A Distal Region in the Interferon-γ Gene Is a Site of Epigenetic Remodeling and Transcriptional Regulation by Interleukin-2
2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês
10.1074/jbc.m401168200
ISSN1083-351X
AutoresJay H. Bream, Deborah L. Hodge, Rivkah Gonsky, Rosanne Spolski, Warren J. Leonard, Stéphanie Krebs, Stephan R. Targan, Akio Morinobu, John J. O’Shea, Howard A. Young,
Tópico(s)T-cell and B-cell Immunology
ResumoInterferon-γ (IFN-γ) is a multifunctional cytokine that defines the development of Th1 cells and is critical for host defense against intracellular pathogens. IL-2 is another key immunoregulatory cytokine that is involved in T helper differentiation and is known to induce IFN-γ expression in natural killer (NK) and T cells. Despite concerted efforts to identify the one or more transcriptional control mechanisms by which IL-2 induces IFN-γ mRNA expression, no such genomic regulatory regions have been described. We have identified a DNase I hypersensitivity site ∼3.5-4.0 kb upstream of the transcriptional start site. Using chromatin immunoprecipitation assays we found constitutive histone H3 acetylation in this distal region in primary human NK cells, which is enhanced by IL-2 treatment. This distal region is also preferentially acetylated on histones H3 and H4 in primary Th1 cells as compared with Th2 cells. Within this distal region we found a Stat5-like motif, and in vitro DNA binding assays as well as in vivo chromosomal immunoprecipitation assays showed IL-2-induced binding of both Stat5a and Stat5b to this distal element in the IFNG gene. We examined the function of this Stat5-binding motif by transfecting human peripheral blood mononuclear cells with -3.6 kb of IFNG-luciferase constructs and found that phorbol 12-myristate 13-acetate/ionomycin-induced transcription was augmented by IL-2 treatment. The effect of IL-2 was lost when the Stat5 motif was disrupted. These data led us to conclude that this distal region serves as both a target of chromatin remodeling in the IFNG locus as well as an IL-2-induced transcriptional enhancer that binds Stat5 proteins. Interferon-γ (IFN-γ) is a multifunctional cytokine that defines the development of Th1 cells and is critical for host defense against intracellular pathogens. IL-2 is another key immunoregulatory cytokine that is involved in T helper differentiation and is known to induce IFN-γ expression in natural killer (NK) and T cells. Despite concerted efforts to identify the one or more transcriptional control mechanisms by which IL-2 induces IFN-γ mRNA expression, no such genomic regulatory regions have been described. We have identified a DNase I hypersensitivity site ∼3.5-4.0 kb upstream of the transcriptional start site. Using chromatin immunoprecipitation assays we found constitutive histone H3 acetylation in this distal region in primary human NK cells, which is enhanced by IL-2 treatment. This distal region is also preferentially acetylated on histones H3 and H4 in primary Th1 cells as compared with Th2 cells. Within this distal region we found a Stat5-like motif, and in vitro DNA binding assays as well as in vivo chromosomal immunoprecipitation assays showed IL-2-induced binding of both Stat5a and Stat5b to this distal element in the IFNG gene. We examined the function of this Stat5-binding motif by transfecting human peripheral blood mononuclear cells with -3.6 kb of IFNG-luciferase constructs and found that phorbol 12-myristate 13-acetate/ionomycin-induced transcription was augmented by IL-2 treatment. The effect of IL-2 was lost when the Stat5 motif was disrupted. These data led us to conclude that this distal region serves as both a target of chromatin remodeling in the IFNG locus as well as an IL-2-induced transcriptional enhancer that binds Stat5 proteins. IFN-γ 1The abbreviations used are: IFN-γ, interferon-γ; NK, natural killer cells; Th1, T helper 1 cells; Stat1, signal transducers and activators of transcription 1; TCR, T cell receptor; IL-2, interleukin-2; FCS, fetal calf serum; PBMC, peripheral blood mononuclear cell; Ab, antibody; EMSA, electrophoretic mobility shift assay; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; ChIP, chromatin immunoprecipitation; RPA, RNase protection assay; P/I, PMA/ionomycin. 1The abbreviations used are: IFN-γ, interferon-γ; NK, natural killer cells; Th1, T helper 1 cells; Stat1, signal transducers and activators of transcription 1; TCR, T cell receptor; IL-2, interleukin-2; FCS, fetal calf serum; PBMC, peripheral blood mononuclear cell; Ab, antibody; EMSA, electrophoretic mobility shift assay; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; ChIP, chromatin immunoprecipitation; RPA, RNase protection assay; P/I, PMA/ionomycin. is a cytokine expressed primarily by, but not limited to, lymphocytes of T and NK cell lineages. NK cells are a major early source of IFN-γ in vivo, and T cell expression of IFN-γ is generally restricted as T helper 1 (Th1) cells (1Szabo S.J. Sullivan B.M. Peng S.L. Glimcher L.H. Annu. Rev. Immunol. 2003; 21: 713-758Crossref PubMed Scopus (768) Google Scholar). These cell types and their predominate cytokine IFN-γ are intimately involved in protection against intracellular pathogens and the development of tumors (2Agnello D. Lankford C.S. Bream J. Morinobu A. Gadina M. O'Shea J.J. Frucht D.M. J. Clin. Immunol. 2003; 23: 147-161Crossref PubMed Scopus (310) Google Scholar). This is evident in humans with gene mutations that interfere with IFN-γ signaling, including the IFN-γ receptor (3Jouanguy E. Altare F. Lamhamedi S. Revy P. Emile J.F. Newport M. Levin M. Blanche S. Seboun E. Fischer A. Casanova J.L. N. Engl. J. Med. 1996; 335: 1956-1961Crossref PubMed Scopus (638) Google Scholar) and Stat1 (4Dupuis S. Jouanguy E. Al Hajjar S. Fieschi C. Al Mohsen I.Z. Al Jumaah S. Yang K. Chapgier A. Eidenschenk C. Eid P. Al Ghonaium A. Tufenkeji H. Frayha H. Al Gazlan S. Al Rayes H. Schreiber R.D. Gresser I. Casanova J.L. Nat. Genet. 2003; 33: 388-391Crossref PubMed Scopus (623) Google Scholar) as well as in IFN-γ-deficient mice (5Dalton D.K. Pitts-Meek S. Keshav S. Figari I.S. Bradley A. Stewart T.A. Science. 1993; 259: 1739-1742Crossref PubMed Scopus (1499) Google Scholar), which are highly susceptible to infectious agents and prone to certain cancers. In contrast, circumstances in which IFN-γ is overproduced or its effects fail to induce negative feedback loops lead to widespread pathology (6Young H.A. Klinman D.M. Reynolds D.A. Grzegorzewski K.J. Nii A. Ward J.M. Winkler-Pickett R.T. Ortaldo J.R. Kenny J.J. Komschlies K.L. Blood. 1997; 89: 583-595Crossref PubMed Google Scholar). Not surprisingly, IFN-γ expression patterns are tightly regulated in a tissue-specific manner by precise induction signals. Much effort has focused on characterizing the proximal IFNG promoter and intervening sequences in an effort to identify potential regulatory regions that are responsible for signal- and cell-specific control of gene expression. This approach has been useful in defining putative regulatory regions near the core IFNG promoter. It is notable, however, that regions proximal to the IFNG gene fail to explain tissue-specific patterns of IFN-γ expression as evidenced by transgenic mice expressing only the proximal IFNG promoter linked to a reporter gene (7Aune T.M. Penix L.A. Rincon M.R. Flavell R.A. Mol. Cell. Biol. 1997; 17: 199-208Crossref PubMed Scopus (79) Google Scholar, 8Soutto M. Zhou W. Aune T.M. J. Immunol. 2002; 169: 6664-6667Crossref PubMed Scopus (46) Google Scholar). In addition, transgenic mice that contain the full-length human IFNG gene and ∼2.7 kb of upstream sequence have similar signal-specific regulatory patterns to the endogenous gene, yet the responses are relatively weak (8Soutto M. Zhou W. Aune T.M. J. Immunol. 2002; 169: 6664-6667Crossref PubMed Scopus (46) Google Scholar). Therefore, it is likely that regions more distal from the core IFNG promoter are involved in determining tissue-specific and optimal signal-specific expression of IFN-γ (9Zhu H. Yang J. Murphy T.L. Ouyang W. Wagner F. Saparov A. Weaver C.T. Murphy K.M. J. Immunol. 2001; 167: 855-865Crossref PubMed Scopus (39) Google Scholar). In naïve T cells, induction signals for activation and IFN-γ expression are provided optimally through T cell receptor (TCR) and co-stimulatory receptor (CD28) engagement (10Ho I.C. Glimcher L.H. Cell. 2002; 109: S109-S120Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Following primary activation, differentiation of effector CD4+ T helper (Th) cell populations occurs over several days and is directed in large part by cytokines such as IL-2, IL-4, and IL-12 (11Murphy K.M. Reiner S.L. Nat. Rev. Immunol. 2002; 2: 933-944Crossref PubMed Scopus (1358) Google Scholar). Not only do IL-2 and IL-12 induce Th1 cell development, they also directly induce expression of IFN-γ. This is most apparent in NK cells, because no preceding activation/differentiation signals are required for cytokine responsiveness and transactivation of the IFNG gene (12Biron C.A. Nguyen K.B. Pien G.C. Cousens L.P. Salazar-Mather T.P. Annu. Rev. Immunol. 1999; 17: 189-220Crossref PubMed Scopus (1760) Google Scholar). Therefore, NK cell models offer an advantage in studying regulation of IFN-γ expression by isolating the effects of autonomous cytokine receptor signaling events. IL-12 acts via the transcription factor signal transducer and activator of transcription 4 (Stat4) that is thought to mediate trans-activation of the IFNG gene by binding to sequence motifs therein or enhancing the binding of AP-1 to the core IFNG promoter (13Nakahira M. Ahn H.J. Park W.R. Gao P. Tomura M. Park C.S. Hamaoka T. Ohta T. Kurimoto M. Fujiwara H. J. Immunol. 2002; 168: 1146-1153Crossref PubMed Scopus (209) Google Scholar). The requirement of Stat4 in regulating IFN-γ is underscored in Stat4-/- T cells and NK cells that are refractory to IL-12-induced IFN-γ expression. In both mice (14Nguyen K.B. Watford W.T. Salomon R. Hofmann S.R. Pien G.C. Morinobu A. Gadina M. O'Shea J.J. Biron C.A. Science. 2002; 297: 2063-2066Crossref PubMed Scopus (407) Google Scholar) and humans (15Barbulescu K. Becker C. Schlaak J.F. Schmitt E. Meyer Zum Buschenfelde K.H. Neurath M.F. J. Immunol. 1998; 160: 3642-3647PubMed Google Scholar), 2Morinobu, A., Kanno, Y., and O'Shea, J. J. (2004) J. Biol. Chem.279, 40640-40646. 2Morinobu, A., Kanno, Y., and O'Shea, J. J. (2004) J. Biol. Chem.279, 40640-40646. Stat4 has been shown to interact with the IFNG gene in the proximal promoter and/or first intron (16Xu X. Sun Y.L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (406) Google Scholar, 17Gonsky R. Deem R.L. Young H.A. Targan S.R. Eur. J. Immunol. 2003; 33: 1152-1162Crossref PubMed Scopus (14) Google Scholar). These elements are based on non-consensus STAT motifs, and their functional role is still not firmly established. Additionally, the human intronic STAT binding region is not present in murine genomic DNA, suggesting its importance in regulating IFNG gene expression may be a recent evolutionary event. Despite concentrated efforts and that fact that IFN-γ is highly regulated by cytokines, the identification of functional STAT binding elements in the IFNG gene has proved to be challenging. In the case of IL-2, the exact mechanisms by which this cytokine regulates IFN-γ have been surprisingly neglected, despite reports dating to 1983 indicating that IL-2 induces IFN-γ expression (18Kasahara T. Hooks J.J. Dougherty S.F. Oppenheim J.J. J. Immunol. 1983; 130: 1784-1789PubMed Google Scholar, 19Farrar W.L. Ruscetti F.W. Young H.A. J. Immunol. 1985; 135: 1551-1554PubMed Google Scholar, 20Reem G.H. Yeh N.H. Science. 1984; 225: 429-430Crossref PubMed Scopus (252) Google Scholar). IL-2 is an important factor in the differentiation of both Th1 and Th2 cells (11Murphy K.M. Reiner S.L. Nat. Rev. Immunol. 2002; 2: 933-944Crossref PubMed Scopus (1358) Google Scholar). Accordingly, this cytokine is routinely added to in vitro culture systems to enhance Th cell development and is thought to act via Stat5 (21Zhu J. Cote-Sierra J. Guo L. Paul W.E. Immunity. 2003; 19: 739-748Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). In IFN-γ-secreting cells, induction of IFN-γ mRNA by IL-2 is rapid and occurs at the level of transcription, as demonstrated by nuclear run-on analysis. The induction occurs in the presence of cycloheximide, indicating that protein synthesis is not necessary for the induction of transcription (22Young H.A. Birchenall-Sparks M. Kovacs E. Dorman L. Ruscetti F.W. J. Interferon Res. 1988; 8: 527-538Crossref PubMed Scopus (9) Google Scholar). Although IL-2 has long been associated with IFN-γ expression, the molecular mechanisms involved are still unknown. In this report, we identify and characterize a distal region of the IFNG gene that is a target for cytokine-driven epigenetic modifications, which correlate with competency of tissue-specific IFN-γ expression profiles. Contained within this same region is a distal cis-acting element that binds Stat5 proteins and enhances transcription of the IFNG promoter in an IL-2-dependent fashion. These data indicate that this distal 5′-flanking region of the IFNG gene is a critical part of the functional IFNG gene and provides the first indication of the molecular mechanisms involved in IL-2 regulation of IFN-γ expression. Antibodies and Cytokines—Antibodies directed against Stat5a, Stat5b, and Stat3 were purchased from R&D Systems and were used in supershift analyses as well as chromatin immunoprecipitation assays. Recombinant human IL-2 was obtained from Hoffmann-La Roche. Recombinant mouse IL-12 was generously provided by Genetics Institute (Cambridge, MA). Recombinant human IL-4 was purchased from R&D Systems. Anti-CD3ϵ, anti-CD28, anti-IL-4, anti-IL-12, and anti-IFN-γ antibodies were purchased from BD Pharmingen. Anti-acetylated H3, anti-acetylated H4, and normal rabbit IgG were from Upstate Biotechnology (Lake Placid, NY). Tissue Culture—NK92 cells were maintained in RPMI 1640 medium (BioWhittaker, Walkersville, MD), supplemented with 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 200 units/ml recombinant human IL-2, and 10 ng/ml recombinant human IL-15. Cells were cultured to a density of 5 × 105/ml in a 37 °C incubator with 5% CO2. Prior to stimulation, NK92 cells were starved overnight in medium containing 2% FCS, 2 mml-glutamine, and 100 units/ml of penicillin-streptomycin (starvation medium). Human PBMC were isolated from healthy volunteers over Ficoll-Hypaque, and for T cell differentiation studies, naïve CD4+ T cells were purified with the human CD4+/45RO- naïve T cell subset column kit or human CD4+ T cell subset column kit (both from R&D Systems, Minneapolis, MN) followed by positive selection using CD45RA beads (Mylteni Biotec Inc., Auburn, CA). Purity was typically >90%. Cells were stimulated with plate-bound anti-CD3 (10 μg/ml) plus anti-CD28 (5 μg/ml) Abs and IL-2 (50 units/ml) for 3 days, and expanded in a new flask for a further 3 days with exogenous IL-2. To generate Th1 cells, IL-12 (10 ng/ml) was added throughout the culture period and anti-IL-4 Ab (10 μg/ml) was added for the first 3 days. For Th2 cells, IL-4 (25 ng/ml) was added throughout the culture period, and anti-IL-12 Ab (5 μg/ml) was added for the first 3 days. Promoter Cloning and Reporter Constructs—Human genomic DNA (200 ng) was used as a template for all PCR reactions in 10- to 50-μl volumes. The full -3.662 kb to +38 bp IFNG 5′-flanking region was amplified in 10-μl PCR reactions containing 10 mm Tris-HCl (pH 8.3), 50 mm KCl, 1.5 mm MgCl2, 200 μm each of dATP, dGTP, dTTP, 80 ng of each oligonucleotide primer, and 1.5 units of High Fidelity Taq polymerase (Invitrogen). The primers used to amplify the -3.662 to +38 region are as follows with the MluI linkers underlined: 5′-GTA CGC GTG TGG AAA CAA GTG CAA AAC G-3′ and 5′-GTA CGC GTC TAA TAG CTG ATC TTC AGA TG-3′. For amplification of the -3.6-kb fragment, the conditions were as follows: 95 °C for 5 min, then 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 60 s, and extension at 68 °C for 5 min. These PCR reactions were performed using a Hybaid Touchdown thermal cycler (Hybaid Limited, UK). The -3.6-kb IFNG upstream region was amplified from genomic DNA with MluI linkers and ligated into a TA vector (Invitrogen). Following MluI and PvuI double digestion, the -3.6-kb insert was extracted from the 0.8% agarose gel, purified, and ligated into the PGL3 Basic Luciferase vector (Promega, Madison, WI). Restriction digests revealed clones with a 5′ to 3′ orientation, and sequencing confirmed the STAT element. Site-directed mutagenesis was performed on the -3.6-kb IFNG-Luc construct to disrupt the Stat5 motif from TTCAAGGAA to TTCAACCTT. DNA sequencing confirmed the mutagenesis. The mutations introduced in the Stat5 element are the same as those used in EMSA experiments, which failed to bind IL-2-induced Stat5 proteins and do not complete for Stat5 binding by excess cold competition. Electrophoretic Mobility Shift Assay—Complementary single-stranded oligonucleotides were commercially synthesized (Invitrogen) to span ∼10 bp on either side of the Stat element (in bold). Another oligonucleotide with a 4-bp mutation in the Stat site (underlined) was made, and the sequences are as follows: 5′-AGT TAT TAG AAA TTT CAA GGA AGT GAC AAC AGA G-3′ (Stat WT) and 5′-AGT TAT TAG AAA TTT CAACCT TGT GAC AAC AGA G-3′ (Stat Mutant). Complementary strands were annealed by combining 2 μg of each oligonucleotide and 6 μl of 10× annealing buffer (500 mm Tris, 100 mm MgCl2, and 50 mm dithiothreitol) in a 60-μl reaction, denatured in a boiling water bath for 5 min, and then allowed to cool to room temperature. Double-stranded DNA overhangs were labeled with Klenow enzyme by a fill-in reaction, [α-32P]dCTP and free dNTPs (Amersham Biosciences). The DNA-protein binding reaction was conducted in a 20-μl reaction mixture consisting of 5-10 μg of nuclear protein extract, 1 μg of poly(dI-dC) (Sigma), 4 μl of 5× binding buffer (60 mm Hepes, 7.5 mm MgCl2, 300 mm KCl, 1 mm EDTA, 2.5 mm dithiothreitol, 50% glycerol, and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) and 1.5 × 104 cpm of 32P-labeled oligonucleotide probe. For supershift analysis to identify Stat proteins interacting with the IFNG promoter, 1 μl of antibody was added to the reaction prior to the addition of the probes and incubated on ice for 20 min. In cold oligonucleotide competition experiments, 10- to 100-fold excess of unlabeled oligonucleotide probe was added 10 min prior to adding the radiolabeled oligonucleotide probe. Cytoplasmic and Nuclear Extraction—Nuclear extracts were prepared from PHA-stimulated human peripheral blood T cells or NK cells, as described previously (23Bream J.H. Ping A. Zhang X. Winkler C. Young H.A. Genes Immun. 2002; 3: 165-169Crossref PubMed Scopus (63) Google Scholar). Briefly, following no stimulation (NS) or treatment with cytokines for 30-60 min, cell pellets were resuspended in lysis buffer (50 mm KCl, 25 mm Hepes, pH 7.8, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 100 μm dithiothreitol) and subsequently incubated on ice for 5 min. Cellular suspensions were collected by centrifugation at 2,000 rpm, and the supernatant was harvested as the cytoplasmic protein fraction. Nuclei were washed in wash buffer (lysis buffer without Nonidet P-40) and harvested by centrifugation at 2,000 rpm. Nuclear pellets were resuspended in extraction buffer (500 mm KCl, 25 mm Hepes, pH 7.8, 5% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 100 μm dithiothreitol), frozen in dry ice, thawed slowly on ice, and finally centrifuged at 14,000 rpm for 10 min. The supernatant was harvested, and nuclear proteins were quantified with the bicinchoninic acid protein assay reagent (Pierce). Transfection Assays—PBMCs were transfected following overnight culture in RPMI 1640 medium containing 10% FCS with or without 10-100 units/ml rhIL-2 (Chiron Therapeutics, Emeryville, CA). Cells were then washed and resuspended in 250 μl of fresh medium at 2 × 107 cells/ml and electroporated in the presence of 50 μg of reporter construct (600 V, for 9 pulses of 500 μs, with 100 μs between pulses) using 4-mm (gap width) cuvettes in a BTX Electro Square Porator ECM 830 (Genetronics, Inc., San Diego, CA). A control plasmid containing the β-actin promoter driving a Renilla luciferase (provided by Dr. Christopher Wilson, University of Washington) was co-transfected as an internal standard, and values were normalized to correct for transfection efficiency (generally 30-50%). After electroporation the cells were diluted in fresh medium, allowed to rest for 1 h prior to plating, and then stimulated with 20 ng/ml PMA plus 400 nm calcium ionophore for 4 h. Luminescence was measured using a Promega luciferase assay kit and counted on a six-detector PerkinElmer Life Sciences 1450 Microbeta liquid scintillation counter with coincidence counting deactivated. Nuclei Isolation and DNase I Hypersensitivity Analysis—NK92 cells were rested in RPMI medium plus 10% FCS without IL-2 and IL-15 for 12 h. Approximately 100 × 106 cells were used for each stimulation. The cells were stimulated with IL-2 (100 units/ml) and IL-12 (10 units/ml) alone and in combination for 1 h. For nuclei isolation NK92 cells were spun at 600 × g, washed once in 1× phosphate-buffered saline, and spun again. The cell pellet was resuspended in 3 ml of cold lysis buffer consisting of 0.35 m sucrose, 60 mm KCl, 15 mm NaCl, 2 mm EDTA, 0.5 mm EGTA, 15 mm Hepes, pH 7.4, 0.6% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 0.15 mm spermine, 0.5 mm spermidine and incubated on ice for 5 min. The lysate was spun at 800 × g for 5 min at 4 °C. The nuclear pellet was washed twice in 2 ml of nuclei wash buffer (lysis buffer without Nonidet P-40) and spun at 800 × g for 5 min at 4 °C. The nuclei were resuspended in 500 μl of nuclei storage buffer consisting of 60 mm KCl, 15 mm NaCl, 0.1 mm EDTA, 0.1 mm EGTA, 75 mm Hepes, pH 7.5, glycerol (40% by vol.), 0.1 mm phenylmethylsulfonyl fluoride, 0.15 mm spermidine, 0.5 mm dithiothreitol, and stored at -70 °C until needed. Nuclei were spun at 800 × g and resuspended in a nuclease digest buffer consisting of 10 mm Tris-HCl, pH 7.5, 10 mm NaCl, 5 mm MgCl2, and 0.1 mm CaCl2. The nuclei were digested with increasing concentrations of DNase I (Roche Applied Science) that ranged from 0 to 0.4 unit per reaction for 10 min at 37 °C. The DNase digestion was stopped by the addition of an equal volume of stop solution consisting of 20 mm Tris-HCl, pH 7.5, 10 mm EDTA, 0.6 m NaCl, 1% SDS, and 400 μg/ml proteinase K, and the digests were incubated at 55 °C for overnight. The genomic DNA was purified from each reaction by phenol/chloroform and subsequent ethanol precipitation. The precipitated DNA was resuspended in 10 mm Tris, pH 7.4, and quantitated by optical density. Approximately 20 μg of DNase-treated DNA was digested with HindIII and separated by size on a 0.8% agarose gel using 1 × TBE (Tris/borate/EDTA) buffer. The DNA was transferred to a nylon membrane, and Southern analysis was performed using a 32P-labeled human IFN-γ-specific XcmI-HindIII DNA fragment as the probe. The DNase I hypersensitivity sites were visualized by exposing the blot to x-ray film for 24-36 h. Chromatin Immunoprecipitation Assays—ChIP assays were performed according to the manufacturer's instructions (Upstate Biotechnology, Lake Placid, NY). Briefly, 2 × 106 cells were fixed with 1% formaldehyde, washed with cold phosphate-buffered saline, and lysed in buffer containing 10 μg/ml aprotinin (ICN Biomedicals, Aurora, OH), 10 μg/ml leupeptin (Bachem, Torrance, CA), and 2.5 μm 4-nitrophenyl 4-guanidinobenzoate hydrochloride (Sigma). Nuclei were sonicated for a total of 60 s to shear DNA (Heat Systems, Farmingdale, NY), the lysates were pelleted, and supernatants were diluted. Diluted lysates were pre-cleared with salmon sperm DNA/protein A-agarose for 30 min prior to immunoprecipitation with specified antibodies. A proportion (2%) of the diluted supernatant was kept as “input” to quantify genomic DNA. After immunoprecipitation, the protein-DNA complexes were incubated with Protein A beads, and the protein-DNA complexes were eluted in 1% SDS/0.1 m NaHCO3, and cross-links were reversed at 65 °C. DNA was recovered by phenol-chloroform extraction and ethanol precipitation then subjected to PCR and/or real-time PCR analysis. PCR was carried out with AmpliTaq Gold (Applied Biosystems) for 35 cycles (40 s at 95 °C, 40 s at 59 °C, and 40 s at 72 °C), and the products were visualized by ethidium bromide staining. The immunoprecipitated DNA samples were normalized to “input” DNA samples. PCR primers for the distal IFNG promoter were as follows: 5′-GTTTCACTGCCAGTCTGG-3′ and 5′-ACTACACCCTATCAGGCTTTA-3′; primers for the proximal IFNG promoter region were 5′-GTTTCACTGCCAGTCTGG-3′ and 5′-ACTACACCCTATCAGGCTTTA-3′. Ribonuclease Protection Assay—RNase protection assays (RPA) were performed according to the manufacturer's specifications (Ambion, Austin, TX) with ∼5 μg of RNA per reaction. RNA hybridization products were separated by size on a 6% denaturing polyacrylamide gel. IL-2 Induces the Expression of IFN-γ mRNA—The process of Th cell differentiation is complicated and involves diverse extracellular signals through multiple classes of receptors, including the T cell receptor (TCR), co-stimulatory molecules, and cytokine receptors (1Szabo S.J. Sullivan B.M. Peng S.L. Glimcher L.H. Annu. Rev. Immunol. 2003; 21: 713-758Crossref PubMed Scopus (768) Google Scholar, 2Agnello D. Lankford C.S. Bream J. Morinobu A. Gadina M. O'Shea J.J. Frucht D.M. J. Clin. Immunol. 2003; 23: 147-161Crossref PubMed Scopus (310) Google Scholar, 11Murphy K.M. Reiner S.L. Nat. Rev. Immunol. 2002; 2: 933-944Crossref PubMed Scopus (1358) Google Scholar). This process typically: 1) occurs over an extended period of time (5-10 days), 2) seems to occur with cell division (24Reiner S.L. Mullen A.C. Hutchins A.S. Pearce E.L. Immunol. Res. 2003; 27: 463-468Crossref PubMed Scopus (5) Google Scholar), and 3) includes IL-2 in the cultures, either endogenously produced by T cells and/or by exogenous addition. We wanted to isolate the effects of IL-2 on IFNG gene regulation per se from the process of Th cell differentiation. Thus, we have primarily used fresh NK cells and an NK cell line (NK92) as a model to study cytokine-induced epigenetic and transcriptional modifications to the IFNG gene. We first confirmed that IL-2 treatment of NK92 cells leads to IFN-γ mRNA expression (Fig. 1A). Strong IFN-γ mRNA expression in NK92 was evident at 3 h and was slightly diminished after 6 h of IL-2 treatment. Our group and others have also reported that co-treatment of activated T cells or NK cells with IL-2 and IL-12 leads to a highly synergistic effect on IFN-γ expression (25Bream J.H. Curiel R.E. Yu C.R. Egwuagu C.E. Grusby M.J. Aune T.M. Young H.A. Blood. 2003; 102: 207-214Crossref PubMed Scopus (43) Google Scholar, 26Chan S.H. Perussia B. Gupta J.W. Kobayashi M. Pospisil M. Young H.A. Wolf S.F. Young D. Clark S.C. Trinchieri G. J. Exp. Med. 1991; 173: 869-879Crossref PubMed Scopus (957) Google Scholar). A time course experiment was performed on NK92 cells under IL-2, IL-12, and IL-2 plus IL-12 conditions to confirm these data (Fig. 1B). The results confirm that IL-2 and IL-12 induce IFN-γ mRNA expression rapidly and when added together, synergistically induce IFN-γ transcripts. These data are similar to what was observed in activated T cells (data not shown). Fig. 1C shows by real-time PCR the response to individual cytokine treatment and the synergistic response to IL-2 and IL-12 stimulation in NK92 cells. Thus, we determined that the NK92 cell line was an appropriate model to study IL-2-regulation of IFN-γ expression. Identification of a DNase I Hypersensitivity Site ∼3.5-4 kb Upstream of the IFNG Gene—DNase I HS mapping strategies have been used to successfully identify genomic regions that are functionally linked to gene expression. This approach allows for the investigation of the chromatin structure of the native gene in intact nuclei. It has been shown previously that DNase I HSs in the IFNG gene are found only in cells capable of transcribing the gene (27Hardy K.J. Peterlin B.M. Atchison R.E. Stobo J.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8173-8177Crossref PubMed Scopus (65) Google Scholar). Furthermore, the appearance or predominance of these sites in T cells is stimulation-sensitive, indicating a dynamic relationship between cell-specific competency for expression and transcription that is activation-dependent (28Hardy K.J. Manger B. Newton M. Stobo J.D. J. Immunol. 1987; 138: 2353-2358PubMed Google Scholar). Because the proximal 5′-flanking region of the IFNG gene fails to account for normal IFN-γ regulation (8Soutto M. Zhou W. Aune T.M. J. Immunol. 2002; 169: 6664-6667Crossref PubMed Scopus (46) Google Scholar), we sought to determine if more distal regions of the IFNG gene become remodeled upon cytokine stimulation. To identify potential regulatory regions in the IFNG gene we performed DNase I HS analysis using NK92 cells (Fig. 2). The results show the appearance of two bands by Southern blot analysis. The band of ∼12 kb represents the HindIII-HindIII restriction fragment, visible only after phenol-chloroform extraction and complete HindIII digestion. The 3′ HindIII site in the first intron is located at position +669 and the 5′ HindIII site is at position -11,780 relative to the transcriptional start site according to our nomenclature (29Bream J.H. Carrington M. O'Toole S. Dean M. Gerrard B. Shin H.D. Kosack D. Modi W. Young H.A. Smith M.W. Immunogenetics. 2000; 51: 50-58Crossref PubMed Scopus (71) Google Scholar). The lower band represents a single DNase I HS that was estimated to be ∼4.5 kb upstream of the 3′ HindIII restriction site in the first intron (Fig. 2, see arrows) and ∼3.5-4 kb 5′ to the transcriptional start site. The 4-kb HS was observed with the highest DNase I concentrations in unstimulated cells (lane 4), but when NK92 cells were treated fo
Referência(s)