Abstract
A role for DNA demethylation in transcriptional regulation of genes expressed in differentiated somatic cells remains controversial. Here, we define a small region in the promoter-enhancer of the interleukin-2 (Il2) gene that demethylates in T lymphocytes following activation, and remains demethylated thereafter. This epigenetic change was necessary and sufficient to enhance transcription in reporter plasmids. The demethylation process started as early as 20 minutes after stimulation and was not prevented by a G1 to S phase cell cycle inhibitor that blocks DNA replication. These results imply that this demethylation process proceeds by an active enzymatic mechanism.
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References
Dutton, R.W., Bradley, L.M. & Swain, S.L. T cell memory. Annu. Rev. Immunol. 16, 201–223 (1998).
Agarwal, S. & Rao, A. Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9, 765–775 (1998).
Fitzpatrick, D.R. et al. Distinct methylation of the interferon γ (IFN-γ) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-γ promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells. J. Exp. Med. 188, 103–117 (1998).
Fitzpatrick, D.R., Shirley, K.M. & Kelso, A. Cutting edge: stable epigenetic inheritance of regional IFN-γ promoter demethylation in CD44highCD8+ T lymphocytes. J. Immunol. 162, 5053–5057 (1999).
Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).
Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).
Powell, J.D., Bruniquel, D. & Schwartz, R.H. TCR engagement in the absence of cell cycle progression leads to T cell anergy independent of p27(Kip1). Eur. J. Immunol. 31, 3737–3746 (2001).
Cervoni, N. & Szyf, M. Demethylase activity is directed by histone acetylation. J. Biol. Chem. 276, 40778–40787 (2001).
Bruniquel, D., Borie, N., Hannier, S. & Triebel, F. Regulation of expression of the human lymphocyte activation gene-3 (LAG-3) molecule, a ligand for MHC class II. Immunogenetics 48, 116–124 (1998).
Crane-Robinson, C., Myers, F.A., Hebbes, T.R., Clayton, A.L. & Thorne, A.W. Chromatin immunoprecipitation assays in acetylation mapping of higher eukaryotes. Methods Enzymol. 304, 533–547 (1999).
Matsuo, K. et al. An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA. EMBO J. 17, 1446–1453 (1998).
Jones, P.L. & Wolffe, A.P. Relationships between chromatin organization and DNA methylation in determining gene expression. Semin. Cancer Biol. 9, 339–347 (1999).
Wolffe, A.P., Jones, P.L. & Wade, P.A. DNA demethylation. Proc. Natl. Acad. Sci. USA 96, 5894–5896 (1999).
Lin, I.G., Tomzynski, T.J., Ou, Q. & Hsieh, C.L. Modulation of DNA binding protein affinity directly affects target site demethylation. Mol. Cell. Biol. 20, 2343–2349 (2000).
Stunkel, W., Ait-Si-Ali, S., Jones, P.L. & Wolffe, A.P. Programming the transcriptional state of replicating methylated dna. J. Biol. Chem. 276, 20743–20749 (2001).
Lyons, A.B. & Parish, C.R. Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137 (1994).
Storek, J., Schmid, I., Ferrara, S. & Saxon, A. A novel B cell stimulation/proliferation assay using simultaneous flow cytometric detection of cell surface markers and DNA content. J. Immunol. Methods 151, 261–267 (1992).
Bird, J.J. et al. Helper T cell differentiation is controlled by the cell cycle. Immunity 9, 229–237 (1998).
Kress, C., Thomassin, H. & Grange, T. Local DNA demethylation in vertebrates: how could it be performed and targeted? FEBS Lett. 494, 135–140 (2001).
Ward, S.B. et al. Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions. Nucleic Acids Res. 26, 2923–2934 (1998).
Fiering, S. et al. Single cell assay of a transcription factor reveals a threshold in transcription activated by signals emanating from the T-cell antigen receptor. Genes Dev. 4, 1823–1834 (1990).
Garrity, P.A., Chen, D., Rothenberg, E.V. & Wold, B.J. Interleukin-2 transcription is regulated in vivo at the level of coordinated binding of both constitutive and regulated factors. Mol. Cell. Biol. 14, 2159–2169 (1994).
Gudmundsdottir, H., Wells, A.D. & Turka, L.A. Dynamics and requirements of T cell clonal expansion in vivo at the single-cell level: effector function is linked to proliferative capacity. J. Immunol. 162, 5212–5223 (1999).
Weiss, A., Keshet, I., Razin, A. & Cedar, H. DNA demethylation in vitro: involvement of RNA. Cell 86, 709–718 (1996).
Fremont, M. et al. Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA. Nucleic Acids Res. 25, 2375–2380 (1997).
Bhattacharya, S.K., Ramchandani, S., Cervoni, N. & Szyf, M. A mammalian protein with specific demethylase activity for mCpG DNA. Nature 397, 579–583 (1999).
Ramchandani, S., Bhattacharya, S.K., Cervoni, N. & Szyf, M. DNA methylation is a reversible biological signal. Proc. Natl. Acad. Sci. USA 96, 6107–6112 (1999).
Schwarz, S. et al. A CpG-rich RNA and an RNA helicase tightly associated with the DNA demethylation complex are present mainly in dividing chick embryo cells. Eur. J. Cell Biol. 79, 488–494 (2000).
Zhu, B. et al. 5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence. Nucleic Acids Res. 28, 4157–4165 (2000).
Detich, N., Theberge, J. & Szyf, M. Promoter-specific activation and demethylation by MBD2/demethylase. J. Biol. Chem. 277, 35791–35794 (2002).
Zhu, B. et al. 5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex. Proc. Natl. Acad. Sci. USA 97, 5135–5139 (2000).
Jost, J.P. et al. 5-Methylcytosine DNA glycosylase participates in the genome-wide loss of DNA methylation occurring during mouse myoblast differentiation. Nucleic Acids Res. 29, 4452–4461 (2001).
Zhu, B. et al. Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene. Proc. Natl. Acad. Sci. USA 98, 5031–5036 (2001).
Mayer, W., Niveleau, A., Walter, J., Fundele, R. & Haaf, T. Demethylation of the zygotic paternal genome. Nature 403, 501–502 (2000).
Sullivan, C.H. & Grainger, R.M. Delta-crystallin genes become hypomethylated in postmitotic lens cells during chicken development. Proc. Natl. Acad. Sci. USA 84, 329–333 (1987).
Saluz, H.P., Jiricny, J. & Jost, J.P. Genomic sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylation of the overlapping estradiol/glucocorticoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc. Natl. Acad. Sci. USA 83, 7167–7171 (1986).
Richter, A., Lohning, M. & Radbruch, A. Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell cycle progression. J. Exp. Med. 190, 1439–1450 (1999).
Avni, O. et al. T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat. Immunol. 3, 643–651 (2002).
Siebenlist, U. et al. Promoter region of interleukin-2 gene undergoes chromatin structure changes and confers inducibility on chloramphenicol acetyltransferase gene during activation of T cells. Mol. Cell. Biol. 6, 3042–3049 (1986).
Rao, S., Procko, E. & Shannon, M.F. Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene. J. Immunol. 167, 4494–4503 (2001).
Attema, J.L. et al. The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation. J. Immunol. 169, 2466–2476 (2002).
Mostoslavsky, R. et al. Kappa chain monoallelic demethylation and the establishment of allelic exclusion. Genes Dev. 12, 1801–1811 (1998).
Thomassin, H., Flavin, M., Espinas, M.L. & Grange, T. Glucocorticoid-induced DNA demethylation and gene memory during development. EMBO J. 20, 1974–1983 (2001).
Miller, C., Ragheb, J.A. & Schwartz, R.H. Anergy and cytokine-mediated suppression as distinct superantigen-induced tolerance mechanisms in vivo. J. Exp. Med. 190, 53–64 (1999).
Leo, O., Foo, M., Sachs, D.H., Samelson, L.E. & Bluestone, J.A. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA 84, 1374–1378 (1987).
Gross, J.A., St John, T. & Allison, J.P. The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression. J. Immunol. 144, 3201–3210 (1990).
Huang, H., Pannetier, C., Hu-Li, J. & Paul, W.E. Transient transfection of primary T helper cells by particle-mediated gene transfer. J. Immunol. Methods 215, 173–177 (1998).
Acknowledgements
We thank G. Felsenfeld, R. Germain and F. Magdinier for critical reading of the manuscript, and B. Winders, J.K. Friend and M. Hsie for technical assistance.
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Bruniquel, D., Schwartz, R. Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 4, 235–240 (2003). https://doi.org/10.1038/ni887
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DOI: https://doi.org/10.1038/ni887
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