The Cell Cycle Control Element of Histone H4 Gene Transcription Is Maximally Responsive to Interferon Regulatory Factor Pairs IRF-1/IRF-3 and IRF-1/IRF-7*

Ronglin Xie, André J. van Wijnen, Caroline van der Meijden, Mai X. Luong, Janet L. Stein, and Gary S. SteinDagger

From the Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Received for publication, November 15, 2000, and in revised form, February 2, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interferon regulatory factors (IRFs) are transcriptional mediators of interferon-responsive signaling pathways that are involved in antiviral defense, immune response, and cell growth regulation. To investigate the role of IRF proteins in the regulation of histone H4 gene transcription, we compared the transcriptional contributions of IRF-1, IRF-2, IRF-3, and IRF-7 using transient transfection assays with H4 promoter/luciferase (Luc) reporter genes. These IRF proteins up-regulate reporter gene expression but IRF-1, IRF-3, and IRF-7 are more potent activators of the H4 promoter than IRF-2. Forced expression of different IRF combinations reveals that IRF-2 reduces IRF-1 or IRF-3 dependent activation, but does not affect IRF-7 function. Thus, IRF-2 may have a dual function in histone H4 gene transcription by acting as a weak activator at low dosage and a competitive inhibitor of other strongly activating IRFs at high levels. IRF-1/IRF-3 and IRF-1/IRF-7 pairs each mediate the highest levels of site II-dependent promoter activity and can up-regulate transcription by 120-150-fold. We also find that interferon gamma  up-regulates IRF-1 and site II-dependent promoter activity. This up-regulation is not observed when the IRF site is mutated or if cells are preloaded with IRF-1. Our results indicate that IRF-1, IRF-2, IRF-3, and IRF-7 can all regulate histone H4 gene expression. The pairwise utilization of distinct IRF factors provides a flexible transcriptional mechanism for integration of diverse growth-related signaling pathways.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interferon regulatory factors (IRFs)1 form a large family of transcription factors involved in antiviral defense, immune activation, and cell growth regulation. IRFs were initially identified as regulators of interferon genes in response to viral infection. However, it has subsequently been shown that there are at least nine cellular IRF proteins (IRF-1, IRF-2, IRF-3, IRF-4/Pip/ICSAT, IRF-5, IRF-6, IRF-7, ICSBP/IRF8, and ISGF3gamma /p48/IRF9), as well as virally encoded forms, with broad biological functions (1, 2). All members of the IRF family share significant homology in the N-terminal 115 amino acids, which comprise the DNA-binding domain. For the IRF-3, IRF-4, IRF-5, IRF-8, and IRF-9 proteins, the homology extends into the C-terminal region with which these IRFs interact with other proteins or family members. Current data indicate that IRFs can function as transcriptional activators (e.g. IRF-1, IRF-3, and IRF-9), repressors (e.g. IRF-8), or both (e.g. IRF-2, IRF-4, and IRF-7). Studies with IRF-expressing cell lines and IRF knockout mice reveal that IRF family members have distinct roles in various biological processes, including cytokine signaling, responses to pathogens, cell growth regulation, and hematopoietic development (1-3).

IRF-1 and IRF-2 are transcription factors that interact with the same DNA sequence element (designated ISRE/IRF-E) in the promoters of type I interferon (IFN) and other cytokine inducible genes (4-8). IRF-1 is up-regulated by type I interferons and the type II interferon, IFN-gamma (2). IRF-2 is up-regulated by IRF-1 and antagonizes IRF-1 activation by competing with IRF-1 for its DNA-binding site (4, 9-12). IRF-2 also functions as a transcriptional activator (13) and has been shown to activate the genes for histone H4 (14, 15), Epstein-Barr virus nuclear antigen-1 (EBNA-1) (16), and murine muscle vascular cell adhesion molecule-1 (17). In addition, IRF-1 and IRF-2 can co-occupy the Class II transactivator type IV promoter element IRF-E and synergistically activate this promoter (18).

IRF-1 and IRF-2 are key regulators of cell growth, cell cycle, and apoptosis, and function as an anti-oncogene and oncogene, respectively (14, 19-22). Our laboratory has established that IRF-1 and IRF-2 can each functionally interact with and transcriptionally activate the H4 promoter (14, 15). Furthermore, the gene for p21WAF1/cip1, a member of the family of cyclin-dependent kinase (CDK) inhibitors, which plays a primary role in cell cycle control, is regulated in response to DNA damage by both IRF-1 and p53 (23-25). These observations suggest that the transcriptional properties of IRF-1 and IRF-2 are linked to their cell growth regulatory potential.

Cell cycle control of histone gene transcription at the onset of S phase is required for the functional coupling of histone gene expression and DNA replication (26, 27). Transcriptional control of the human histone H4 gene designated FO108 (28) has been extensively studied. The H4 gene is regulated by two multipartite proximal promoter elements (sites I and II), which together with two distal auxiliary domains (sites III and IV) modulate histone H4 promoter activity (27). Site II mediates cell cycle control of histone H4 transcription by interacting with three distinct factors, including IRF-2/HiNF-M, CDP-cut/HiNF-D, and HiNF-P (14, 29-33). The cell cycle element (CCE), 5'-CTTTCGGTTTT-3', which is located in the distal part of site II (34) and controls transcription at the G1/S phase transition (29), is known to interact functionally with both IRF-1 and IRF-2 (14, 15).

Recently, other IRF proteins (e.g. IRF-3 and IRF-7) have been shown to contribute to transcriptional control via IRF-binding sites. For example, the formation of distinct heterodimers between activated IRF-3 and IRF-7 may lead to differential regulation of the IFN-alpha and IFN-beta genes (2, 35) which were initially characterized as responsive to IRF-1 and IRF-2. Both IRF-3 and IRF-7 are constitutively present in several cell types and can be activated in response to different biological stimuli, including viral infection, type I interferons, and/or DNA damage (2). These recent findings necessitate evaluation of the extent to which distinct combinations of IRF proteins may regulate histone H4 gene expression. To investigate the role of multiple IRF members in histone H4 gene transcription, we performed transfection studies with H4 promoter-luciferase reporter genes and a panel of IRF expression vectors. Our results suggest that IRF-1, IRF-2, IRF-3, and IRF-7 can all actively regulate histone H4 gene expression and that specific IRF pairs (i.e. IRF-1/IRF-3 and IRF-1/IRF-7) are strong activators.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of H4-luciferase Reporter Gene Constructs and IRF Expression Vectors-- The wild type H4 promoter/luciferase reporter gene construct wtH4/Luc was derived from pFO108 wt/CAT (30, 31, 36), which contains the proximal promoter region of the H4 gene (nucleotides -240 to -38 relative to the ATG start codon; mRNA cap site at nucleotide -30) and spans sites I and II. The CAT gene was removed by PstI and HindIII cleavage and replaced by a 1.65-kilobase PstI/HindIII fragment spanning the luciferase (Luc) gene. The Luc gene was amplified from pGL3 (Promega, Madison, WI) by polymerase chain reaction amplification with two primers: forward PstI primer, 5'-gactgcagGCATTCCGGTACTGTTG-3'; reverse HindIII primer, 5'-gcaagcttACACGGCGATCTTTCC-3'; lowercase nucleotides were added to create restriction sites. The H4/Luc construct in which the IRF-binding site is mutated (IRF mutH4/Luc) was prepared from pMSP16-CAT (33) and the CAT gene was exchanged for the Luc reporter as described above. The 4X IRF/H4-Site II/Luc plasmid was constructed by inserting an oligonucleotide cassette containing a tandemly repeated IRF-binding site (5'-gatccGCTTTCGGTTTTCAGCTTTCGGTTTTCAGATCCGCTTTCGGTTTTCAGCTTTCGGTTTTCa-3'; 5'-gatctGAAAACCGAAAGCTGAAAACCGAAAGCGGATCTGAAAACCGAAAGCTGAAAACCGAAAGCg-3'; BamHI/BglII overhangs) into the BamHI site of pFP201CAT (29, 37) and was converted to a luciferase reporter using the same polymerase chain reaction-derived fragment described above. The FP201 segment of the H4 promoter spans nucleotides -97 to -38. All oligonucleotides were synthesized using a Beckman 1000M DNA synthesizer and all inserts were sequenced (ABI 100 model 377) to verify correct orientation and absence of polymerase chain reaction or chemical synthesis-related mutations. The 3X H4 distal site II wild type promoter-luciferase reporter gene construct (3X H4 distal Site II/Luc) which contains three copies of an oligonucleotide spanning the distal segment of H4 site II (5'-CGCTTTCGGTTTTCAATCTGGTCCGATAC-3') fused to the TATA box of the H2-L gene was a kind gift from Dr. Keiko Ozato (38). The companion construct with mutated IRF-binding sites (3X H4 distal site II IRF-mutant/Luc) was constructed by digesting the wild type plasmid with XhoI and BglII to remove the multimerized site II and then inserting the mutant multimer oligonucleotide (5'-CGCTTCAGGTTTTCAATCTGGTCCGATAC-3'). IRF expression constructs (pcDNA/IRF-1, pcDNA/IRF-2, and 6X-His-tagged human IRF-3 and IRF-7) were kindly provided by Dr. T. Maniatis (35, 39). The CDK2/Luc construct, containing the 2.4-kilobase human cdk2 promoter inserted into the pGL2-basic plasmid, was a kind gift of Dr. Dov Shiffman (40).

In Vitro Translation and Electrophoretic Mobility Shift Assay-- The expression constructs pcDNA IRF-1, pcDNA IRF-3, and pcDNA IRF-7 were subjected to coupled in vitro transcription and translation with unlabeled methionine or [35S]methionine in a rabbit reticulocyte lysate system according to the manufacturer's instructions (Promega). Aliquots of 35S-labeled IRF-1 (5 µl), IRF-3 (20 µl), and IRF-7 (20 µl) were separated by SDS-PAGE in a 10% gel, which was subsequently dried and exposed for autoradiography. The bands for IRF-1, IRF-3, and IRF-7 were removed and incorporation of [35S]methionine for each protein was measured using an LS 6500 multipurpose scintillation counter (Beckman, Fullerton, CA). The measured radioactivity (in cpm) was used to calculate the molar ratios of IRF-1, IRF-3, and IRF-7. Electrophoretic mobility shift assays were performed as described previously (14) with unlabeled in vitro translated IRF proteins. Each reaction contained 10 fmol of 32P-labeled double-stranded CCE oligomer, IRF protein, 2 µg of poly(dG-dC)·(dG-dC), 1 µg of poly (dI-dC)·(dI-dC), and 1 pmol of unlabeled competitor oligomers where indicated. CCE-wt is 5'-GATCCCGGCGCGCTTTCGGTTTTCA and CCE-mut is 5'-GATCCCGGCGCGCTTTCAGGTTTTCA. The binding reactions were separated in a 4% polyacrylamide gel.

Cell Culture and Transfection Experiments-- Actively proliferating cultures of NIH3T3 cells were maintained at subconfluency in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin (Sigma), and 0.2 µM L-glutamine, at 37 °C in humidified air containing 5% CO2. Cells were seeded in 6-well culture plates at a density of 1.5 × 105 cells/well, and were transiently transfected 24 h later at ~70% confluency by the Superfect transfection method (Qiagen, Valencia, CA). We co-transfected 0.8 µg of each H4/Luc reporter gene construct with different amounts of each IRF expression vector. The amount of DNA in each well was kept constant by supplementing the transfection mixture with the empty expression vector. Cells were also transfected with 50 ng/well of the pRL-CMV construct (Promega), which contains a cytomegalovirus promoter upstream of the Renilla luciferase gene, as an internal control for transfection efficiency (41). Cell lysates were prepared for luciferase assay or for Western blot analysis 24 h after transfection. To monitor the effect of IFN-gamma , cells transfected with reporter gene constructs at 0.8 µg/well were incubated with 0-1.0 ng/ml IFN-gamma at 24 h post-transfection, and analyzed 12 h after treatment. Each transfection was performed in triplicate and repeated at least three times.

Measurement of Reporter Gene Activity by Dual-luciferase Assay-- Cells were washed twice with 1 × PBS buffer 24 h after transfection and lysed with 1 × lysis buffer (Promega). Luciferase assays were carried out according to the manufacturer's specifications using a dual-luciferase reporter assay system (Promega) and a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). The activity of Renilla luciferase was used to normalize for variation in transfection efficiency by calculating the ratio of firefly and Renilla luciferase activities.

Western Blot Analysis and Densitometry-- Cell lysates were centrifuged at 14,000 × g (4 °C for 30 min), and protein concentrations were determined using the Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. Equal amounts of total cellular protein were mixed with loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 2% beta -mercaptoethanol, and bromphenol blue), boiled for 5 min, and subjected to 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA). The membranes were saturated with phosphate-buffered saline containing 0.05% Tween 20 (1 × PBS-T buffer) and 5% fat-free dry milk (42) for 1 h at room temperature and incubated overnight with primary IRF antibodies at 1:1,000 dilution (alpha -IRF-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 1:3,000 dilution (alpha -IRF-2 (14)), or alpha -His antibodies at 1:1,500 dilution (for detection of IRF-3 or IRF-7; Qiagen) in 1% fat-free dry milk in 1 × PBS-T buffer. After washing with 1 × PBS-T buffer containing 1% milk, blots were further incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) diluted 1:10,000 in milk/PBS-T buffer. Blots were then washed five times with the same buffer before visualization of immunoreactive protein bands by enhanced chemiluminescence detection (ECL kit; Amersham Pharmacia Biotech Inc., Piscataway, NJ). Densitometry was performed by using the Alpha Imager 2000 densitometer (Alpha Innotech Corp., San Leandro, CA) according to the manufacturer's instructions. The protein level of each IRF member in untransfected cells was set as control and the relative protein level of each IRF member in transfected cells was determined by dividing the densitometry measurements of IRF transfected cells by the densitometry measurements of the control.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IRF-3 and IRF-7 Bind to the Cell Cycle Element (CCE) of Histone H4 Site II-- The CCE within site II of the histone H4 promoter has previously been shown to interact with IRF-1 and IRF-2 (14) and has high similarity to the IRF-E and ISRE consensus elements. To determine whether more recently identified IRF family members (e.g. IRF-3 and IRF-7) are also capable of binding to the histone H4 promoter, we performed protein-DNA interaction studies with IRF proteins produced by coupled in vitro transcription and translation. The IRF-1, IRF-3, and IRF-7 proteins were analyzed by SDS-PAGE in a 10% gel for radiometric quantitation (Fig. 1, A and B). Approximately equimolar amounts of these IRF proteins were evaluated by electrophoretic mobility shift assay for binding to an oligonucleotide spanning the CCE in the distal segment of histone H4 site II (Fig. 1C). All three proteins (Fig. 1), as well as IRF-2 (Ref. 14 and data not shown), form complexes with the CCE and these complexes are competed specifically by the unlabeled wild type but not the mutant CCE oligonucleotides. The relative intensities of the signals of protein-DNA complexes suggest that IRF proteins have different affinities for the same site (IRF-1 = IRF-2 > IRF-3 = IRF-7) (Fig. 1 and data not shown). Our results indicate that in addition to IRF-2 (14), IRF-1, IRF-3, and IRF-7 are also capable of binding to the CCE in the histone H4 promoter.


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Fig. 1.   IRF-3 and IRF-7 interact with distal site II of the histone H4 gene promoter. A, 35S-labeled IRF-1, IRF-3, and IRF-7 proteins (see "Materials and Methods") were separated by SDS-PAGE in a 10% gel. Mr standards are shown on the left. B, equimolar amounts of IRF-1, IRF-3, and IRF-7 were used in electrophoretic mobility shift assays and shown in the gel. Molar ratio was calculated by measuring the [35S]methionine radioactivity and the number of methionines in each IRF protein. C, electrophoretic mobility shift assays with in vitro transcribed and translated IRF-1, IRF-3, and IRF-7 (as indicated above each panel) show sequence specific binding to the CCE/site II of histone H4 promoter. Competition analysis of the IRF·CCE complexes was carried out with 50-fold excess unlabeled wild-type (CCE-wt) and mutant (CCE-mut) oligonucleotides.

Multiple IRF Proteins (IRF-1, IRF-2, IRF-3, and IRF-7) Regulate Site II-dependent H4 Promoter Activity-- We have previously shown that the transcription factor IRF-2 can activate histone H4 gene expression (14) and is involved in cell cycle regulation of histone H4 gene transcription (15). To determine whether IRF-1, IRF-3, and IRF-7 function as activators or repressors of histone H4 gene expression, we performed co-transfection assays with IRF expression vectors and histone H4 gene promoter/luciferase reporter gene constructs (Figs. 2 and 3). We tested IRF-dependent activation in the context of the wild type histone H4 promoter spanning sites I and II, as well as with a mutant H4 promoter in which the IRF binding element in site II was altered by a two-nucleotide substitution that prevents IRF binding (33). The results show that IRF-3 and IRF-7 are each capable of activating transcription by 5-6-fold (Fig. 3A). For comparison, IRF-1 and IRF-2 increase transcription by ~11- and 2-fold, respectively. When the IRF-binding site was mutated, activation of the histone H4 promoter by IRF factors was completely abrogated (Fig. 3B). These results show that IRF-3 and IRF-7 can activate the histone H4 promoter via the IRF recognition motif in site II.


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Fig. 2.   Organization of the H4 gene proximal promoter and five H4 gene promoter-luciferase constructs. A, promoter organization of the human histone H4 gene designated FO108 (28). B, schematic diagram of the H4 promoter-luciferase reporter constructs used in this study: 1) wtH4/Luc, 2) IRF mutH4/Luc, 3) 4X IRF/H4-site II/Luc, 4) 3X H4 distal site II/Luc, 5) 3X H4 distal site II IRF mutant/Luc.


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Fig. 3.   The effects of four IRFs on histone H4 transcription assayed with different promoter constructs. NIH3T3 cells were transfected with 0.8 µg/well of different luciferase promoter constructs: A, wtH4/Luc; B, IRF mutH4/Luc; C, 4X IRF/H4-site II/Luc; D, 3X H4 distal site II/Luc. The cells were co-transfected with 0.4 µg/well of pcDNA/IRF-1, pcDNA/IRF-2, pcDNA/IRF-3, pcDNA/IRF-7, and pcDNA as control. Renilla luciferase construct (50 ng/well) was used as an internal control for each sample. Samples were analyzed by the dual-luciferase assay 24 h after transfection as described under "Materials and Methods." The graphs were based on at least three independent experiments with triplicate samples.

To assess further the role of H4 site II promoter elements in mediating activation by IRF factors, we prepared two promoter constructs in which either the IRF element or the entire distal site II segment was tandemly repeated upstream of distinct minimal promoters, i.e. human histone H4 or the mouse MHC class I H2-L TATA box regions, respectively (Fig. 3, C and D). Our results indicate that IRF-1 can synergistically activate H4-related transcription in the presence of multimerized IRF elements (compare 36-60-fold activation in Fig. 3, C and D, with 11-fold activation in Fig. 3A). In contrast, IRF-2, IRF-3, and IRF-7 show approximately the same levels of activity for both the wild type histone H4 promoter and the multimerized IRF promoter constructs. Thus, it appears that IRF-1, but not other IRFs, can synergize with itself to up-regulate H4 site II-related transcription.

Dose-dependent Activation of H4 Site II-dependent Transcription by IRF Proteins-- The differences in site II-dependent transcriptional activation observed for the four IRF proteins may be influenced by the relative levels of these factors. Therefore, we analyzed reporter gene activity (3X H4 distal site II/Luc) at different levels of each IRF factor by monitoring transcription as a function of time after transfection (Fig. 4) or concentration of expression vector (Fig. 5). The results show that IRF-related transcriptional activation by IRF-1, IRF-3, and IRF-7, but not IRF-2, is dramatically increased as these proteins accumulate at later times (e.g. 10-24 h) after transfection (Fig. 4). To relate IRF-dependent activation directly to protein levels, we performed Western blot analysis of cells transfected with different amounts of expression constructs and also monitored IRF activity by measuring reporter gene expression in parallel. Western blot analysis demonstrates that increasing the amount of IRF expression vector results in the expected elevation of cellular IRF proteins (Fig. 5, right panels). Reporter gene assays reveal that IRF-1-dependent activation through site II reaches saturation at a relatively modest level (0.4 µg) of IRF-1 expression vector, whereas with increasing IRF-3 and IRF-7 levels, transcriptional activity continues to increase (Fig. 5, left panels). These data are consistent with the apparent affinities of the IRF factors for site II (see Fig. 1) in that the protein with the highest affinity (i.e. IRF-1) may reach binding site saturation inside the cell at lower protein concentrations than IRF-3 and IRF-7.


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Fig. 4.   Time course effects of IRF proteins on H4 site II promoter activity. NIH3T3 cells were transfected with 3X H4 distal site II/Luc construct (0.8 µg/well) together with pcDNA expression vectors containing IRF-1, -2, -3, -7, or pcDNA alone (0.4 µg/well). Renilla luciferase construct (50 ng/well) was used as the internal control. Samples were taken at time points 0, 4, 6, 8, 10, 12, and 24 h after transfection and analyzed by the dual-luciferase assay. Results shown were based on three independent experiments, each comprised of triplicate samples.


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Fig. 5.   IRF proteins up-regulate H4 promoter activity in a dose-dependent manner. NIH3T3 cells were co-transfected with 3X H4 distal site II promoter/Luc plasmid (0.8 µg/well) and increasing amounts of IRF-1, IRF-2, IRF-3, and IRF-7 expression constructs. Dual luciferase assays performed 24 h after transfection show promoter activity in response to increasing amounts of IRF expression (left panels). The same samples were analyzed by Western blot assays with specific IRF antibodies for IRF-1 and IRF-2 (right panel, the upper part of A and B) or with antibody to the His epitope tag for IRF-3 and IRF-7 (right panel, C and D). The graphs on the right show IRF expression levels relative to the levels found for untransfected cells, and are based on quantitation of Western blot data (see "Materials and Methods"). The reported values represent an average of two (for Western blots) or three (for luciferase assays) independent experiments of triplicate samples.

Interestingly, increasing concentrations of IRF-2 result in enhancement of H4-site II driven promoter activity at low dosage, but not at higher levels (Fig. 5B). At the highest concentrations tested, IRF-2 does not display transactivation potential. These data indicate that IRF-2 is only transcriptionally active in a very narrow concentration range. Taken together, our IRF titration results indicate that these proteins have highly distinct concentration-dependent activity profiles. These distinct profiles suggest that differences in transcription observed on the wild type H4 promoter and site II related test promoters (see Fig. 3) can be accounted for by both IRF protein concentration and intrinsic transactivation potential.

IRF-1/IRF-3 and IRF-1/IRF-7 Pairs Are Strong Activators of H4 Site II-dependent Transcription-- Recently it has been shown that IRF-3 and IRF-7 can function together in the regulation of the IFNbeta promoter (35). To assess whether IRF-3 and IRF-7 are capable of co-regulating histone H4 gene transcription together with IRF-1 or IRF-2, we performed co-transfection assays with pairwise combinations of IRF proteins (Fig. 6). The experiments show that co-expression of increasing amounts of IRF-2 in the presence of a fixed amount of IRF-1, IRF-3, or IRF-7 results in either a minor decrease or no effect on promoter activity (Fig. 6, A, C, and D). When IRF-2 concentrations are maintained at a constant level in the presence of increasing amounts of IRF-1, IRF-3, or IRF-7, enhancement of promoter activity is observed (Fig. 6B). These findings are consistent with limited competition of the IRF-2 protein with IRF-1, IRF-3, or IRF-7, each of which has higher activation potential at H4 site II than IRF-2. Strikingly, all pairwise combinations of IRF-1 with either IRF-3 or IRF-7 yield significantly stronger activation of reporter gene expression than any one of these three factors by itself (Fig. 6). For comparison, the combination of IRF-3 and IRF-7 (Fig. 6, C and D) results in quantitatively modest levels of reporter gene transcription. We conclude from these data (Fig. 6) that IRF-1/IRF-3 and IRF-1/IRF-7 pairs are the strongest activators of H4 site II-dependent transcription.


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Fig. 6.   Combinatorial effects of IRF protein expression on the up-regulation of the H4 site II promoter. NIH3T3 cells were transfected with 3X H4 distal site II/Luc, a fixed amount (0.4 µg/well) of IRF-1 (panel A), IRF-2 (panel B), IRF-3 (panel C), or IRF-7 (panel D), and increasing amounts (0, 0.1, 0.2, and 0.4 µg/well) of the remaining three IRF factors, except for IRF-2 in panel A, where the amounts were 0, 0.05, 0.1, 0.2, 0.3, and 0.4 µg/well. At least four independent experiments with triplicates were performed and normalized as described in Fig. 3.

Interferon-gamma Selectively Up-regulates Histone H4 Site II-dependent Transcription-- Upon establishing that IRF-1 in combination with IRF-3 and IRF-7 is a strong enhancer of cell cycle controlled histone H4 gene transcription, we addressed the possibility that H4 gene promoter activity may be responsive to signaling mechanisms that up-regulate IRF-1. To address whether H4 site II is capable of integrating IRF-related cellular responses, we transfected cells with H4/Luc constructs and treated cells with interferon-gamma (Fig. 7). When transfected mouse NIH3T3 cells were treated with mouse interferon-gamma (0.5 ng/ml), we observed a strong 4-5-fold up-regulation of H4 site II-driven luciferase activity (Fig. 7, A and B) which did not occur when the IRF element within Site II was mutated (Fig. 7B). Consistent with the species specificity of interferon-gamma signaling, the same concentration of human interferon-gamma did not influence reporter gene activity in mouse NIH3T3 cells (Fig. 7A). We also did not observe effects of mouse interferon-gamma on a promoter-less construct (pGL2) or an unrelated promoter (CDK2/Luc) (Fig. 7B). Interferon-gamma did not further enhance site II-dependent transcription when cells were preloaded with IRF-1 or IRF-7 (Fig. 7C). This desensitization of interferon-gamma is consistent with IRF-1 being a downstream mediator of interferon-gamma effects on site II. Consistent with this concept, we find that interferon-gamma significantly increases IRF-1 protein levels in NIH3T3 cells (Fig. 7D). Taken together, interactions of IRF-1 and other IRF factors with the cell cycle regulatory element in site II of the histone H4 gene may support interferon-gamma -dependent cell signaling mechanisms.


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Fig. 7.   Activation of histone H4 gene promoter and IRF-1 expression in response to IFN-gamma . NIH3T3 cells were transfected with 3X H4 distal site II/Luc wild type or IRF-mutant constructs and treated with interferon-gamma . A, dose-dependent activation of the wild type promoter in response to mouse (mIFN () or human (hIFN (black-square) interferon-gamma . B, effect of mouse IFN-gamma treatment (0.5 ng/ml) on activity of vector control (pGL2), histone H4 wild type, histone H4 IRF-mutant, and Cdk2 gene promoters. C, effects of IRF-1, IRF-2, IRF-3, and IRF-7 or control pcDNA vector in combination with mouse IFN-gamma (0.5 ng/ml) on H4 promoter activity. The reported values are an average of three independent experiments of triplicate samples. D, Western blot analysis of IRF-1 levels in the experiment shown in B; actin protein levels are shown for comparison.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell cycle control of histone H4 gene transcription involves a 3-fold enhancement of promoter activity at the G1/S phase transition and is mediated by the CCE. The results of this study show that the CCE represents a universal IRF recognition motif capable of interacting with all IRF members analyzed to date (i.e. IRF-1, -2, -3, and -7). We find that these IRF factors can each up-regulate histone H4 gene transcription and that IRF-1 is a stronger activator than IRF-2, IRF-3, or IRF-7. More interestingly, pairwise combinations of IRF-1 with either IRF-3 or IRF-7 mediate the strongest level of activation. Thus, dimeric IRF interactions with the CCE may function to integrate signals of two distinct classes of IRF factors. The multitude of IRF factors involved in diverse immunological, genotoxic, and cell growth regulatory functions may provide a broad spectrum of gene regulatory options to control H4 gene transcription during the cell cycle.

We have previously shown that IRF-2 is identical to HiNF-M, the major DNA-binding protein that interacts with the CCE within H4 site II in HeLa S3 cells. Cell cycle analyses using mouse fibroblasts in which the genes for IRF-1 and/or IRF-2 have been ablated show that IRF-2 is important for cell cycle regulation of histone H4 gene expression (15). The deregulation of histone gene expression in these cells may be due to direct effects on the promoter or to a general effect of the IRF-2 oncoprotein on cell growth. Studies using human HeLa cervical carcinoma cells have revealed that abolishing the IRF/site II interaction by mutating the CCE has a major effect on basal level transcription (29, 33), but does not affect the timing of transcription (43). These data suggest that the role of IRF-2 in histone gene expression may be related to the growth regulatory phenotype of the cell and is possibly subject to compensatory mechanisms. The main finding in this study is that different IRF members can substitute for IRF-2 in modulating transcription, which may reflect the in vivo complexity of cell cycle regulatory mechanisms at the site II element.

IRF-2 is a weak activator of histone H4 gene transcription (14) and is also known to activate other genes, including EBNA/Qp and vascular cell adhesion molecule-1 (16, 17, 44). For some genes, IRF-2 has been shown to inhibit IRF-1 activation (4, 13), ostensibly by competing with IRF-1 for IRF-binding sites. The data presented here indicate that IRF-2 has a biphasic transcriptional activity curve in response to elevations in IRF-2 levels. Activation of the H4 gene is only observed in cells expressing low levels of IRF-2, and IRF-2 becomes transcriptionally neutral at high dosage. Thus, the dual role of IRF-2 in both transcriptional activation and inhibition of different genes may in part be attributable to cellular IRF-2 levels. The biphasic response of IRF-2 in modulating H4 gene transcription is consistent with data on IRF-2 dependent activation of the EBNA/Qp promoter in different cell types, which displays a similar dependence of promoter activity on IRF-2 concentrations (45).

In this study, we have shown that IRF-1 by itself is the strongest transactivator of histone H4-related transcription when compared with IRF-2, IRF-3, and IRF-7. IRF-1 has been shown to regulate the genes for IL-4, IL-5, IL-7 receptor, IL-12, guanylate-binding protein, EBNA/Qp, cyclin D1, ICAM 1, p53, ICE, 2-5A synthetase, p21, c-Myb, IRF-2, as well as H4 (10, 13-16, 19, 21, 22, 25, 46-48). The strength by which IRF-1 can activate transcription via the CCE suggests that the H4 gene is a physiological target of IRF-1 at least in some biological circumstances. IRF-1 dependent control of H4 gene expression would be consistent with the cell growth and/or apoptotic properties of this protein (1, 2). One novel finding of our study is that IRF-3 and IRF-7 are also capable of mediating CCE-related control of H4 gene transcription and that these proteins, when paired with IRF-1, are the strongest modulators of promoter activity. Hence, the H4 gene appears to be targeted by heterodimers comprised of IRF-1/IRF-3 and IRF-1/IRF-7.

IRF-3 and IRF-7 are phosphorylated in virus-infected cells. Phosphorylation is required for nuclear-cytoplasmic transport of IRF-3 and IRF-7, transcriptional activation, and association of IRF-3 with p300 (35, 49-54). IRF-7 was first described to bind and repress the Qp promoter region of the Epstein-Barr virus encoded gene EBNA-1 which contains an ISRE-like element (44, 55). Recent results indicate that IRF-3 and IRF-7 can be detected in both uninfected and virus-infected cells and that IRF-7 expression is up-regulated by type I IFNs, lipopolysaccharide, and virus infection (35, 56). Our results clearly indicate that IRF-3 and IRF-7 together with IRF-1 activate the histone H4 gene promoter in the absence of viral infection.

Recent studies have indicated that IRF family members form homo- and/or heterodimers and upon binding to DNA can regulate the same gene. For example, IRF-8 forms multiple protein complexes with both IRF-1 and IRF-2 (57, 58), while IRF-3 and IRF-7 form dimers upon binding to the IFNbeta promoter (52, 56, 59, 60). Similar complexes were reported for IRF-1 and/or IRF-2 (16, 38, 58, 61, 62). Our protein/DNA interaction data, as well as results from transient co-expression experiments, suggest that IRF family members may also form dimers at the CCE to regulate histone H4 gene expression. These combined observations reinforce the idea that protein-protein complexes play central roles in the ability of IRFs to bind their cognate target genes and mediate biological functions. We conclude that the multiplicity of IRF family members apart from roles in virus-mediated signaling perform essential functions in the regulation of cell growth control in normal and tumor cells.

IFN-gamma is a multifunctional cytokine with a highly cell-type dependent activity that plays an important role in immunity and cell growth control (63-67). For example, IFN-gamma mediates activation of an antiviral state and regulates cell growth (64, 68). Furthermore, IFN-gamma is capable of inducing cell cycle arrest and apoptosis in primary hepatocytes, but not in hepatoma cell lines (HepG2, H4IIE, and Hepa1-6) (63). Interferon-gamma can stimulate cell growth depending on serum levels in malignant human T cells (68). Our data demonstrate that CCE dependent activity of the histone H4 gene promoter but not CDK2 promoter activity is selectively elevated in response to IFN-gamma through IRF-1. IRF-1 dependent activation of interferon-gamma responsive genes has also been observed for other genes (1-5). The biological effects of IFN-gamma are mediated through a heterodimeric transmembrane receptor which is capable of stimulating the JAK-STAT pathway (69) and result in activation of IRF-1. Not withstanding the known role of interferon-gamma in cell growth suppression of different cell types (69), these data suggest that IRFs, IFN-gamma , and the CCE of the histone H4 gene are components of a cell signaling mechanism that may contribute to regulation of histone H4 gene transcription during the cell cycle in a cell-type or serum-dependent manner.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK50222 and GM32010.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Correspondence should be addressed to authors at: Dept. of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-5625; Fax: 508-856-6800; E-mail: gary.stein@umassmed.edu.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M010391200

    ABBREVIATIONS

The abbreviations used are: IRF, interferon regulatory factor; Luc, luciferase; IFN, interferon, EBNA-1, Epstein-Barr virus nuclear antigen-1; CDK, cyclin-dependent kinase; CCE, cell cycle element; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Vaughan, P. S., van Wijnen, A. J., Stein, J. L., and Stein, G. S. (1997) J. Mol. Med. 75, 348-359[CrossRef][Medline] [Order article via Infotrieve]
2. Mamane, Y., Heylbroeck, C., Genin, P., Algarte, M., Servant, M. J., LePage, C., DeLuca, C., Kwon, H., Lin, R., and Hiscott, J. (1999) Gene 237, 1-14[CrossRef][Medline] [Order article via Infotrieve]
3. Nguyen, H., Hiscott, J., and Pitha, P. M. (1997) Cytokine Growth Factor Rev. 8, 293-312[CrossRef][Medline] [Order article via Infotrieve]
4. Harada, H., Fujita, T., Miyamoto, M., Kimura, Y., Maruyama, M., Furia, A., Miyata, T., and Taniguchi, T. (1989) Cell 58, 729-739[Medline] [Order article via Infotrieve]
5. Miyamoto, M., Fujita, T., Kimura, Y., Maruyama, M., Harada, H., Sudo, Y., Miyata, T., and Taniguchi, T. (1988) Cell 54, 903-913[Medline] [Order article via Infotrieve]
6. Fujita, T., Kimura, Y., Miyamoto, M., Barsoumian, E. L., and Taniguchi, T. (1989) Nature 337, 270-272[CrossRef][Medline] [Order article via Infotrieve]
7. Pine, R. (1992) J. Virol. 66, 4470-4478[Abstract]
8. Hiscott, J., Nguyen, H., and Lin, R. (1995) Semin. Virology 6, 161-173[CrossRef]
9. Harada, H., Willison, K., Sakakibara, J., Miyamoto, M., Fujita, T., and Taniguchi, T. (1990) Cell 63, 303-312[Medline] [Order article via Infotrieve]
10. Tanaka, N., Kawakami, T., and Taniguchi, T. (1993) Mol. Cell. Biol. 13, 4531-4538[Abstract]
11. Taniguchi, T., Harada, H., and Lamphier, M. (1995) J. Cancer Res. Clin. Oncol. 121, 516-520[Medline] [Order article via Infotrieve]
12. Taniguchi, T., Lamphier, M. S., and Tanaka, N. (1997) Biochim. Biophys. Acta 1333, M9-17[CrossRef][Medline] [Order article via Infotrieve]
13. Yamamoto, H., Lamphier, M. S., Fujita, T., Taniguchi, T., and Harada, H. (1994) Oncogene 9, 1423-1428[Medline] [Order article via Infotrieve]
14. Vaughan, P. S., Aziz, F., van Wijnen, A. J., Wu, S., Harada, H., Taniguchi, T., Soprano, K. J., Stein, J. L., and Stein, G. S. (1995) Nature 377, 362-365[CrossRef][Medline] [Order article via Infotrieve]
15. Vaughan, P. S., van der Meijden, C. M. J., Aziz, F., Harada, H., Taniguchi, T., van Wijnen, A. J., Stein, J. L., and Stein, G. S. (1998) J. Biol. Chem. 273, 194-199[Abstract/Free Full Text]
16. Schaefer, B. C., Paulson, E., Strominger, J. L., and Speck, S. H. (1997) Mol. Cell. Biol. 17, 873-886[Abstract]
17. Jesse, T. L., LaChance, R., Iademarco, M. F., and Dean, D. C. (1998) J. Cell Biol. 140, 1265-1276[Abstract/Free Full Text]
18. Xi, H., Eason, D. D., Ghosh, D., Dovhey, S., Wright, K. L., and Blanck, G. (1999) Oncogene 18, 5889-5903[CrossRef][Medline] [Order article via Infotrieve]
19. Harada, H., Kitagawa, M., Tanaka, N., Yamamoto, H., Harada, K., Ishihara, M., and Taniguchi, T. (1993) Science 259, 971-974[Medline] [Order article via Infotrieve]
20. Tanaka, H., and Samuel, C. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7995-7999[Abstract]
21. Tamura, T., Ishihara, M., Lamphier, M. S., Tanaka, N., Oishi, I., Aizawa, S., Matsuyama, T., Mak, T. W., Taki, S., and Taniguchi, T. (1995) Nature 376, 596-599[CrossRef][Medline] [Order article via Infotrieve]
22. Willman, C. L., Sever, C. E., Pallavicini, M. G., Harada, H., Tanaka, N., Slovak, M. L., Yamamoto, H., Harada, K., Meeker, T. C., and List, A. F. (1993) Science 259, 968-971[Medline] [Order article via Infotrieve]
23. Tanaka, N., Ishihara, M., Lamphier, M. S., Nozawa, H., Matsuyama, T., Mak, T. W., Aizawa, S., Tokino, T., Oren, M., and Taniguchi, T. (1996) Nature 382, 816-818[CrossRef][Medline] [Order article via Infotrieve]
24. Sherr, C. J., and Roberts, J. M. (1995) Genes Dev. 9, 1149-1163[CrossRef][Medline] [Order article via Infotrieve]
25. Coccia, E. M., Del Russo, N., Stellacci, E., Orsatti, R., Benedetti, E., Marziali, G., Hiscott, J., and Battistini, A. (1999) Oncogene 18, 2129-2137[CrossRef][Medline] [Order article via Infotrieve]
26. Stein, G. S., Stein, J. L., and Marzluff, W. F. (1984) Histone Genes , John Wiley & Sons, Inc., New York
27. Stein, G. S., Stein, J. L., van Wijnen, A. J., and Lian, J. B. (1996) Cell Biol. Int. 20, 41-49[CrossRef][Medline] [Order article via Infotrieve]
28. Sierra, F., Stein, G., and Stein, J. (1983) Nucleic Acids Res. 11, 7069-7086[Abstract]
29. Ramsey-Ewing, A., van Wijnen, A. J., Stein, G. S., and Stein, J. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4475-4479[Abstract]
30. van Wijnen, A. J., Ramsey-Ewing, A. L., Bortell, R., Owen, T. A., Lian, J. B., Stein, J. L., and Stein, G. S. (1991) J. Cell. Biochem. 46, 174-189[Medline] [Order article via Infotrieve]
31. van Wijnen, A. J., van den Ent, F. M., Lian, J. B., Stein, J. L., and Stein, G. S. (1992) Mol. Cell. Biol. 12, 3273-3287[Abstract]
32. van der Meijden, C. M. J., Vaughan, P. S., Staal, A., Albig, W., Doenecke, D., Stein, J. L., Stein, G. S., and van Wijnen, A. J. (1998) Biochim. Biophys. Acta 1442, 82-100[Medline] [Order article via Infotrieve]
33. Aziz, F., van Wijnen, A. J., Vaughan, P. S., Wu, S., Shakoori, A. R., Lian, J. B., Soprano, K. J., Stein, J. L., and Stein, G. S. (1998) Mol. Biol. Rep. 25, 1-12[CrossRef][Medline] [Order article via Infotrieve]
34. Pauli, U., Chrysogelos, S., Stein, G., Stein, J., and Nick, H. (1987) Science 236, 1308-1311[Medline] [Order article via Infotrieve]
35. Wathelet, M. G., Lin, C. H., Parekh, B. S., Ronco, L. V., Howley, P. M., and Maniatis, T. (1998) Mol. Cell 1, 507-518[Medline] [Order article via Infotrieve]
36. Kroeger, P., Stewart, C., Schaap, T., van Wijnen, A., Hirshman, J., Helms, S., Stein, G., and Stein, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3982-3986[Abstract]
37. van Wijnen, A. J., Owen, T. A., Holthuis, J., Lian, J. B., Stein, J. L., and Stein, G. S. (1991) J. Cell. Physiol. 148, 174-189[Medline] [Order article via Infotrieve]
38. Masumi, A., Wang, I. M., Lefebvre, B., Yang, X. J., Nakatani, Y., and Ozato, K. (1999) Mol. Cell. Biol. 19, 1810-1820[Abstract/Free Full Text]
39. Palombella, V. J., and Maniatis, T. (1992) Mol. Cell. Biol. 12, 3325-3336[Abstract]
40. Shiffman, D., Brooks, E. E., Brooks, A. R., Chan, C. S., and Milner, P. G. (1996) J. Biol. Chem. 271, 12199-12204[Abstract/Free Full Text]
41. Behre, G., Smith, L. T., and Tenen, D. G. (1999) BioTechniques 26, 24-6[Medline] [Order article via Infotrieve], 28
42. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1987) Current Protocols in Molecular Biology , John Wiley & Sons, Inc., New York
43. Aziz, F., van Wijnen, A. J., Stein, J. L., and Stein, G. S. (1998) J. Cell. Physiol. 177, 453-464[CrossRef][Medline] [Order article via Infotrieve]
44. Nonkwelo, C., Ruf, I. K., and Sample, J. (1997) J. Virol. 71, 6887-6897[Abstract]
45. Zhang, L., and Pagano, J. S. (1999) Mol. Cell. Biol. 19, 3216-3223[Abstract/Free Full Text]
46. Manzella, L., Gualdi, R., Perrotti, D., Nicolaides, N. C., Girlando, G., Giuffrida, M. A., Messina, A., and Calabretta, B. (2000) Exp. Cell Res. 256, 248-256[CrossRef][Medline] [Order article via Infotrieve]
47. Salkowski, C. A., Kopydlowski, K., Blanco, J., Cody, M. J., McNally, R., and Vogel, S. N. (1999) J. Immunol. 163, 1529-1536[Abstract/Free Full Text]
48. Tanaka, N., Ishihara, M., Kitagawa, M., Harada, H., Kimura, T., Matsuyama, T., Lamphier, M. S., Aizawa, S., Mak, T. W., and Taniguchi, T. (1994) Cell 77, 829-839[Medline] [Order article via Infotrieve]
49. Lin, R., Heylbroeck, C., Pitha, P. M., and Hiscott, J. (1998) Mol. Cell. Biol. 18, 2986-2996[Abstract/Free Full Text]
50. Au, W. C., Moore, P. A., LaFleur, D. W., Tombal, B., and Pitha, P. M. (1998) J. Biol. Chem. 273, 29210-29217[Abstract/Free Full Text]
51. Weaver, B. K., Kumar, K. P., and Reich, N. C. (1998) Mol. Cell. Biol. 18, 1359-1368[Abstract/Free Full Text]
52. Yoneyama, M., Suhara, W., Fukuhara, Y., Fukuda, M., Nishida, E., and Fujita, T. (1998) EMBO J. 17, 1087-1095[Abstract/Free Full Text]
53. Sato, M., Hata, N., Asagiri, M., Nakaya, T., Taniguchi, T., and Tanaka, N. (1998) FEBS Lett. 441, 106-110[CrossRef][Medline] [Order article via Infotrieve]
54. Navarro, L., Mowen, K., Rodems, S., Weaver, B., Reich, N., Spector, D., and David, M. (1998) Mol. Cell. Biol. 18, 3796-3802[Abstract/Free Full Text]
55. Zhang, L., and Pagano, J. S. (1997) Mol. Cell. Biol. 17, 5748-5757[Abstract]
56. Lin, R., Mamane, Y., and Hiscott, J. (2000) J. Biol. Chem. 275, 34320-34327[Abstract/Free Full Text]
57. Bovolenta, C., Driggers, P. H., Marks, M. S., Medin, J. A., Politis, A. D., Vogel, S. N., Levy, D. E., Sakaguchi, K., Appella, E., and Coligan, J. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5046-5050[Abstract]
58. Sharf, R., Meraro, D., Azriel, A., Thornton, A. M., Ozato, K., Petricoin, E. F., Larner, A. C., Schaper, F., Hauser, H., and Levi, B. Z. (1997) J. Biol. Chem. 272, 9785-9792[Abstract/Free Full Text]
59. Maniatis, T., Falvo, J. V., Kim, T. H., Kim, T. K., Lin, C. H., Parekh, B. S., and Wathelet, M. G. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 609-620[Medline] [Order article via Infotrieve]
60. Marie, I., Durbin, J. E., and Levy, D. E. (1998) EMBO J. 17, 6660-6669[Abstract/Free Full Text]
61. Meraro, D., Hashmueli, S., Koren, B., Azriel, A., Oumard, A., Kirchhoff, S., Hauser, H., Nagulapalli, S., Atchison, M. L., and Levi, B. Z. (1999) J. Immunol. 163, 6468-6478[Abstract/Free Full Text]
62. Kirchhoff, S., Schaper, F., Oumard, A., and Hauser, H. (1998) Biochimie (Paris) 80, 659-664[CrossRef][Medline] [Order article via Infotrieve]
63. Kano, A., Haruyama, T., Akaike, T., and Watanabe, Y. (1999) Biochem. Biophys. Res. Commun. 257, 672-677[CrossRef][Medline] [Order article via Infotrieve]
64. Balkwill, F., and Taylor-Papadimitriou, J. (1978) Nature 274, 798-800[Medline] [Order article via Infotrieve]
65. Bromberg, J. F., Horvath, C. M., Wen, Z., Schreiber, R. D., and Darnell, J. E., Jr. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7673-7678[Abstract/Free Full Text]
66. Coughlin, C. M., Salhany, K. E., Gee, M. S., LaTemple, D. C., Kotenko, S., Ma, X., Gri, G., Wysocka, M., Kim, J. E., Liu, L., Liao, F., Farber, J. M., Pestka, S., Trinchieri, G., and Lee, W. M. (1998) Immunity 9, 25-34[Medline] [Order article via Infotrieve]
67. Kaplan, D. H., Shankaran, V., Dighe, A. S., Stockert, E., Aguet, M., Old, L. J., and Schreiber, R. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7556-7561[Abstract/Free Full Text]
68. Novelli, F., Di Pierro, F., Francia, D. C., Bertini, S., Affaticati, P., Garotta, G., and Forni, G. (1994) J. Immunol. 152, 496-504[Abstract/Free Full Text]
69. Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H., and Schreiber, R. D. (1998) Annu. Rev. Biochem. 67, 227-264[CrossRef][Medline] [Order article via Infotrieve]


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