Discrete Roles for Histone Acetylation in Human T Helper 1 Cell-specific Gene Expression
2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês
10.1074/jbc.m407576200
ISSN1083-351X
AutoresAkio Morinobu, Yuka Kanno, John J. O’Shea,
Tópico(s)Histone Deacetylase Inhibitors Research
ResumoTo better understand the control of T helper (TH) 1-expressed genes, we compared and contrasted acetylation and expression for three key genes, IFNG, TBET, and IL18RAP and found them to be distinctly regulated. The TBET and the IFNG genes, but not the IL18RAP gene, showed preferential acetylation of histones H3 and H4 during TH1 differentiation. Analysis of acetylation of specific histone residues revealed that H3(Lys-9), H4(Lys-8), and H4(Lys-12) were preferentially modified in TH1 cells, suggesting a possible contribution of acetylation of these residues for induction of these genes. On the other hand, the acetylation of IL18RAP gene occurred both in TH1 and TH2 cells the similar kinetics and on the same with residues, demonstrating that selective histone acetylation was not universally the case for all TH1-expressed genes. Histone H3 acetylation of IFNG and TBET genes occurred with different kinetics, however, and was distinctively regulated by cytokines. Interleukin (IL)-12 and IL-18 enhanced the histone acetylation of the IFNG gene. By contrast, histone acetylation of the TBET gene was markedly suppressed by IL-4, whereas IL-12 and IL-18 had only modest effects suggesting that histone acetylation during TH1 differentiation is a process that is regulated by various factors at multiple levels. By treating Th2 cells with a histone deacetylase inhibitor, we restored histone acetylation of the IFNG and TBET genes, but it did not fully restore their expression in TH2 cells, again suggesting that histone acetylation explains one but not all the aspects of TH1-specific gene expression. To better understand the control of T helper (TH) 1-expressed genes, we compared and contrasted acetylation and expression for three key genes, IFNG, TBET, and IL18RAP and found them to be distinctly regulated. The TBET and the IFNG genes, but not the IL18RAP gene, showed preferential acetylation of histones H3 and H4 during TH1 differentiation. Analysis of acetylation of specific histone residues revealed that H3(Lys-9), H4(Lys-8), and H4(Lys-12) were preferentially modified in TH1 cells, suggesting a possible contribution of acetylation of these residues for induction of these genes. On the other hand, the acetylation of IL18RAP gene occurred both in TH1 and TH2 cells the similar kinetics and on the same with residues, demonstrating that selective histone acetylation was not universally the case for all TH1-expressed genes. Histone H3 acetylation of IFNG and TBET genes occurred with different kinetics, however, and was distinctively regulated by cytokines. Interleukin (IL)-12 and IL-18 enhanced the histone acetylation of the IFNG gene. By contrast, histone acetylation of the TBET gene was markedly suppressed by IL-4, whereas IL-12 and IL-18 had only modest effects suggesting that histone acetylation during TH1 differentiation is a process that is regulated by various factors at multiple levels. By treating Th2 cells with a histone deacetylase inhibitor, we restored histone acetylation of the IFNG and TBET genes, but it did not fully restore their expression in TH2 cells, again suggesting that histone acetylation explains one but not all the aspects of TH1-specific gene expression. A critical aspect of the adaptive immune response is that naïve CD4+ T cells can develop into distinct T helper (TH) 1The abbreviations used are: TH, T helper; IL, interleukin; IFN, interferon; Stat, signal transducer and activator of transcription; IL18Rap, IL-18 receptor accessory protein; ChIP, chromatin immunoprecipitation; HDAC, histone deacetylase; Ab, antibody; mAb, monoclonal Ab. 1The abbreviations used are: TH, T helper; IL, interleukin; IFN, interferon; Stat, signal transducer and activator of transcription; IL18Rap, IL-18 receptor accessory protein; ChIP, chromatin immunoprecipitation; HDAC, histone deacetylase; Ab, antibody; mAb, monoclonal Ab. subsets depending on pathogens they encounter, and appropriate TH differentiation is essential for elimination of these pathogens (1Abbas A.K. Murphy K.M. Sher A. Nature. 1996; 383: 787-793Crossref PubMed Scopus (3844) Google Scholar). That is, TH1 cells are involved in cell-mediated immunity and critical in protection against intracellular pathogens, whereas TH2 cells are essential for a humoral immune response and elimination of extracellular microorganisms. TH1 and TH2 cells are principally characterized by the cytokines they secrete with TH1 cells producing interferon (IFN)-γ, tumor necrosis factor-α, and lymphotoxin, which activate macrophages and TH2 cells producing interleukin (IL)-4, IL-5, IL-10, and IL-13, which promote B cell immunoglobulin class switching (2Glimcher L.H. Murphy K.M. Genes Dev. 2000; 14: 1693-1711PubMed Google Scholar).A number of factors, including signals emanating from the T cell receptor are involved in the regulation TH1 and TH2 differentiation, but cytokines themselves are particularly important (3Murphy K.M. Reiner S.L. Nat. Rev. Immunol. 2002; 2: 933-944Crossref PubMed Scopus (1355) Google Scholar, 4Szabo S.J. Sullivan B.M. Peng S.L. Glimcher L.H. Annu. Rev. 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Bian Y. Hamaoka T. Ohta T. Kurimoto M. Fujiwara H. J. Immunol. 2001; 167: 1306-1312Crossref PubMed Scopus (57) Google Scholar). T-bet is another transcription factor that is important for TH1 differentiation, although it is not critical for IFN-γ production by CD8+ cells. T-bet-deficient mice exhibit defective TH1 differentiation and default TH2 response and consequently develop an airway inflammation similar to asthma (22Szabo S.J. Sullivan B.M. Stemmann C. Satoskar A.R. Sleckman B.P. Glimcher L.H. Science. 2002; 295: 338-342Crossref PubMed Scopus (968) Google Scholar, 23Finotto S. Neurath M.F. Glickman J.N. Qin S. Lehr H.A. Green F.H. Ackerman K. Haley K. Galle P.R. Szabo S.J. Drazen J.M. De Sanctis G.T. Glimcher L.H. Science. 2002; 295: 336-338Crossref PubMed Scopus (538) Google Scholar). T-bet also induces expression of IL-12 receptor β2, thus making these cells responsive to IL-12 (24Mullen A.C. High F.A. Hutchins A.S. Lee H.W. Villarino A.V. Livingston D.M. Kung A.L. Cereb N. Yao T.P. Yang S.Y. Reiner S.L. Science. 2001; 292: 1907-1910Crossref PubMed Scopus (679) Google Scholar, 25Afkarian M. Sedy J.R. Yang J. Jacobson N.G. Cereb N. Yang S.Y. Murphy T.L. Murphy K.M. Nat. Immunol. 2002; 3: 549-557Crossref PubMed Scopus (751) Google Scholar). T-bet expression is regulated by T cell receptor occupancy and cytokines, especially IFN-γ and IL-27 (25Afkarian M. Sedy J.R. Yang J. Jacobson N.G. Cereb N. Yang S.Y. Murphy T.L. Murphy K.M. Nat. Immunol. 2002; 3: 549-557Crossref PubMed Scopus (751) Google Scholar, 26Lighvani A.A. Frucht D.M. Jankovic D. Yamane H. Aliberti J. Hissong B.D. Nguyen B.V. Gadina M. Sher A. Paul W.E. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15137-15142Crossref PubMed Scopus (596) Google Scholar, 27Takeda A. Hamano S. Yamanaka A. Hanada T. Ishibashi T. Mak T.W. Yoshimura A. Yoshida H. J. Immunol. 2003; 170: 4886-4890Crossref PubMed Scopus (461) Google Scholar).Increasing evidence indicates that an important aspect of TH1 and TH2 differentiation is chromatin remodeling and the accessibility of the loci encoding IFN-γ and IL-4, respectively (28Smale S.T. Fisher A.G. Annu. Rev. Immunol. 2002; 20: 427-462Crossref PubMed Scopus (139) Google Scholar). For instance, naïve CD4+ T cells acquire distinctive patterns of DNase I hypersensitivity at the IFNG or IL4 loci during TH differentiation according to the respective subsets (29Agarwal S. Rao A. Immunity. 1998; 9: 765-775Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar). Overexpression of T-bet has been reported to enhance IFN-γ gene transcription and induce the DNase I hypersensitivity of the IFNG locus and in this manner is thought to promote TH1 differentiation, although a direct role of chromatin remodeling has not been established (24Mullen A.C. High F.A. Hutchins A.S. Lee H.W. Villarino A.V. Livingston D.M. Kung A.L. Cereb N. Yao T.P. Yang S.Y. Reiner S.L. Science. 2001; 292: 1907-1910Crossref PubMed Scopus (679) Google Scholar, 30Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2692) Google Scholar). Although sites of DNase I hypersensitivity are indicative of the accessibility of transcription factors, histone modification is also associated with chromosome structure accessibility and transcriptional activation of the locus (31Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). Recently, two groups (32Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 10: 10Google Scholar, 33Fields P.E. Kim S.T. Flavell R.A. J. Immunol. 2002; 169: 647-650Crossref PubMed Scopus (282) Google Scholar) have demonstrated increased histone acetylation in IFNG and IL4 loci during TH1/TH2 differentiation, respectively, and involvement of Stat4, T-bet, and Stat6 using knock-out mice. Thus, the role of epigenetic mechanisms in lineage commitment and regulation of distinct patterns of gene expression in TH1 and TH2 cells is an important area of investigation.Although TH1 differentiation is regulated by multiple cytokines and transcription factors (34Robinson D.S. O'Garra A. Immunity. 2002; 16: 755-758Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), exactly how histone acetylation might play a role in the expression of TH1 genes has not been determined. In this study we set out to compare and contrast acetylation and expression for three TH1 genes, IFNG, TBET, and IL18RAP, focusing on the differentiation of human cells. Surprisingly, we found that the acetylation of these three genes is regulated quite differently. We showed that histone acetylation of the TBET gene, like the IFNG gene, occurs preferentially during TH1 differentiation. However the modifications of the genes occurred with different kinetics, and the principal cytokines that govern these modifications are distinct; IFNG gene acetylation is positively regulated by IL-12, IL-18, and Stat4, whereas the TBET gene is predominantly negatively regulated by IL-4. Selective histone acetylation, however, was not universally the case for all genes expressed in TH1 cells. Despite its preferential expression, the IL18RAP gene did not exhibit selective histone acetylation. IFN-γ gene expression correlated well with the extent of histone acetylation of IFNG gene promoter region. However pharmacological restoration of IFNG gene acetylation in TH2 cells did not fully recovered IFN-γ gene expression, indicating that histone acetylation is not sufficient to explain all aspects of preferential gene expression.EXPERIMENTAL PROCEDURESReagents—Human IL-4, IL-12, and IL-18 were purchased from R&D Systems, Minneapolis, MN, and human IL-2 was from Dr. Reynolds, NCI, Frederick, MD. Anti-CD3, anti-CD28, anti-IL-4, anti-IL-12, and anti-IFN-γ antibodies were purchased from Pharmingen. Anti-acetylated H3, anti-acetylated H4, anti-acetylhistone H3(Lys-9), anti-acetylhistone H3(Lys-14), and normal rabbit IgG were from Upstate Biotechnology, Lake Placid, NY. Anti-acetylhistone H4(Lys-8) and antiacetylhistone H4(Lys-12) were from Abcam, Inc, Cambridge MA. Anti-Stat4 was from Zymed Laboratory, San Francisco, CA. Sodium butyrate was purchased from Sigma.Cell Culture—Human peripheral blood mononuclear cells were isolated from healthy volunteer cells over Ficoll-HyPaque, and naïve CD4+ T cells were purified with human CD4+/45RO– naïve T cell subset column kit (R&D Systems) or human CD4+ T cell subset column kit (R&D Systems) followed by positive selection using CD45RA beads (Mylteni Biotec Inc., Auburn, CA). Purity was usually >90%. Cells were stimulated with plate-bound anti-CD3 (10 μg/ml) plus anti-CD28 (5 μg/ml) antibodies (Abs) and IL-2 (50 units/ml) for 3 days and expanded in a new flask for an extra 3 days with exogenous IL-2. To induce 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. To induce 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. IL-18 (10 ng/ml) and anti-IFN-γ Ab (10 μg/ml) were added throughout the culture period when indicated.Gene Expression in TH1 and TH2 Cells—TH1 and TH2 cells were generated as described above, harvested at day 6, and left unstimulated or stimulated with plate-bound CD3 mAb for 5 h, and mRNA was isolated. First strand cDNA synthesis and real time PCR were done as described previously (35Morinobu A. Gadina M. Strober W. Visconti R. Fornace A. Montagna C. Feldman G.M. Nishikomori R. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12281-12286Crossref PubMed Scopus (142) Google Scholar). Commercial assay reagents for IFN-γ and glyceraldehyde-3-phosphate dehydrogenase were purchased from Applied Biosystems (Foster City, CA). The sequences of primer pairs and probes for T-bet were described previously, and those for IL18Rap are 5′-CCCACAGTTACTTGGAGAGGCTTA-3′, 5′-GGCATGTGGTAGCGCATTT-3′, and 6-carboxyfluorescein-5′-AGTTCCTCCCAATTCTAGGTTCTGGGCC-3′-6-carboxytetramethylrhodamine (26Lighvani A.A. Frucht D.M. Jankovic D. Yamane H. Aliberti J. Hissong B.D. Nguyen B.V. Gadina M. Sher A. Paul W.E. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 15137-15142Crossref PubMed Scopus (596) Google Scholar). Levels of gene expression are normalized to glyceraldehyde-3-phosphate dehydrogenase expression.Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was carried out essentially according to manufacturer's instructions (Upstate Biotechnology). Briefly, 1–2 × 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 to shear DNA (Heat Systems, Farmingdale, NY), sedimented, and diluted supernatants were immunoprecipitated with antibodies. A proportion (2%) of the diluted supernatants were kept as "input." The bead-bound protein-DNA complexes were eluted in 1% SDS, 0.1 m NaHCO3, and cross-links were reversed at 65 °C. DNA was recovered by phenolchloroform extraction and ethanol precipitation and subjected to PCR 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 sequences of the primer pairs used are listed in Table I. In selected experiments, real time PCR was performed to quantify the ChIP assay. The sequences of primer pairs and probes used are listed in Table II. The amount of the immunoprecipitated DNA samples was normalized against input DNA samples.Table IOligonucleotide sequences of PCR primer pairs for ChIPGeneForward/reverse primer sequenceshIFNG locusa5′-GTTTCACTGCCAGTCTGG-3′5′-ACTACACCCTATCAGGCTTTA-3′b5′-CACTTGTTCCCAACCACAAG-3′5′-GTGGCATTTGGGTGTTGTAG-3′c5′-GGTGGGAACTCATTCAGTTG-3′5′-GCAACGAAGTTCCGGAATAC-3′d5′-TGATGACCTTCTTTGCTCCAA-3′5′-AGGAGTTCTCCAGCTGAGAT-3′e5′-TAGCTGGCTCAAGGTCAGAA-3′5′-GCTAATGTCATGATGGTCAGT-3′f5′-TTCTACCAGCTGTGGAACGT-3′5′-AAGAGTGGAGACACCAGTAG-3′Intron 15′-CTTCGTTGCTCACTGGGATT-3′5′-TGTGCCTTCCTGTAGGGTAT-3′hTBET locusa5′-AGGCACGTGAGGTTGACTTT-3′5′-GAGTTGGCTGCATCTTGTAG-3′b5′-AATCTGTTACTCTGGCACAAC-3′5′-TGTCTCTAACCTTAGCTGCTT-3′c5′-ACTGTGTAAGCTACTGTTACC-3′5′-CCTGACGTCAGGAGTTCG-3′ Open table in a new tab Table IIOligonuceotide sequences for real time PCR for quantitative ChIPGeneSequences (forward and reverse primers and a probe)hTBET-p5′-GAATGTTCATGGTCAAAGTTGCTT-3′5′-GGCATTCTCCTGGCACTCA-3′6FAM-5′-TGTGCCCGTGCTCCGAGCCT-3′-TAMRAhIFNG-p5′-TGGTGTGAAGTAAAAGTGCCTTCA-3′5′-CGATGAGACAGACCCATTATGC-3′6FAM-5′-AGAATCCCACCAGAATGGCACAGGTG-3′-TAMRAhIL18RAP-p5′-TCCTAAGGCTTCCCCAGGA-3′5′-GTTCTGCAGGCCACACAGAA-3′6FAM-5′-AAGATGCCGCTCTGC-3′-MGB Open table in a new tab RESULTSPreferential Histone Acetylation of the IFNG Gene in Human TH1 Cells—Previous reports in mouse cells have indicated that the proximal promoter region of the IFNG gene becomes hyperacetylated in TH1 cells compared with TH2 cells (32Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 10: 10Google Scholar, 33Fields P.E. Kim S.T. Flavell R.A. J. Immunol. 2002; 169: 647-650Crossref PubMed Scopus (282) Google Scholar). To compare the acetylation of both histone H3 and histone H4 in the human IFNG gene, we prepared a series of primer pairs throughout this locus (Fig. 1A). As shown in Fig. 1B (primer a), the proximal promoter of the human IFNG gene also exhibits histone acetylation in TH1 cells. Interestingly these modifications were observed throughout the IFNG gene, although the differential modification between TH1 and TH2 cells was more evident in the areas surrounding the coding regions than the promoter. Specifically, the proximal promoter region (primer a), the first and second introns (primers b, c, and d), and the 1.4-kb downstream region (primer e) were also hyperacetylated in TH1 cells. To confirm these data, we conducted quantitative ChIP assays using real time PCR in which the IFNG promoter region was amplified (Fig. 1A). Histone H3 and H4 acetylation of the IFNG promoter was higher in TH1 cells than in TH2 cells (Fig. 1C).Stat4 is presumably important for IFNG acetylation, because Stat4-deficient mice show impaired histone acetylation of the IFNG gene (32Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 10: 10Google Scholar, 33Fields P.E. Kim S.T. Flavell R.A. J. Immunol. 2002; 169: 647-650Crossref PubMed Scopus (282) Google Scholar); however a direct role for Stat4 in IFNG gene regulation has not been established. Thus, we set out to determine whether Stat4 is recruited to the regions of the IFNG gene (proximal promoter and the first intron), which have been suggested to be Stat4 binding sites (36Xu X. Sun Y.L. Hoey T. Science. 1996; 273: 794-797Crossref PubMed Scopus (406) Google Scholar). Purified CD4+ T cells were stimulated with anti-CD3 and CD28 mAbs for 2 days, rested overnight, and then restimulated with IL-12 for 1 h. Using the ChIP assay, we found that Stat4 binds to both sites on the IFNG gene in response to IL-12 (Fig. 1D). This then provides the first direct evidence of Stat4 binding to the IFNG gene in differentiating CD4+ T cells and thus raises the possibility that Stat4 is involved in the histone acetylation of the entire IFNG locus.Histone Acetylation of the TBET Locus in Human TH1 Cells—Multiple lines of evidence point to T-bet being not only a TH1-specific gene but also a regulator of the accessibility of the IFNG gene (25Afkarian M. Sedy J.R. Yang J. Jacobson N.G. Cereb N. Yang S.Y. Murphy T.L. Murphy K.M. Nat. Immunol. 2002; 3: 549-557Crossref PubMed Scopus (751) Google Scholar, 30Szabo S.J. Kim S.T. Costa G.L. Zhang X. Fathman C.G. Glimcher L.H. Cell. 2000; 100: 655-669Abstract Full Text Full Text PDF PubMed Scopus (2692) Google Scholar). Thus, we next examined the magnitude and kinetics of histone acetylation of the TBET locus in differentiating human TH cells. T-bet gene expression was 4–5-fold higher in TH1 cells than in TH2 cells in our system (e.g. see below, Fig. 6C). Histone acetylation in three different regions of TBET locus examined was also increased in TH1 cells (Fig. 2B), which was confirmed using real time PCR ChIP assay (Fig. 2C). Thus, we provide the first evidence that TBET loci are hyperacetylated in human TH1 cells. The kinetics of modification of the TBET and IFNG genes is presented below.Fig. 6Effects of IL-4, IL-12, and IL-18 on gene expression levels and histone H3 acetylation of IFNG and TBET genes. CD4+ naïve TH cells were differentiated into T cells under indicated conditions. A and C, basal and CD3-induced levels of IFN-γ and T-bet mRNA expression. Levels of mRNA expression were determined by real time PCR and expressed as a ratio to that of non-stimulated CD4+ T cells cultured with anti-IL-4 Ab only. B and D, quantitative ChIP analysis for histone H3 acetylation of IFNG and TBET promoter regions. Results were expressed as a ratio to that of CD4+ T cells cultured with anti-IL-4 Ab only. Representative results of three separate experiments were shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2Histone acetylation of TBET genes in TH1 and TH2 cells.A diagram of the human TBET locus and the regions (not to scale) amplified for the ChIP assay. B, ChIP assay for acetylated (ac) histone H3 and H4 between TH1 and TH2 cells throughout the TBET gene. C, quantitative ChIP assay for the TBET promoter (TBET-P) region. Results were expressed as a ratio to that of TH1 cells. Data are representative of more than three similar experiments. In, input; C, control antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Histone Acetylation of Specific Lysine Residues of Histone H3 and H4 in Human TH1 Cells—Specific lysine residues on histone tails can be modified including acetylation, and it has been hypothesized that a specific pattern of histone tail modification could determine the transcriptional activity of the gene (31Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7538) Google Scholar). For instance, the acetylation of H3(Lys-9) and H4(Lys-8) correlate well with overall H3 and H4 acetylation of the IFN-β promoter following virus stimulation (37Agalioti T. Chen G. Thanos D. Cell. 2002; 111: 381-392Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). To confirm and extend the data presented above (Figs. 1 and 2), we next asked whether preferential acetylation of H3(Lys-9), H3(Lys-14), H4(Lys-8), and H4(Lys-12) occurred in the IFNG and TBET promoters in TH1 cells. As shown in Fig. 3, H3(Lys-9), H4(Lys-8), and H4(Lys-12) are preferentially acetylated in both IFNG and TBET genes in TH1 cells as determined by quantitative ChIP assay. On the other hand, H3(Lys-14) acetylation did not distinguish TH1 and TH2 cells. Therefore, acetylation of H3(Lys-9), H4(Lys-8), and H4(Lys-12) residues may contribute to the preferential induction of IFN-γ and T-bet genes in TH1 cells, whereas a modification of H3(Lys-14) would presumably not.Fig. 3Preferential acetylation of the IFNG and TBET genes on specific histones H3 and H4 lysine residues in TH1 and TH2 cells. A quantitative PCR of ChIP samples was performed in triplicate using the IFNG promoter (IFNG-P) (A and B) and TBET promoter (TBET-P)(C and D). Antibodies against acetylated Lys-9 and Lys-14 of H3 or Lys-8 and Lys-12 of H4 were used for immunoprecipitation and anti-total-acetyl H3 was also included as a reference control. The immunoprecipitated DNA samples were normalized against input DNA samples and expressed as a ratio to that of total-acetyl H3 in TH1 cells. AcK, acetylated lysine.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Histone Acetylation of the IL18RAP Locus in Human TH1 Cells—We next examined the histone modification of another TH1-expression gene, the IL18RAP gene. The IL18RAP promoter region lies between IL18R1 and IL18RAP genes both of which are TH1-specific IL-12-inducible genes (18Chan W.L. Pejnovic N. Lee C.A. Al-Ali N.A. J. Immunol. 2001; 167: 1238-1244Crossref PubMed Scopus (73) Google Scholar, 38Rogge L. Bianchi E. Biffi M. Bono E. Chang S.Y. Alexander H. Santini C. Ferrari G. Sinigaglia L. Seiler M. Neeb M. Mous J. Sinigaglia F. Certa U. Nat. Genet. 2000; 25: 96-101Crossref PubMed Scopus (195) Google Scholar). As expected, IL18Rap and IL-18 receptor 1 mRNA were higher in TH1 cells (Fig. 4A and data not shown). Surprisingly, however, histone acetylation of the IL18RAP promoter was equivalent in TH1 and TH2 cells as indicated by quantitative ChIP assay (Fig. 4B). The levels of histone acetylation did not differ between TH1 and TH2 cells on both days 2 and 6 of differentiation, suggesting similar kinetics of histone acetylation in these cells. We also compared the residue-specific acetylation of the IL18RAP promoter using the same battery of antibodies described above and observed no significant difference in acetylation of individual residues (Fig. 4, C and D). Thus histone acetylation of the IL18RAP occurs on the same residues in both TH1 and TH2 cells with similar kinetics and to the same extent.Fig. 4Histone acetylation of IL18RAP genes in TH1 and TH2 cells.A basal IL18Rap mRNA expression by TH1 and TH2 cells. Levels of mRNA expression were determined by real time PCR and expressed as a ratio to that of TH1 cells. B, quantitative ChIP assay for acetylation of the IL18RAP promoter (IL18RAP-P) from cells cultured under TH1 or TH2 conditions on days 2 and 6. Values were normalized to the levels obtained from cells cultured under TH1 conditions on day 6. C and D, acetylation of individual lysine residues on histones H3 and H4 for IL18RAP-P in TH1 versus TH2 cells. Quantitative ChIP was carried out as described in the legend to Fig. 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Collectively, these results indicate that the re
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