transcription complexes of gliogenesis genes [33 ]. Our data demonstrated that IFN-{gamma} induces the in vivo association of CBP and STAT-1{alpha}. In addition, ChIP assays demonstrated that IFN-{gamma} decreases the association of CBP/p300 to the MMP-9 promoter, and furthermore, overexpression of CBP relieves IFN-{gamma}-mediated suppression of MMP-9 transcription. Thus, competition of CBP between the PMA-activated mitogen-activated protein kinase kinase (MEK)-1/extracellular signal-regulated kinase (ERK) and NF-{kappa}B pathways and the IFN-{gamma}-activated JAK/STAT pathway is one of the mechanisms contributing to suppression of MMP-9 transcription by IFN-{gamma}.

Our data indicate that IFN-{gamma} does not interfere with chromatin remodeling of the MMP-9 promoter induced by PMA. In our previous study, we found that PMA-induced activation of the MEK-1/ERK and NF-{kappa}B pathways is important for chromatin remodeling of the MMP-9 gene and recruitment of transcription factors on the MMP-9 promoter [21 ]. In addition, our data indicate that IFN-{gamma} does not interfere with PMA activation of the MEK-1/ERK and NF-{kappa}B pathways, as IFN-{gamma} does not affect PMA-induced ERK1/2 phosphorylation, I{kappa}B phosphorylation and degradation, or nuclear translocation of AP-1 and NF-{kappa}B transcription factors (data not shown). Furthermore, the ChIP assay also demonstrated that IFN-{gamma} does not inhibit the binding of AP-1 and NF-{kappa}B transcription factors. As well, we had determined previously that Brg-1 is critical for chromatin-remodeling events on the MMP-9 promoter, and IFN-{gamma} does not affect Brg-1 binding to the MMP-9 promoter. Therefore, IFN-{gamma}-mediated suppression of MMP-9 is not through interfering with adenosine 5'-triphosphate-dependent chromatin-remodeling events on the MMP-9 promoter or activation of signaling pathways that induce chromatin remodeling on the MMP-9 promoter.

We examined the in vivo assembly of transcription complexes on the MMP-9 promoter under the influence of IFN-{gamma}. Although the binding of AP-1, NF-{kappa}B, and Sp1 transcription factors was not affected, IFN-{gamma} decreased the acetylation of H3/H4 induced by PMA. Furthermore, IFN-{gamma} also prevented the release of the Sin3A corepressor from the MMP-9 promoter (data not shown); therefore, retention of corepressors and dissociation of coactivators may contribute to hypoacetylation of the MMP-9 promoter and subsequent transcriptional suppression. It is interesting that IFN-{gamma} also interferes with recruitment of Pol II; as such, IFN-{gamma} also suppresses MMP-9 gene transcription by affecting the general transcription machinery. Osaki et al. [16 ] suggested that IFN-{gamma} suppresses the type II collagen gene by interrupting the general transcription machinery but did not formally demonstrate this effect.

In summary, transcriptional suppression of the MMP-9 gene by IFN-{gamma} is dependent on activated STAT-1{alpha} (Fig. 6 ), which suppresses MMP-9 gene transcription by competitively binding with CBP and p300 proteins in the nucleus, thereby resulting in decreased association of CBP and p300 to the MMP-9 promoter. Furthermore, STAT-1{alpha} also suppresses histone acetylation and recruitment of Pol II. All of these mechanisms interfere with the assembly of functional transcription complexes on the MMP-9 promoter, although recruitment of transcription factors such as NF-{kappa}B, AP-1, and Sp1 and chromatin remodeling is not affected by STAT-1{alpha}. Further studies of STAT-1{alpha}-interacting proteins of transcriptional importance will provide insights into understanding the inhibitory effects of IFN-{gamma} on gene transcription.



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  		Figure 6. Involvement of STAT-1{alpha} in IFN-{gamma}-mediated MMP-9 gene suppression. IFN-{gamma}-activated STAT-1{alpha} suppression of MMP-9 gene transcription is dependent on tyrosine 701 phosphorylation of STAT-1{alpha} but not phosphorylation of serine 727 of STAT-1{alpha}. IFN-{gamma} inhibits the assembly of the MMP-9 transcription complex by suppressing CBP/p300 association and H3/H4 acetylation and inhibiting recruitment of Pol II. See text for details.

 


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants CA-97247 and NS-39954 to E. N. B. We thank Drs. D. Boyd, J. E. Darnell, J. E. Kudlow, M. G. Rosenfeld, and G. R. Stark for providing valuable reagents.

Received February 23, 2005; revised April 20, 2005; accepted April 25, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schroder, K., Hertzog, P. J., Ravasi, T., Hume, D. A. (2003) Interferon-{gamma}: an overview of signals, mechanisms, and functions J. Leukoc. Biol. 75,163-189
  2. Shuai, K., Liu, B. (2003) Regulation of JAK-STAT signaling in the immune system Nat. Rev. Immunol. 3,900-911[CrossRef][Medline]
  3. Wen, Z., Zhong, Z., Darnell, J. E., Jr (1995) Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation Cell 82,241-250[CrossRef][Medline]
  4. Chatterjee-Kishore, M., van den Akker, F., Stark, G. R. (2000) Association of STATs with relatives and friends Trends Cell Biol. 10,106-111[CrossRef][Medline]
  5. Nusinzon, I., Horvath, C. M. (2003) Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1 Proc. Natl. Acad. Sci. USA 100,14742-14747[Abstract/Free Full Text]
  6. Ramana, C. V., Chatterjee-Kishore, M., Nguyen, H., Stark, G. R. (2000) Complex roles of Stat1 in regulating gene expression Oncogene 19,2619-2627[CrossRef][Medline]
  7. Kim, S., Koga, T., Isobe, M., Kern, B. E., Yokochi, T., Chin, Y. E., Karsenty, G., Taniguchi, T., Takayanagi, H. (2003) Stat1 functions as a cytoplasmic attenuator of Runx2 in the transcriptional program of osteoblast differentiation Genes Dev. 17,1979-1991[Abstract/Free Full Text]
  8. Sizemore, N., Agarwal, A., Das, K., Lerner, N., Sulak, M., Rani, S., Ransohoff, R., Shultz, D., Stark, G. R. (2004) Inhibitor of {kappa}B kinase is required to activate a subset of interferon {gamma}-stimulated genes Proc. Natl. Acad. Sci. USA 101,7994-7998[Abstract/Free Full Text]
  9. Ma, Z., Qin, H., Benveniste, E. N. (2001) Transcriptional suppression of matrix metalloproteinase-9 gene expression by IFN-{gamma} and IFN-ß: critical role of STAT-1{alpha} J. Immunol. 167,5150-5159[Abstract/Free Full Text]
  10. Ramana, C. V., Grammatikakis, N., Chernov, M., Nguyen, H., Goh, K. C., Williams, B. R., Stark, G. R. (2000) Regulation of c-myc expression by IFN-{gamma} through Stat1-dependent and -independent pathways EMBO J. 19,263-272[CrossRef][Medline]
  11. Elser, B., Lohoff, M., Kock, S., Giaisi, M., Kirchhoff, S., Krammer, P. H., Li-Weber, M. (2002) IFN-{gamma} represses IL-4 expression via IRF-1 and IRF-2 Immunity 17,703-712[CrossRef][Medline]
  12. Kominsky, S. L., Hobeika, A. C., Lake, F. A., Torres, B. A., Johnson, H. M. (2000) Down-regulation of neu/HER-2 by interferon-{gamma} in prostate cancer cells Cancer Res. 60,3904-3908[Abstract/Free Full Text]
  13. Horvai, A. E., Xu, L., Korzus, E., Brard, G., Kalafus, D., Mullen, T. M., Rose, D. W., Rosenfeld, M. G., Glass, C. K. (1997) Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300 Proc. Natl. Acad. Sci. USA 94,1074-1079[Abstract/Free Full Text]
  14. Zhu, X. S., Ting, J. P. (2001) A 36-amino-acid region of CIITA is an effective inhibitor of CBP: novel mechanism of {gamma} interferon-mediated suppression of collagen {alpha}(2)(I) and other promoters Mol. Cell. Biol. 21,7078-7088[Abstract/Free Full Text]
  15. Nozell, S., Ma, Z., Wilson, C., Shah, R., Benveniste, E. N. (2004) Class II major histocompatibility complex transactivator (CIITA) inhibits matrix metalloproteinase-9 gene expression J. Biol. Chem. 279,38577-38589[Abstract/Free Full Text]
  16. Osaki, M., Tan, L., Choy, B. K., Yoshida, Y., Cheah, K. S., Auron, P. E., Goldring, M. B. (2003) The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-{gamma}-mediated inhibition in human chondrocytes: requirement for Stat1{alpha}, Jak1 and Jak2 Biochem. J. 369,103-115[CrossRef][Medline]
  17. Sternlicht, M. D., Werb, Z. (2001) How matrix metalloproteinases regulate cell behavior Annu. Rev. Cell Dev. Biol. 17,463-516[CrossRef][Medline]
  18. Opdenakker, G., Van den Steen, P. E., Van Damme, J. (2001) Gelatinase B: a tuner and amplifier of immune functions Trends Immunol. 22,571-579[CrossRef][Medline]
  19. Sato, H., Seiki, M. (1993) Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells Oncogene 8,395-405[Medline]
  20. Levine, M., Tjian, R. (2003) Transcription regulation and animal diversity Nature 424,147-151[CrossRef][Medline]
  21. Ma, Z., Shah, R. C., Chang, M. J., Benveniste, E. N. (2004) Coordination of cell signaling, chromatin remodeling, histone modifications, and regulator recruitment in human matrix metalloproteinase 9 gene transcription Mol. Cell. Biol. 24,5496-5509[Abstract/Free Full Text]
  22. Albert, T., Wells, J., Funk, J. O., Pullner, A., Raschke, E. E., Stelzer, G., Meisterernst, M., Farnham, P. J., Eick, D. (2001) The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements J. Biol. Chem. 276,20482-20490[Abstract/Free Full Text]
  23. Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., Brown, M. (2000) Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription Cell 103,843-852[CrossRef][Medline]
  24. Agalioti, T., Lomvardas, S., Parekh, B., Yie, J., Maniatis, T., Thanos, D. (2000) Ordered recruitment of chromatin modifying and general transcription factors to the IFN-ß promoter Cell 103,667-678[CrossRef][Medline]
  25. Gum, R., Lengyel, E., Juarez, J., Chen, J. H., Sato, H., Seiki, M., Boyd, D. (1996) Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences J. Biol. Chem. 271,10672-10680[Abstract/Free Full Text]
  26. McKendry, R., John, J., Flavell, D., Muller, M., Kerr, I. M., Stark, G. R. (1991) High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both {alpha} and {gamma} interferons Proc. Natl. Acad. Sci. USA 88,11455-11459[Abstract/Free Full Text]
  27. Varinou, L., Ramsauer, K., Karaghiosoff, M., Kolbe, T., Pfeffer, K., Muller, M., Decker, T. (2003) Phosphorylation of the Stat1 transactivation domain is required for full-fledged IFN-{gamma}-dependent innate immunity Immunity 19,793-802[CrossRef][Medline]
  28. Zhang, J. J., Vinkemeier, U., Gu, W., Chakravarti, D., Horvath, C. M., Darnell, J. E., Jr (1996) Two contact regions between Stat1 and CBP/p300 in interferon {gamma} signaling Proc. Natl. Acad. Sci. USA 93,15092-15096[Abstract/Free Full Text]
  29. Marambaud, P., Wen, P. H., Dutt, A., Shioi, J., Takashima, A., Siman, R., Robakis, N. K. (2003) A CBP binding transcriptional repressor produced by the PS1/{varepsilon}-cleavage of N-cadherin is inhibited by PS1 FAD mutations Cell 114,635-645[CrossRef][Medline]
  30. Sanceau, J., Boyd, D. D., Seiki, M., Bauvois, B. (2002) Interferons inhibit tumor necrosis factor-{alpha}-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition with NF-{kappa}B J. Biol. Chem. 277,35766-35775[Abstract/Free Full Text]
  31. Lee, Y. J., Han, Y., Lu, H. T., Nguyen, V., Qin, H., Howe, P. H., Hocevar, B. A., Boss, J. M., Ransohoff, R. M., Benveniste, E. N. (1997) TGF-ß suppresses IFN-{gamma} induction of class II MHC gene expression by inhibiting class II transactivator messenger RNA expression J. Immunol. 158,2065-2075[Abstract]
  32. Goodman, R. H., Smolik, S. (2000) CBP/p300 in cell growth, transformation, and development Genes Dev. 14,1553-1577[Free Full Text]
  33. Sun, Y., Nadal-Vicens, M., Misono, S., Lin, M. Z., Zubiaga, A., Hua, X., Fan, G., Greenberg, M. E. (2001) Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms Cell 104,365-376[CrossRef][Medline]



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