Active Demethylation of the Foxp3 Locus Leads to the Generation of Stable Regulatory T Cells within the Thymus This information is current as Aras Toker, Dirk Engelbert, Garima Garg, Julia K. of September 25, 2021. Polansky, Stefan Floess, Takahisa Miyao, Udo Baron, Sandra Düber, Robert Geffers, Pascal Giehr, Sonja Schallenberg, Karsten Kretschmer, Sven Olek, Jörn Walter, Siegfried Weiss, Shohei Hori, Alf Hamann and Jochen Huehn Downloaded from J Immunol published online 18 February 2013 http://www.jimmunol.org/content/early/2013/02/17/jimmun ol.1203473 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/02/19/jimmunol.120347 Material 3.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! 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Published February 18, 2013, doi:10.4049/jimmunol.1203473 The Journal of Immunology Active Demethylation of the Foxp3 Locus Leads to the Generation of Stable Regulatory T Cells within the Thymus Aras Toker,* Dirk Engelbert,† Garima Garg,* Julia K. Polansky,* Stefan Floess,* Takahisa Miyao,‡ Udo Baron,x Sandra Du¨ber,{ Robert Geffers,‖ Pascal Giehr,# Sonja Schallenberg,** Karsten Kretschmer,** Sven Olek,x Jo¨rn Walter,# Siegfried Weiss,{ Shohei Hori,‡ Alf Hamann,† and Jochen Huehn* Stable expression of Foxp3 in regulatory T cells (Tregs) depends on DNA demethylation at the Treg-specific demethylated region (TSDR), a conserved, CpG-rich region within the Foxp3 locus. The TSDR is selectively demethylated in ex vivo Tregs purified from secondary lymphoid organs, but it is unclear at which stage of Treg development demethylation takes place. In this study, we show that commitment to a stable lineage occurred during early stages of murine thymic Treg development by engraving of Downloaded from lineage-specific epigenetic marks in parallel with establishment of a Treg-specific gene expression profile. TSDR demethylation was achieved through an active mechanism and involved enzymes of the ten-eleven-translocation family and hydroxylation of methylated cytosines, a modification that is implicated as an initiating step of mitosis-independent DNA demethylation pathways and has not yet been observed at specific loci during immune cell differentiation. Together, our results demonstrate that initiating TSDR demethylation during early stages of thymic Treg development commences stabilization of Foxp3 expression and guaran- tees full functionality and long-term lineage stability of Tregs. The Journal of Immunology, 2013, 190: 000–000. http://www.jimmunol.org/ 2 D4+ regulatory T cells (Tregs) play an essential role in (CD4SP; i.e., CD4+CD8 ) stage (9, 10). Thymic Treg develop- maintaining immune homeostasis and preventing autoim- ment requires that CD4SP thymocytes encounter cognate Ag C mune reactivity of potentially self-reactive lymphocytes presented by thymic APCs in the context of MHC class II (11–14), that have escaped central tolerance mechanisms (1). For proper and CD4SP thymocytes appear to be predisposed to upregulate development and function, Tregs crucially depend on the forkhead Foxp3 expression (15). According to the two-step model of thymic box transcription factor Foxp3, and loss of Foxp3 function in hu- Treg development, this TCR signaling event induces upregulation mans and rodents results in devastating autoimmunity (2–7). of IL-2R a-chain (CD25), rendering these thymocytes receptive to by guest on September 25, 2021 The vast majority of Foxp3+ Tregs is generated during T cell subsequent cytokine signals that foster their development into development in the thymus (8). Development of thymic Tregs fully functional Foxp3+ Tregs (16, 17). IL-2 plays a predominant mainly takes place in medullary regions at the CD4 single-positive role in this second step of thymic Treg development. However, its loss can be compensated by other cytokines that signal through receptors containing the common g subunit, such as IL-7 and IL- *Experimental Immunology, Helmholtz Centre for Infection Research, Braunsch- weig 38124, Germany; †Experimental Rheumatology, Medical Clinic–Rheumatology 15 (16, 18, 19). and Clinical Immunology, Charite´ University Medicine, Berlin 10117, Germany; Stability of Foxp3 expression correlates to DNA demethylation ‡Research Unit for Immune Homeostasis, RIKEN Research Centre for Allergy and Immunology, Yokohama 230-0045, Japan; xEpiontis GmbH, Berlin 12489, at a conserved intronic CpG-rich region within the Foxp3 gene Germany; {Molecular Immunology, Helmholtz Centre for Infection Research, locus, designated Treg-specific demethylated region (TSDR) (20). ‖ Braunschweig 38124, Germany; Genome Analytics, Helmholtz Centre for Infection Demethylation at the TSDR (also known as CNS2) is not required Research, Braunschweig 38124, Germany; #Department of Genetics/Epigenetics, Saarland University, Saarbru¨cken 66123, Germany; and **Deutsche Forschungsge- for initiation of Foxp3 expression, but for its long-term mainte- meinschaft–Center for Regenerative Therapies Dresden 01307, Dresden, Germany nance (21–23). Accordingly, stable Tregs display a fully demeth- + Received for publication December 20, 2012. Accepted for publication January 24, ylated TSDR, whereas the TSDR of conventional CD4 T cells 2013. and in vitro–induced Tregs is heavily methylated (22). In addition, This work was supported by the German Research Foundation (SFB738 to J.H. and forced TSDR demethylation confers stability to in vitro–induced SFB650 and Ha1505-10/1 to A.H.), the German Ministry for Education and Research (Deutsches Epigenom Programm to J.W. and A.H.), and by Grants-in-Aid for Sci- Tregs (21). Hence, understanding the mechanisms that cause TSDR entific Research from the Ministry of Education, Culture, Sports, Science and Tech- demethylation in developing Tregs could open up ways to manip- nology of Japan (19059014 and 20689012 to S.H.). ulate TSDR methylation and allow safe application of in vitro– The sequences presented in this article have been submitted to the Gene Expression generated Tregs for therapeutic approaches (24). Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE42021. In this study, we provide direct evidence that commitment to Address correspondence and reprint requests to Prof. Jochen Huehn, Helmholtz a stable Foxp3-expressing lineage is initiated already during early Centre for Infection Research, Inhoffenstrasse 7, Braunschweig 38124, Germany. E-mail address: [email protected] stages of thymic Treg development and is completed by TSDR + The online version of this article contains supplemental material. demethylation in mature thymic Foxp3 Tregs, ensuring full Abbreviations used in this article: FTOC, fetal thymic organ culture; 5hmC, 5- functionality and long-term lineage stability. In developing Tregs, hydroxymethylcytosine; 5mC, 5-methylcytosine; RTE, recent thymic emigrant; SP, CpGs of the TSDR are demethylated through an active mechanism single-positive; Tet, ten-eleven-translocation; Treg, regulatory T cell; TSDR, regula- that involves the recently discovered intermediate of active DNA tory T cell–specific demethylated region. demethylation pathways, 5-hydroxymethylcytosine (5hmC), and Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 enzymes of the ten-eleven-translocation (Tet) family. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203473 2 STABLE Treg INDUCTION IN THE THYMUS Materials and Methods lowed: low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Mice Tris [pH 8.1], and 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton X- 100, 2 mM EDTA, 20 mM Tris [pH 8.1,] and 500 mM NaCl), LiCl buffer BALB/c and C57BL/6J mice were purchased from Janvier. BALB/c 3 (25 mM LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, 1 mM EDTA, and Thy1.1 and Foxp3GFP reporter mice (kindly provided by Alexander 10 mM Tris [pH 8]), and TE buffer (10 mM Tris and 1 mM EDTA). After- Rudensky, Memorial Sloan-Kettering Cancer Center, New York, NY) were wards, Ab-bound fragments were collected with hot elution buffer (1% SDS bred at the Helmholtz Centre for Infection Research (Braunschweig, and 100 mM NaHCO3) and purified using the Nucleospin Extract II kit Germany). Indu-Rag1fl/fl 3 Rosa26-CreERT2 mice were obtained by (Macherey-Nagel) according to the procedure recommended for the purifi- crossing Indu-Rag1fl/fl mice (25) with Rosa26-CreERT2 mice (kindly cation of samples containing SDS. The amount of immunoprecipitated DNA provided by Prof. Anton Berns, The Netherlands Cancer Institute, and input DNA was quantified by real-time PCR using SYBR Green Amsterdam, The Netherlands) and bred at the Helmholtz Centre for In- (Invitrogen) with the following primer pair: TSDR forward: 59-AACC- fection Research. Lymphocyte development was induced in Indu-Rag1fl/fl