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Unconventional Association of the Polycomb Group Proteins with Cytokine Genes in Differentiated T Helper Cells

2008; Elsevier BV; Volume: 283; Issue: 19 Linguagem: Inglês

10.1074/jbc.m709886200

1083-351X

Eyal Jacob, Reut Hod Dvorai, Sagie Schif‐Zuck, Orly Avni,

Immune Cell Function and Interaction

The cytokine transcription profiles of developing T helper 1 and T helper 2 cells are imprinted and induced appropriately following stimulation of differentiated cells. Epigenetic regulation combines several mechanisms to ensure the inheritance of transcriptional programs. We found that the expression of the polycomb group proteins, whose role in maintaining gene silencing is well documented, was induced during development in both T helper lineages. Nevertheless, the polycomb proteins, YY1, Mel-18, Ring1A, Ezh2, and Eed, bound to the Il4 and Ifng loci in a differential pattern. In contrast to the prevailing dogma, the binding activity of the polycomb proteins in differentiated T helper cells was associated with cytokine transcription. The polycomb proteins bound to the cytokine genes under resting conditions, and their binding was induced dynamically following stimulation. The recruitment of the polycomb proteins Mel-18 and Ezh2 to the cytokine promoters was inhibited in the presence of cyclosporine A, suggesting the involvement of NFAT. Considering their binding pattern at the cytokine genes and their known function in higher order folding of regulatory elements, we propose a model whereby the polycomb proteins, in some contexts, positively regulate gene expression by mediating long-distance chromosomal interactions. The cytokine transcription profiles of developing T helper 1 and T helper 2 cells are imprinted and induced appropriately following stimulation of differentiated cells. Epigenetic regulation combines several mechanisms to ensure the inheritance of transcriptional programs. We found that the expression of the polycomb group proteins, whose role in maintaining gene silencing is well documented, was induced during development in both T helper lineages. Nevertheless, the polycomb proteins, YY1, Mel-18, Ring1A, Ezh2, and Eed, bound to the Il4 and Ifng loci in a differential pattern. In contrast to the prevailing dogma, the binding activity of the polycomb proteins in differentiated T helper cells was associated with cytokine transcription. The polycomb proteins bound to the cytokine genes under resting conditions, and their binding was induced dynamically following stimulation. The recruitment of the polycomb proteins Mel-18 and Ezh2 to the cytokine promoters was inhibited in the presence of cyclosporine A, suggesting the involvement of NFAT. Considering their binding pattern at the cytokine genes and their known function in higher order folding of regulatory elements, we propose a model whereby the polycomb proteins, in some contexts, positively regulate gene expression by mediating long-distance chromosomal interactions. When naive T helper (Th) 3The abbreviations used are: Th, T helper; PcG, Polycomb group; TrxG, Trithorax group; DH, DNase I hypersensitivity; PRC, PcG repressive complex; HDACs, histone deacetylases; HMTase, histone methyltransferase; DNMT, DNA methyltransferase; CsA, cyclosporine A; HATs, histone acetyltransferases; CNS, conserved non-coding sequence; PI, propidium iodide; ChIP, chromatin immunoprecipitation assay; IFN, interferon; IL, interleukin; TCR, T cell receptor; STAT, signal transducer and activator of transcription; NFAT, nuclear factor of activated T cells. 3The abbreviations used are: Th, T helper; PcG, Polycomb group; TrxG, Trithorax group; DH, DNase I hypersensitivity; PRC, PcG repressive complex; HDACs, histone deacetylases; HMTase, histone methyltransferase; DNMT, DNA methyltransferase; CsA, cyclosporine A; HATs, histone acetyltransferases; CNS, conserved non-coding sequence; PI, propidium iodide; ChIP, chromatin immunoprecipitation assay; IFN, interferon; IL, interleukin; TCR, T cell receptor; STAT, signal transducer and activator of transcription; NFAT, nuclear factor of activated T cells. cells (CD4+) encounter antigen in the periphery, they can differentiate into several lineages distinguished by their cytokine production. The most studied are the Th1 and Th2 lineages. The hallmark cytokine of Th1 cells is IFNγ while Th2 cells transcribe IL-4, IL-5, and IL-13. IFNγ exerts protective functions in microbial infections and is observed clinically in cases of autoimmune disease. In contrast, IL-4 is strongly apparent during parasitic infections and is associated with deleterious allergic reactions. Lineage polarization can be achieved in vitro by manipulating the cytokine milieu: IL-12 and IL-4 strongly potentiate Th1 and Th2 differentiation, respectively, via the transcription factors STAT4 and STAT6. The lineage-specific transcription factors T-bet and GATA3 are critical for Th1 and Th2 differentiation, respectively; when ectopically expressed these proteins not only promote transcription of the relevant cytokines but also suppress expression of the inappropriate cytokines (1Ansel K.M. Djuretic I. Tanasa B. Rao A. Annu. Rev. Immunol. 2006; 24: 607-656Crossref PubMed Scopus (504) Google Scholar, 2Lee G.R. Kim S.T. Spilianakis C.G. Fields P.E. Flavell R.A. Immunity. 2006; 24: 369-379Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The antigen-inducible transcription factor NFAT1 is important for the expression of both Th1 and Th2 cytokine genes (3Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2125) Google Scholar). Although NFAT1 is activated and enters the nucleus equivalently in both lineages, we have previously shown that NFAT1 binds the cytokine genes, in vivo, in a restricted manner: NFAT1 binds the Ifng regulatory elements only in stimulated Th1 cells and the Il4 regulatory elements only in stimulated Th2 cells (4Agarwal S. Avni O. Rao A. Immunity. 2000; 12: 643-652Abstract Full Text Full Text PDF PubMed Google Scholar). Looking for an explanation, we and others found that the presence of the polarizing cytokines, IL-12 and IL-4, as well as their downstream transcription factors is necessary to establish a differential pattern of histone acetylation at the cytokine genes (5Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar, 6Fields P.E. Kim S.T. Flavell R.A. J. Immunol. 2002; 169: 647-650Crossref PubMed Google Scholar, 7Messi M. Giacchetto I. Nagata K. Lanzavecchia A. Natoli G. Sallusto F. Nat. Immunol. 2003; 4: 78-86Crossref PubMed Scopus (288) Google Scholar, 8Yamashita M. Ukai-Tadenuma M. Kimura M. Omori M. Inami M. Taniguchi M. Nakayama T. J. Biol. Chem. 2002; 277: 42399-42408Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The hyperacetylated status of the cytokine genes correlates with the selective binding of NFAT1, and therefore we suggested that the differentiation process of Th cells reinforces chromatin structural changes, that facilitate differential accessibility to acute transcription factors (5Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar). The polarizing cytokines and downstream transcription factors are also crucial for the establishment of other epigenetic features of the cytokine genes in developing Th cells (1Ansel K.M. Djuretic I. Tanasa B. Rao A. Annu. Rev. Immunol. 2006; 24: 607-656Crossref PubMed Scopus (504) Google Scholar, 2Lee G.R. Kim S.T. Spilianakis C.G. Fields P.E. Flavell R.A. Immunity. 2006; 24: 369-379Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). However, even in the absence of the polarizing cytokines, differentiated Th1 and Th2 cells memorize their previous cytokine expression profiles and transiently transcribe the appropriate cytokines upon T cell receptor (TCR) stimulation. The activity of STATs is inducible; therefore it is unlikely that they maintain constitutively the poised transcriptional status of the cytokine genes in memory cells (7Messi M. Giacchetto I. Nagata K. Lanzavecchia A. Natoli G. Sallusto F. Nat. Immunol. 2003; 4: 78-86Crossref PubMed Scopus (288) Google Scholar, 9Yamashita M. Shinnakasu R. Nigo Y. Kimura M. Hasegawa A. Taniguchi M. Nakayama T. J. Biol. Chem. 2004; 279: 39454-39464Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The simplest and most plausible idea is that lineage-specific transcription factors downstream of the STATs sustain the open chromatin configuration. The question of whether GATA3 is necessary for Il4 transcription in differentiated Th2 cells was studied by several groups using conditional ablation approaches (10Zhu J. Min B. Hu-Li J. Watson C.J. Grinberg A. Wang Q. Killeen N. Urban Jr., J.F. Guo L. Paul W.E. Nat. Immunol. 2004; 5: 1157-1165Crossref PubMed Scopus (450) Google Scholar, 11Pai S.Y. Truitt M.L. Ho I.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1993-1998Crossref PubMed Scopus (264) Google Scholar, 12Yamashita M. Ukai-Tadenuma M. Miyamoto T. Sugaya K. Hosokawa H. Hasegawa A. Kimura M. Taniguchi M. DeGregori J. Nakayama T. J. Biol. Chem. 2004; 279: 26983-26990Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In all of these cases, a substantial percentage (11Pai S.Y. Truitt M.L. Ho I.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1993-1998Crossref PubMed Scopus (264) Google Scholar, 12Yamashita M. Ukai-Tadenuma M. Miyamoto T. Sugaya K. Hosokawa H. Hasegawa A. Kimura M. Taniguchi M. DeGregori J. Nakayama T. J. Biol. Chem. 2004; 279: 26983-26990Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), or even all (10Zhu J. Min B. Hu-Li J. Watson C.J. Grinberg A. Wang Q. Killeen N. Urban Jr., J.F. Guo L. Paul W.E. Nat. Immunol. 2004; 5: 1157-1165Crossref PubMed Scopus (450) Google Scholar) of the IL-4-producing differentiated Th2 cells maintain their ability to produce IL-4 after in vitro deletion of Gata3 (although the expression levels were decreased (10Zhu J. Min B. Hu-Li J. Watson C.J. Grinberg A. Wang Q. Killeen N. Urban Jr., J.F. Guo L. Paul W.E. Nat. Immunol. 2004; 5: 1157-1165Crossref PubMed Scopus (450) Google Scholar)). In parallel, T-bet (13Szabo 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 Google Scholar), which is important for the establishment of the permissive chromatin configuration of the Ifng gene in developing Th1 cells (5Avni O. Lee D. Macian F. Szabo S.J. 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Van Eynde A. Bernard D. Vanderwinden J.M. Bollen M. Esteller M. Di Croce L. de Launoit Y. Fuks F. Nature. 2005; 439: 871-874Crossref PubMed Scopus (1568) Google Scholar). Whether these modifications are the cause or the consequence of the transcriptional maintenance program is unclear as yet (24Ringrose L. Paro R. Development. 2007; 134: 223-232Crossref PubMed Scopus (360) Google Scholar). This study started with the aim of identifying epigenetic regulators that are expressed similarly in both Th1 and Th2 cells, but differentially bind the cytokine genes. While the PcG proteins meet these criteria, their bulk binding activity was found, unexpectedly, to correlate with the competence to transcribe the cytokine gene and even stronger with active transcription. The expression of the PcG proteins was induced during the development of Th1 and Th2 cells. Their binding activity in the differentiated cells was dynamically modified following stimulation and was inhibited in the presence of cyclosporine A (CsA). We suggest that the known function of the PcG proteins in pairing DNA elements (19Bantignies F. Cavalli G. Curr. Opin. Cell Biol. 2006; 18: 275-283Crossref PubMed Scopus (108) Google Scholar, 22Levine S.S. King I.F. Kingston R.E. Trends Biochem. Sci. 2004; 29: 478-485Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 23Muller J. Kassis J.A. Curr. Opin. Genet. Dev. 2006; 16: 476-484Crossref PubMed Scopus (0) Google Scholar, 25Schuettengruber B. Chourrout D. Vervoort M. Leblanc B. Cavalli G. Cell. 2007; 128: 735-745Abstract Full Text Full Text PDF PubMed Scopus (1045) Google Scholar, 26Schwartz Y.B. Pirrotta V. Nat. Rev. Genet. 2007; 8: 9-22Crossref PubMed Scopus (678) Google Scholar, 27Sparmann A. van Lohuizen M. Nat. Rev. Cancer. 2006; 6: 846-856Crossref PubMed Scopus (1015) Google Scholar, 47Francis N.J. Kingston R.E. Nat. Rev. Mol. Cell. Biol. 2001; 2: 409-421Crossref PubMed Scopus (296) Google Scholar), is not necessarily associated with gene repression, but rather, under some circumstances, it may support active gene expression or maintain transcriptional status by mediating long-distance chromosomal interactions (2Lee G.R. Kim S.T. Spilianakis C.G. Fields P.E. Flavell R.A. Immunity. 2006; 24: 369-379Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Mice—3-4-week-old female BALB/c mice were purchased from Harlan Biotech, Israel and maintained under pathogen-free conditions in the animal facility of the Faculty of Medicine, Technion-Israel Institute of Technology. In Vitro Th Cell Differentiation—Th cell differentiation was carried out as previously described (5Avni O. Lee D. Macian F. Szabo S.J. Glimcher L.H. Rao A. Nat. Immunol. 2002; 3: 643-651Crossref PubMed Google Scholar). Briefly, CD4+ T cells were purified from the spleen and lymph nodes of 3-4-week-old mice with magnetic beads (Dynal). For Th differentiation, cells were stimulated with 1 μg/ml anti-CD3ϵ antibodies and 1 μg/ml anti-CD28 antibodies (145.2C11 and 37.51, respectively, Pharmingen) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, l-glutamine, penicillin-streptomycin, nonessential amino acids, sodium pyruvate, vitamins, HEPES, and 2-mercaptoethanol in a flask coated with 0.3 mg/ml goat anti-hamster antibodies (ICN). For Th1 differentiation, the cells were stimulated in the presence of 10 ng/ml recombinant mouse IL-12 (R&D systems) and 10 μg/ml purified anti-IL-4 antibodies (11B11). For Th2 differentiation, cells were stimulated in the presence of 1000 units/ml mouse IL-4 (added as a supernatant of the 13L6 cell line), 5 μg/ml purified anti-IFNγ antibodies (XMG1.2), and 3 μg/ml purified anti-IL-12 antibodies (C178). After 2 days, the medium was expanded (4-fold) in the absence of anti-TCR and anti-CD28 antibodies, but in the continued (although reduced) presence of cytokines and antibodies, which included 12 units/ml IL-2. The medium was then expanded every other day. After 8 days, differentiated Th cells were left unstimulated or were stimulated with PMA (15 nm) and ionomycin (0.75 μm). When indicated, 2 μm CsA was added 0.5 h before stimulation. Chromatin Immunoprecipitation (ChIP)—ChIP analysis was based on the described protocol (48Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Google Scholar), with several modifications. Briefly, cells (10-24 × 107) were cross-linked on ice for 20 min, by adding a one-tenth volume of 11% formaldehyde solution (0.1 m NaCl, 1 mm EDTA, 0.5 mm EGTA, 50 mm HEPES, pH 8.0) directly to the media. Glycine was then added to a final concentration of 0.125 m. Following incubation in 100 mm Tris-HCl, pH 9.4, 10 mm dithiothreitol, the cells were washed and resuspended in 0.5 ml of lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl, pH 8.1, 1 mm phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 10 mm iodoacetamide) and sonicated three times with 1 min intervals at 4 °C. The samples were centrifuged at 14,000 rpm at 12 °C, and the cleared supernatants were diluted with equal amounts of dilution buffer (1% Triton X-100, 2 mm EDTA, 150 mm NaCl, 20 mm Tris-HCl, pH 8.1, and protease inhibitors). Aliquots containing ∼3 × 107 cells were stored at -80 °C. The samples were thawed in 1 ml of dilution buffer and precleared with 45 μl of a slurry of salmon sperm DNA-coated protein A- or G-Sepharose beads (ssDNA-beads) for 2 h at 4 °C (the beads were incubated first with 100 μg/ml ssDNA). After 10 min of high speed centrifugation, the cleared samples were incubated overnight at 4 °C with 10 μg of specific antibody followed by a 3-h incubation with ssDNA-beads. Specific antibodies used were: anti-Mel-18 (Santa Cruz Biotechnology; sc-8905 or sc-10744), anti-ENX-1 (Ezh2, Santa Cruz Biotechnology; sc-25383), anti-YY1 (Santa Cruz Biotechnology; sc-1703), anti-RING1 (Santa Cruz Biotechnology; sc-28736), and anti-EED (Santa Cruz Biotechnology; sc-28701). After immunoprecipitation, washes, and reverse cross-linking, the samples were extracted twice with phenol/chloroform, once with chloroform and ethanol precipitated in the presence of 30 μg of glycogen. 20 μl of the resulting 160-μl samples were used in a PCR reaction with 28-30 cycles (1 min at 95 °C, 1 min at 48 °C, and 1 min at 72 °C, completed by 10 min at 72 °C). The following primers were used: Il-4 P, 5′-TTGGTCTGATTTCACAGG-3′ and 5′-ATCAATAGCTCTGTGCCG-3′ (240-bp product); Il-4 enhancer (VA), 5′-AGGGCACTTAAACATTGC-3′ and 5′-ACGCCTAAGCACAATTCC-3′ (239-bp product); Il-4 DH site IV, 5′-CTCTTCTTCCCTTGATCG-3′ and 5′-GCACTTGGTATATGAGGC-3′ (219-bp product); Il-4 Site II, 5′-GGGTGTGAATAAGCCATATTG-3′ and 5′-CCCAGCGTTTACATG AGC-3′ (175-bp product); Il-4 LCR RHS7/RAD50-C, 5′-CCACACACTGGGATGTGTAGCTCA-3′ and 5′-AGACCCAGCTCCTCAGAAGGTAGT (250-bp product); Ifng P, 5′GCTCTGTGGATGAGAAAT-3′ and 5′-AAGATGGTGACAGATAGG-3′ (250-bp product); Ifng CNS-1, 5′-CTTTGAAGGATACCATGG-3′ and 5′-AGGTTTCCTCTTAAGGGC-3′ (224-bp product). As controls, PCR using Ifng promoter or VA primers was performed directly on input DNA purified from chromatin before immunoprecipitation. Selected input samples were also amplified with each pair of specific primers. PCR products were resolved on 3% NuSieve/agarose gels and visualized with ethidium bromide. Quantitative PCR was performed using Absolute Blue SYBR-Green ROX mix (Thermo Scientific, ABgene), according to the manufacturer's instructions, and an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Dissociation curves after amplification showed that all primer pairs generated single products. The amount of PCR product amplified was calculated relative to a standard curve of the input. The value of control immunoprecipitation was subtracted from the specific immunoprecipitation. The following primers were used: Ifng P5′-GAGAATCCCACAAGAATGGCA-3′ and 5′-CAGCTATGGTTTTGTGGCATGT-3′ (105-bp product); Il-4 P5′-CTCATTTTCCCTTGGTTTCAGC-3′ and 5′-CAATAGCTCTGTGCCGTCAGTG-3′ (123-bp product). Immunofluorescence Microscopy—Cells were fixed onto poly-l-lysine slides (Sigma) in 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.5% Triton X-100 for 5 min on ice. The cells were washed three times with phosphate-buffered saline, and nonspecific binding was blocked by incubation in 5% donkey serum (0.5% Nonidet P-40, phosphate-buffered saline) for 30 min at room temperature. Then the cells were incubated with anti-PcG antibodies (4 μg/ml, 5% donkey serum in phosphate-buffered saline) for 1 h at room temperature, followed by Cy-2-conjugated donkey anti-goat antibodies or rabbit IgG (7.5 μg/ml, Jackson Laboratories). For controls, the primary antibody was omitted. The DNA was counterstained with Vectashield Mounting Medium with propidium iodide (PI) (Vector, Burlingame). Images were recorded with a Zeiss LSM Meta confocal (Zeiss, Oberkochen, Germany) hooked to an inverted motorized microscope Zeiss Axiovert 200 m using a Zeiss EC Plan-NEOFLUAR 40×/1.3 oil DIC M27 objective, zoom 2. For fluorescence detection, 488-nm and 561-nm lasers were used. Images were acquired using LSM 510 version 4.2 and processed by LSM Image Browser; they represent one middle Z-stack of the cells (1 μm). RT-PCR—Total RNA was extracted, reverse-transcribed, and amplified with the following primer sets: Il4, 5′-CATCGGCATTTTGAACGAGGTCA-3′ and 5′-CTTATCGATGAATCCAGGCATCG-3′ (Genomic: 4,610-bp product, cDNA: 240-bp product); Ifng, 5′-CATTGAAAGCCTAGAAAGTCTG-3′ and 5′-CTCATGAATGCATCCTTTTTCG-3′ (Genomic: 1,548-bp product, cDNA: 267-bp product); Gata3, 5′-GAACACTGAGCTGCCTGGCGCCGT-3′ and 5′-CTTTGCGGGATAGTTTAGCAA-3′ (Genomic: 812-bp product, cDNA: 391-bp product); T-bet 5′-GCTACCCGCCCGTGGATGG-3′ and 5′-CCGGTGTTGGGGGAGTCTGG-3′ (Genomic: 13,279-bp product, cDNA: 384-bp product). YY1 Binds to the Cytokine Genes in a Differential Manner—Most PcG proteins do not possess a DNA binding domain. YY1 is a generally expressed PcG protein that interacts with both PRC1 and PRC2 and has sequence-specific DNA binding activity (49Gordon S. Akopyan G. Garban H. Bonavida B. Oncogene. 2006; 25: 1125-1142Crossref PubMed Scopus (511) Google Scholar, 50Shi Y. Lee J.S. Galvin K.M. Biochim. Biophys. Acta. 1997; 1332: F49-F66Crossref PubMed Scopus (436) Google Scholar, 51Thomas M.J. Seto E. Gene. 1999; 236: 197-208Crossref PubMed Scopus (394) Google Scholar). Therefore, YY1 might play a role in the recruitment of PcG complexes to DNA. YY1 has been found in association with key chromatin remodeling factors, among them HATs, HDACs, and DNMTs (39Rezai-Zadeh N. Zhang X. Namour F. Fejer G. Wen Y.D. Yao Y.L. Gyory I. Wright K. Seto E. Genes Dev. 2003; 17: 1019-1029Crossref PubMed Scopus (139) Google Scholar, 49Gordon S. Akopyan G. Garban H. Bonavida B. Oncogene. 2006; 25: 1125-1142Crossref PubMed Scopus (511) Google Scholar, 50Shi Y. Lee J.S. Galvin K.M. Biochim. Biophys. Acta. 1997; 1332: F49-F66Cro