Eur. J. Immunol. 2015. 45: 11–16
DOI: 10.1002/eji.201444577
HIGHLIGHTS
Yohko Kitagawa ∗1 , Naganari Ohkura ∗1,2 and Shimon Sakaguchi1,3 1
Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Japan 2 Department of Frontier Research in Tumor Immunology, Graduate School of Medicine, Osaka University, Suita, Japan 3 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Thymus-derived Treg cells, which express the transcription factor Foxp3, form a functionally stable cell lineage indispensable for the maintenance of immunological selftolerance and homeostasis. Foxp3 is critically required for Treg-cell function, in particular for their suppressive function. Recent studies have implicated the contribution of Treg-cell-specific epigenetic modifications as a means to ensure the stable expression of Foxp3 and other molecules associated with Treg-cell function. Unexpectedly, epigenetic modifications introduced in the course of thymic Treg-cell development were found to be independent of Foxp3 expression. These findings require reconsideration of the current model of Treg-cell development based on Foxp3 induction. With reference to other examples of lineage specification, we discuss possible models for thymic Treg-cell development.
Keywords: Epigenetics
r
Foxp3 r Regulatory T cells
r
Thymus
Introduction Epigenetic modifications are of general importance in generating and maintaining functionally distinct cell lineages, as they allow the acquisition and stable maintenance of lineage-specific gene expression that is heritable from parent to daughter cells [1, 2]. Lineage-specific epigenetic modifications have been reported in immune cells, including Treg cells [2–4]. Considering that immune cells, including T and B cells, respond to various antigens in specific ways under different immunological settings, some cell types, such as helper T cells, may need some degree of flexibility and adaptability in defending the body from a diverse array of invading microbes and immunological insults. In contrast, cell lineage stability of Treg cells is essential for the long-term stable control of immunological homeostasis, given that Treg-cell deficiency or functional instability has been shown to cause autoimmune and other immunological diseases in both mice and humans [5–8].
Correspondence: Prof. Shimon Sakaguchi e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Even temporary loss of Treg cells has been shown to have detrimental effects, causing wasting disease and terminal autoimmune disease in adult mice [9, 10]. From these observations, the outstanding questions regarding epigenetic modifications and lineage establishment are as follows: What kind of developmental cues induce such epigenetic changes? How is the locus specificity of any epigenetic change determined? How much do epigenetic modifications actually contribute to the establishment of a cell lineage, in comparison with the contribution of, e.g. transcription factors? Natural Treg cells are the best example addressing these questions because the majority of natural Treg cells are produced in the thymus as a functionally mature population specialized for immune suppression, and thereafter persist in the periphery with stable function. To understand the molecular basis of Treg-cell function, numerous studies have identified transcription factors required for the expression of Treg-cell-type genes. Of these, Foxp3 has
∗
These authors contributed equally to this work.
www.eji-journal.eu
Mini-Review
Epigenetic control of thymic Treg-cell development
11
12
Yohko Kitagawa et al.
been considered as the master regulator of Treg cells, as it plays a central role in generating and maintaining Treg-cell-specific gene expression by cooperating with other transcription factors such as Runx1 and Gata3 [11–16]. Foxp3 expression is essential for the suppressive function of Treg cells because Foxp3 deficiency has been shown to abrogate Treg-cell-mediated suppression, while its ectopic expression in conventional T cells has been shown to confer suppressive activity upon these cells, both in mice and humans [8, 11, 17, 18]. As a molecular mechanism to ensure stable Treg-cell function, recent studies have revealed that both thymusderived and peripherally induced Treg cells possess a specific DNA hypomethylation pattern, which is associated with enhancement and stabilization of Foxp3 expression as well as induction of Foxp3-independent gene expression [4, 19, 20]. A limited number (several hundreds) of Treg-cell-specific hypomethylated regions have been found in the whole genome in mice and are mainly associated with gene activation, in contrast to the main function of Foxp3, which acts as a gene repressor [21]. Moreover, this Treg-cell-specific DNA hypomethylation has been shown to occur independently of Foxp3 expression during thymic Treg-cell development [4]. These findings, when taken together, raise the question as to how such epigenetic changes occur in the course of thymic Treg-cell development. In this review, we first summarize our current understanding and general perspective of epigenetic control of cell differentiation in lymphocytes and other cells, and then more specifically discuss the roles of epigenetic modifications in Treg-cell development in the thymus. We propose a possible model in which preestablishment of the chromatin landscape that allows Foxp3-dependent and -independent gene regulation is a critical molecular event that determines Treg-cell lineage specification.
Epigenetic control of cell differentiation: General perspectives As cell differentiation progresses from embryonic stem cells into various lineage-committed cells, there is a general trend that pluripotency genes are silenced with repressive epigenetic marks, while lineage-restricted genes become epigenetically more “open” in the corresponding cell types [22–24]. Such epigenetic modifications, including DNA methylation, histone modifications, and nucleosome positioning, contribute to lineage determination and commitment by altering the accessibility of the target gene loci for transcription factors and maintaining this transcriptional accessibility status over a long period of time (reviewed in [25, 26]). Some examples of epigenetic modifications that contribute to the exposure of DNA and facilitate the binding of transcription factors, thereby initiating gene transcription, include DNA demethylation that removes a methyl group from cytosines, certain histone modifications that reduce the attraction between the histone octamer and DNA, and nucleosome sliding, ejection, or displacement, which detaches DNA from nucleosomes [27, 28]. Furthermore, these epigenetic modifications are copied from parent to
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2015. 45: 11–16
daughter cells, allowing stable maintenance of lineage-specific gene expression throughout numerous cell divisions [29]. Therefore, once lineage-specific epigenetic modifications are installed, they can “lock” at least part of the gene expression profile and consequently facilitate lineage commitment. A key question to be addressed then is whether epigenetic modifications are prerequisites or consequences of cell lineage specification. As an example of the former, during brain development in mice, astrocyte differentiation has been shown to be dependent on the DNA hypomethylation of the STAT3 recognition site in Gfap (glial fibrillary acidic protein) promoter and on STAT3 signaling [30]. STAT3 cannot recognize the methylated STAT3-binding sequence in neuroepithelial cells on embryonic day 11.5, but demethylation occurs by embryonic day 14.5, making the cells epigenetically poised to receive differentiation-inducing signals [30]. In contrast, DNA demethylation at Il4 gene locus has been shown to occur after the initiation of Il4 transcription during murine Th2 differentiation, likely facilitated by the master regulator Gata3, suggesting that DNA demethylation in this case is a consequence of a cell fate decision and contributes to the reinforcement of lineage commitment (reviewed in [31]). Thus, although the roles of epigenetic modifications are not yet fully understood in many cell types, some cases of lineage specification may require prior modulation of the chromatin landscape in order for the master transcription factors to be able to initiate gene regulation, while other cell differentiation programs may solely depend on master transcription factors, which modulate the chromatin landscape to regulate gene expression. In cases where epigenetic modifications need to occur prior to the expression of master transcription factors, one of the determinants of lineage specification is the inducer of such epigenetic modifications. Among molecules involved in epigenetic modifications, the most downstream ones are enzymes that catalyze the modifying processes. For example, there are DNA methyltransferases for DNA methylation, Tet family members for DNA demethylation, and histone acetyltransferases and histone deacetylases for histone modifications [25, 26]. Furthermore, there are groups of enzymes known as chromatin remodelers, which utilize ATP to alter nucleosome positioning. These epigenetic modifying enzymes often act in concert to modulate the overall chromatin architecture (reviewed in [32]). However, these enzymes are ubiquitously expressed and it is likely that their activity and specificity during lineage specification is regulated by their recruitment to particular genomic regions. Therefore, where these enzymes are recruited to determines lineage specificity, and the way in which they are recruited to particular genomic regions holds the key for understanding the mechanism of lineage specification. One possible model to position general epigenetic modifying enzymes at specific loci is the use of “pioneer” factors that specify target genes. In order to change the chromatin architecture of a locus from a condensed to a loosened structure, such pioneer factors can bind either to nearby accessible sites which are already epigenetically open, or directly to epigenetically closed sites, and
www.eji-journal.eu
Eur. J. Immunol. 2015. 45: 11–16
HIGHLIGHTS
Figure 1. Possible model of lineage specification programs initiated by pioneer factors. Some lineage specification programs may involve gene regulation by modulation of the chromatin landscape, which is initiated by the binding of pioneer factors, followed by the recruitment of epigenetic modifying enzymes such as Tet and histone acetyltransferases to demethylate DNA and modify histones, respectively, resulting in a more open chromatin structure. These chromatin remodeling and permissive epigenetic modifications allow the binding of transcription factors (TF), which activate gene transcription.
then modify the epigenetic features at these sites. In both scenarios, the pioneer factors need to be able to induce epigenetic changes by themselves, or to recruit factors with such capabilities, so that the target loci become accessible to general transcription factors as their consensus sequences become revealed. In fact, FoxA and Gata4 can be considered as pioneer factors for embryonic hepatocyte differentiation; these factors have been shown to bind to their target regions in compacted chromatin in liver precursor cells and open up the compacted chromatin [33]. FoxA has also been shown to bind to methylated cell type specific target sites during neural cell differentiation and subsequently induce DNA demethylation, which is essential for the transcription of target genes [34]. Taken together, these examples suggest that, at least in some cases of lineage specification, chromatin remodeling and other epigenetic modifications by pioneer factors that recruit the modifying enzymes allow the appropriate positioning of transcription factors, which then initiate lineage-specific gene regulation (Fig. 1).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Epigenetic regulation in Treg-cell differentiation T cells originate from multipotent HSCs, which differentiate into common lymphoid progenitors, and then into T cells. The path of differentiation is accompanied by progressive lineage restriction, consequent loss of the potential to differentiate into other lineages, and acquisition of specific lineage identity with specific transcriptional programs and functions (reviewed in [35, 36]). Similarly to the cases of aforementioned hepatocyte and neural cell differentiation, lineage commitment is accompanied by permissive epigenetic modifications in functionally relevant genes and repressive modifications of the genes associated with alternative lineages [2, 3, 37]. For example, during differentiation of the lymphoid lineage from HSCs, lymphoid lineage-associated genes such as Lck have been shown to undergo DNA demethylation, whereas myeloid lineageassociated genes become methylated [2, 3]. Epigenetic modifications continue further along the road of specification, from that of
www.eji-journal.eu
13
14
Yohko Kitagawa et al.
lymphocytes into T and B cells, and that of T cells into CD4+ and CD8+ T cells [38–40]. These findings demonstrate that there is an accumulation of epigenetic modifications during the sequential differentiation of HSCs to mature T-cell subpopulations, including Treg cells. Treg cells, as a subset of CD4+ T cells, possess specific epigenetic features in addition to those in conventional CD4+ T cells. For example, DNA hypomethylation is specifically observed at Treg signature gene loci, such as Foxp3, Ctla4, Ikzf4, and Ikzf2 [4] and permissive histone marks are specifically present in Treg cells at the Foxp3 promoter region [20]. Particularly, DNA hypomethylation at Foxp3 CNS2 (conserved noncoding region 2), an enhancer region, is important for Treg-cell lineage specification, as it enhances Foxp3 transcription by allowing the binding of transcription factors, which otherwise would not recognize the sequence [41]. Unlike the T cells that express Foxp3 without possessing Treg-cell-specific DNA hypomethylation, such as TGFβ-induced Foxp3+ T cells, Treg cells with these open chromatin structures at Treg signature genes are able to maintain their protein expression as they divide in vivo, suggesting a pivotal contribution of epigenetic modifications to Treg-cell lineage stability [4, 42]. Furthermore, STAT5 and NFAT activated by IL-2 and T cell receptor (TCR) signaling, respectively, were found to interact with the hypomethylated Foxp3 CNS2 to counteract the destabilizing effects of pro-inflammatory cytokines on Treg-cell identity [43, 44]. These results demonstrate the roles of Treg-cell-specific epigenetic modifications in the maintenance of Treg-cell lineage commitment. In addition, there are several findings that suggest their contribution to thymic Treg-cell development. First, Treg-cell-specific DNA demethylation occurs as Treg cells develop in the thymus, independently of Foxp3 expression; moreover, Foxp3-null Treg cells with hypomethylated Treg signature genes still express a substantial proportion of the Treg-cell-type gene profile [4]. Second, it has been shown that thymic Treg precursors are already primed such that general TCR stimulation and IL-2 signaling are sufficient to induce DNA demethylation and Foxp3 expression, which is a phenomenon observed specifically in thymic Treg precursors but not in conventional CD4SP thymocytes [45]. This suggests that some forms of epigenetic modulation occur to the chromatin landscape prior to Foxp3 induction in Treg-cell precursors, making them poised to differentiate into Treg cells. Third, genomewide analyses of gene expression, Foxp3-binding sites, and DNA methylation status in steady-state and activated Treg cells have revealed that DNA hypomethylated sites are associated with gene activation in steady-state Treg cells, whereas Foxp3 is involved in gene repression in activated Treg cells [21]. Consistently, deletion of DNA methylation maintenance enzyme, Dnmt1, in T cells has been shown to induce Foxp3 expression even in CD8+ T cells upon TCR stimulation, suggesting a role for DNA demethylation in Foxp3 induction [46]. Taken together, these findings suggest that thymic Treg-cell development involves preconditioning of the chromatin landscape in precursor cells, followed by the induction of Foxp3 and DNA demethylation. It is speculated that a pioneer factor may bind to Treg signature genes and alter some epigenetic C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2015. 45: 11–16
features in precursor cells, so that upon further TCR and IL-2 signaling, Tet enzymes recruited by the pioneer factor may actively demethylate Treg signature gene loci, and transcription factors, such as cRel and STAT5, downstream of these signaling cascades, bind to Treg signature genes for transcription initiation. It is likely that Foxp3 then acts as a transcriptional repressor at a late stage of the Treg-cell differentiation program to complete Treg lineage specification. In contrast to the idea that a Treg-cell-specific chromatin architecture needs to be generated to induce and maintain the expression of Treg signature genes, including Foxp3, recent studies have demonstrated that the chromatin landscape required for Foxp3-mediated gene regulation is commonly observed in peripheral conventional CD4+ T cells and Treg cells. Analysis of DNase hypersensitive regions in conventional CD4+ T cells and Treg cells showed that Foxp3 does not actively modulate the chromatin landscape to bind to its cognate motifs, but instead binds to sites that are accessible in both conventional CD4+ T cells and Treg cells and are prebound by its co-factors or structurally similar factors [47]. Furthermore, overexpression of Foxp3 with any one of its five co-factors (Eos, Irf4, Satb1, Lef1, and Gata1) in peripheral conventional CD4+ T cells was found to induce expression of a large proportion of Treg-cell-specific genes, far more than that achieved by Foxp3 alone [15]. These findings suggest that preestablishment of the chromatin landscape prior to Foxp3 induction and the expression of co-factors that interact with Foxp3 are critical components for Foxp3-dependent gene regulation. Since Foxp3binding sites are accessible in conventional CD4+ T cells, it is likely that a part of the chromatin landscape required for Tregcell development is established during CD4+ T-cell development or perhaps even earlier, allowing cells to be poised to receive signals for thymic Treg-cell differentiation. Then, upon receiving Treg-cell differentiation-inducing signals, such as relatively strong TCR stimulation by recognition of self-peptide or MHC ligands in the thymus, further modification of the chromatin landscape and induction of Foxp3 are thought to complete the Treg-cell lineage commitment by allowing stable expression of both Foxp3dependent and Foxp3-independent genes (Fig. 2).
Future perspectives With consideration to the pivotal roles of Treg cells in the control of immunological homeostasis, understanding how Treg-cell stability is generated during their development is critical. Foxp3 function in Treg cells has been comprehensively studied in the last decade, and it has revealed that there is more to Treg-cell development than just Foxp3 expression. Since the establishment of a Treg-cellspecific DNA hypomethylation pattern occurs independently of Foxp3 expression, studying the molecular mechanisms underlying its installation could pave the way for the identification of novel factors involved in Treg-cell development. It could also help us elucidate the signals and progenitor conditions required for Tregcell differentiation, which may ultimately lead to the generation of Treg cells in vitro from conventional T cells for use in cell therapies, www.eji-journal.eu
HIGHLIGHTS
Eur. J. Immunol. 2015. 45: 11–16
Figure 2. Distinct roles of epigenetic modification and Foxp3 expression in thymic Treg-cell development. Thymic Treg-cell development involves gene regulation by two mechanisms; gene activation by Tregcell-specific epigenetic modification, which facilitates the binding of transcription factors, including Foxp3 (left), and gene repression by Foxp3, which binds to a preestablished chromatin landscape in an activationdependent manner (right).
and to the discovery of new therapeutic targets for autoimmune and other immunological diseases.
5 Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases. J. Immunol. 1995. 155: 1151–1164. 6 Bennett, C. L., Christie, J., Ramsdell, F., Brunkow, M. E., Ferguson, P. J., Whitesell, L., Kelly, T. E. et al., The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by muta-
Acknowledgments: We thank J. B. Wing for critical reading of the manuscript and the members of our laboratory for discussion. This work is supported by Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency to S.S., research support program for combined research fields to N.O., and grants-in-aid for JSPS Fellows 261560 from Japanese Society for the Promotion of Science to Y.K.
tions of FOXP3. Nat. Genet. 2001. 27: 20–21. 7 Brunkow, M. E., Jeffery, E. W., Hjerrild, K. A., Paeper, B., Clark, L. B., Yasayko, S. A., Wilkinson, J. E. et al., Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 2001. 27: 68–73. 8 Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 2003. 4: 330–336. 9 Sakaguchi, S., Naturally arising CD4+ regulatory t cells for immuno-
Conflict of interest: The authors declare no financial or commercial conflict of interest.
logic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 2004. 22: 531–562. 10 Kim, J. M., Rasmussen, J. P. and Rudensky, A. Y., Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat.
References
Immunol. 2007. 8: 191–197. 11 Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell
1 Jaenisch, R. and Bird, A., Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 2003. 33(Suppl): 245–254.
development by the transcription factor Foxp3. Science 2003. 299: 1057– 1061. 12 Sugimoto, N., Oida, T., Hirota, K., Nakamura, K., Nomura, T., Uchiyama,
2 Bock, C., Beerman, I., Lien, W. H., Smith, Z. D., Gu, H., Boyle, P., Gnirke, A.
T. and Sakaguchi, S., Foxp3-dependent and -independent molecules spe-
et al., DNA methylation dynamics during in vivo differentiation of blood
cific for CD25+CD4+ natural regulatory T cells revealed by DNA microar-
and skin stem cells. Mol. Cell 2012. 47: 633–647.
ray analysis. Int. Immunol. 2006. 18: 1197–1209.
3 Ji, H., Ehrlich, L. I., Seita, J., Murakami, P., Doi, A., Lindau, P., Lee, H.
13 Hill, J. A., Feuerer, M., Tash, K., Haxhinasto, S., Perez, J., Melamed, R.,
et al., Comprehensive methylome map of lineage commitment from
Mathis, D. and Benoist, C., Foxp3 transcription-factor-dependent and -
haematopoietic progenitors. Nature 2010. 467: 338–342.
independent regulation of the regulatory T cell transcriptional signature.
4 Ohkura, N., Hamaguchi, M., Morikawa, H., Sugimura, K., Tanaka, A.,
Immunity 2007. 27: 786–800.
Ito, Y., Osaki, M. et al., T cell receptor stimulation-induced epigenetic
14 Ono, M., Yaguchi, H., Ohkura, N., Kitabayashi, I., Nagamura, Y., Nomura,
changes and Foxp3 expression are independent and complementary
T., Miyachi, Y. et al., Foxp3 controls regulatory T-cell function by inter-
events required for Treg cell development. Immunity 2012. 37: 785–799.
acting with AML1/Runx1. Nature 2007. 446: 685–689.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
15
16
Yohko Kitagawa et al.
Eur. J. Immunol. 2015. 45: 11–16
15 Fu, W., Ergun, A., Lu, T., Hill, J. A., Haxhinasto, S., Fassett, M. S., Gazit, R.
34 Serandour, A. A., Avner, S., Percevault, F., Demay, F., Bizot, M., Lucchetti-
et al., A multiply redundant genetic switch ’locks in’ the transcriptional
Miganeh, C., Barloy-Hubler, F. et al., Epigenetic switch involved in acti-
signature of regulatory T cells. Nat. Immunol. 2012. 13: 972–980. 16 Rudra, D., deRoos, P., Chaudhry, A., Niec, R. E., Arvey, A., Samstein, R. M., Leslie, C. et al., Transcription factor Foxp3 and its protein partners form a complex regulatory network. Nat. Immunol. 2012. 13: 1010–1019. 17 Bacchetta, R., Passerini, L., Gambineri, E., Dai, M., Allan, S. E., Perroni, L., Dagna-Bricarelli, F. et al., Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J. Clin. Invest. 2006. 116: 1713–1722.
vation of pioneer factor FOXA1-dependent enhancers. Genome Res. 2011. 21: 555–565. 35 Rothenberg, E. V., Moore, J. E. and Yui, M. A., Launching the Tcell-lineage developmental programme. Nat. Rev. Immunol. 2008. 8: 9– 21. 36 Koch, U. and Radtke, F., Mechanisms of T cell development and transformation. Annu. Rev. Cell Dev. Biol. 2011. 27: 539–562. 37 Vigano, M. A., Ivanek, R., Balwierz, P., Berninger, P., van Nimwegen, E.,
18 Aarts-Riemens, T., Emmelot, M. E., Verdonck, L. F. and Mutis, T., Forced
Karjalainen, K. and Rolink, A., An epigenetic profile of early T-cell devel-
overexpression of either of the two common human Foxp3 isoforms can
opment from multipotent progenitors to committed T-cell descendants.
induce regulatory T cells from CD4(+)CD25(-) cells. Eur. J. Immunol. 2008.
Eur. J. Immunol. 2014. 44: 1181–1193.
38: 1381–1390. 19 Floess, S., Freyer, J., Siewert, C., Baron, U., Olek, S., Polansky, J., Schlawe, K. et al., Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007. 5: e38. 20 Schmidl, C., Klug, M., Boeld, T. J., Andreesen, R., Hoffmann, P., Edinger, M. and Rehli, M., Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity. Genome Res. 2009. 19: 1165–1174. 21 Morikawa, H., Ohkura, N., Vandenbon, A., Itoh, M., Nagao-Sato, S., Kawaji, H., Lassmann, T. et al., Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation. Proc. Natl. Acad. Sci. USA 2014. 111: 5289–5294. 22 Xie, W., Schultz, M. D., Lister, R., Hou, Z., Rajagopal, N., Ray, P., Whitaker, J. W. et al., Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 2013. 153: 1134–1148. 23 Chen, T. and Dent, S. Y., Chromatin modifiers and remodellers: regulators of cellular differentiation. Nat. Rev. Genet. 2014. 15: 93–106. 24 Mohn, F., Weber, M., Rebhan, M., Roloff, T. C., Richter, J., Stadler, M. B., Bibel, M. et al., Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 2008. 30: 755–766. 25 Bird, A., DNA methylation patterns and epigenetic memory. Genes Dev. 2002. 16: 6–21. 26 Williams, K., Christensen, J. and Helin, K., DNA methylation: TET proteins-guardians of CpG islands? EMBO Rep. 2012. 13: 28–35.
38 Taniuchi, I. and Ellmeier, W., Transcriptional and epigenetic regulation of CD4/CD8 lineage choice. Adv. Immunol. 2011. 110: 71–110. 39 Barneda-Zahonero, B., Roman-Gonzalez, L., Collazo, O., Mahmoudi, T. and Parra, M., Epigenetic regulation of B lymphocyte differentiation, transdifferentiation, and reprogramming. Comp. Funct. Genomics 2012. 2012: 564381. 40 Wilson, C. B., Makar, K. W., Shnyreva, M. and Fitzpatrick, D. R., DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin. Immunol. 2005. 17: 105–119. 41 Polansky, J. K., Schreiber, L., Thelemann, C., Ludwig, L., Kruger, M., Baumgrass, R., Cording, S. et al., Methylation matters: binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells. J. Mol. Med. 2010. 88: 1029–1040. 42 Ohkura, N., Kitagawa, Y. and Sakaguchi, S., Development and maintenance of regulatory T cells. Immunity 2013. 38: 414–423. 43 Li, X., Liang, Y., LeBlanc, M., Benner, C. and Zheng, Y., Function of a Foxp3 cis-element in protecting regulatory T cell identity. Cell 2014. 158: 734–748. 44 Feng, Y., Arvey, A., Chinen, T., van der Veeken, J., Gasteiger, G. and Rudensky, A. Y., Control of the inheritance of regulatory T cell identity by a cis element in the Foxp3 locus. Cell 2014. 158: 749–763. 45 Toker, A., Engelbert, D., Garg, G., Polansky, J. K., Floess, S., Miyao, T., Baron, U. et al., Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus. J. Immunol. 2013. 190: 3180–3188.
27 Pina, B., Bruggemeier, U. and Beato, M., Nucleosome positioning modu-
46 Josefowicz, S. Z., Wilson, C. B. and Rudensky, A. Y., Cutting edge: TCR
lates accessibility of regulatory proteins to the mouse mammary tumor
stimulation is sufficient for induction of Foxp3 expression in the absence
virus promoter. Cell 1990. 60: 719–731. 28 Boyes, J. and Bird, A., DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 1991. 64: 1123–1134. 29 Probst, A. V., Dunleavy, E. and Almouzni, G., Epigenetic inheritance dur-
of DNA methyltransferase 1. J. Immunol. 2009. 182: 6648–6652. 47 Samstein, R. M., Arvey, A., Josefowicz, S. Z., Peng, X., Reynolds, A., Sandstrom, R., Neph, S. et al., Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 2012. 151: 153–166.
ing the cell cycle. Nat. Rev. Mol. Cell Biol. 2009. 10: 192–206. 30 Takizawa, T., Nakashima, K., Namihira, M., Ochiai, W., Uemura, A., Yanagisawa, M., Fujita, N. et al., DNA methylation is a critical cellintrinsic determinant of astrocyte differentiation in the fetal brain. Dev. Cell 2001. 1: 749–758. 31 Ansel, K. M., Djuretic, I., Tanasa, B. and Rao, A., Regulation of Th2 differ-
Full correspondence: Prof. Shimon Sakaguchi, Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan Fax: +81-6-6879-4464 e-mail: [email protected]
entiation and Il4 locus accessibility. Annu. Rev. Immunol. 2006. 24: 607–656. 32 Cedar, H. and Bergman, Y., Linking DNA methylation and histone modification: patterns and paradigms. Nat. Rev. Genet. 2009. 10: 295–304. 33 Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M. and Zaret, K. S., Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol. Cell 2002. 9: 279–289.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 17/9/2014 Revised: 17/10/2014 Accepted: 22/10/2014 Accepted article online: 28/10/2014
www.eji-journal.eu
DOI: 10.1002/eji.201444577
HIGHLIGHTS
Yohko Kitagawa ∗1 , Naganari Ohkura ∗1,2 and Shimon Sakaguchi1,3 1
Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Japan 2 Department of Frontier Research in Tumor Immunology, Graduate School of Medicine, Osaka University, Suita, Japan 3 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Thymus-derived Treg cells, which express the transcription factor Foxp3, form a functionally stable cell lineage indispensable for the maintenance of immunological selftolerance and homeostasis. Foxp3 is critically required for Treg-cell function, in particular for their suppressive function. Recent studies have implicated the contribution of Treg-cell-specific epigenetic modifications as a means to ensure the stable expression of Foxp3 and other molecules associated with Treg-cell function. Unexpectedly, epigenetic modifications introduced in the course of thymic Treg-cell development were found to be independent of Foxp3 expression. These findings require reconsideration of the current model of Treg-cell development based on Foxp3 induction. With reference to other examples of lineage specification, we discuss possible models for thymic Treg-cell development.
Keywords: Epigenetics
r
Foxp3 r Regulatory T cells
r
Thymus
Introduction Epigenetic modifications are of general importance in generating and maintaining functionally distinct cell lineages, as they allow the acquisition and stable maintenance of lineage-specific gene expression that is heritable from parent to daughter cells [1, 2]. Lineage-specific epigenetic modifications have been reported in immune cells, including Treg cells [2–4]. Considering that immune cells, including T and B cells, respond to various antigens in specific ways under different immunological settings, some cell types, such as helper T cells, may need some degree of flexibility and adaptability in defending the body from a diverse array of invading microbes and immunological insults. In contrast, cell lineage stability of Treg cells is essential for the long-term stable control of immunological homeostasis, given that Treg-cell deficiency or functional instability has been shown to cause autoimmune and other immunological diseases in both mice and humans [5–8].
Correspondence: Prof. Shimon Sakaguchi e-mail: [email protected] C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Even temporary loss of Treg cells has been shown to have detrimental effects, causing wasting disease and terminal autoimmune disease in adult mice [9, 10]. From these observations, the outstanding questions regarding epigenetic modifications and lineage establishment are as follows: What kind of developmental cues induce such epigenetic changes? How is the locus specificity of any epigenetic change determined? How much do epigenetic modifications actually contribute to the establishment of a cell lineage, in comparison with the contribution of, e.g. transcription factors? Natural Treg cells are the best example addressing these questions because the majority of natural Treg cells are produced in the thymus as a functionally mature population specialized for immune suppression, and thereafter persist in the periphery with stable function. To understand the molecular basis of Treg-cell function, numerous studies have identified transcription factors required for the expression of Treg-cell-type genes. Of these, Foxp3 has
∗
These authors contributed equally to this work.
www.eji-journal.eu
Mini-Review
Epigenetic control of thymic Treg-cell development
11
12
Yohko Kitagawa et al.
been considered as the master regulator of Treg cells, as it plays a central role in generating and maintaining Treg-cell-specific gene expression by cooperating with other transcription factors such as Runx1 and Gata3 [11–16]. Foxp3 expression is essential for the suppressive function of Treg cells because Foxp3 deficiency has been shown to abrogate Treg-cell-mediated suppression, while its ectopic expression in conventional T cells has been shown to confer suppressive activity upon these cells, both in mice and humans [8, 11, 17, 18]. As a molecular mechanism to ensure stable Treg-cell function, recent studies have revealed that both thymusderived and peripherally induced Treg cells possess a specific DNA hypomethylation pattern, which is associated with enhancement and stabilization of Foxp3 expression as well as induction of Foxp3-independent gene expression [4, 19, 20]. A limited number (several hundreds) of Treg-cell-specific hypomethylated regions have been found in the whole genome in mice and are mainly associated with gene activation, in contrast to the main function of Foxp3, which acts as a gene repressor [21]. Moreover, this Treg-cell-specific DNA hypomethylation has been shown to occur independently of Foxp3 expression during thymic Treg-cell development [4]. These findings, when taken together, raise the question as to how such epigenetic changes occur in the course of thymic Treg-cell development. In this review, we first summarize our current understanding and general perspective of epigenetic control of cell differentiation in lymphocytes and other cells, and then more specifically discuss the roles of epigenetic modifications in Treg-cell development in the thymus. We propose a possible model in which preestablishment of the chromatin landscape that allows Foxp3-dependent and -independent gene regulation is a critical molecular event that determines Treg-cell lineage specification.
Epigenetic control of cell differentiation: General perspectives As cell differentiation progresses from embryonic stem cells into various lineage-committed cells, there is a general trend that pluripotency genes are silenced with repressive epigenetic marks, while lineage-restricted genes become epigenetically more “open” in the corresponding cell types [22–24]. Such epigenetic modifications, including DNA methylation, histone modifications, and nucleosome positioning, contribute to lineage determination and commitment by altering the accessibility of the target gene loci for transcription factors and maintaining this transcriptional accessibility status over a long period of time (reviewed in [25, 26]). Some examples of epigenetic modifications that contribute to the exposure of DNA and facilitate the binding of transcription factors, thereby initiating gene transcription, include DNA demethylation that removes a methyl group from cytosines, certain histone modifications that reduce the attraction between the histone octamer and DNA, and nucleosome sliding, ejection, or displacement, which detaches DNA from nucleosomes [27, 28]. Furthermore, these epigenetic modifications are copied from parent to
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2015. 45: 11–16
daughter cells, allowing stable maintenance of lineage-specific gene expression throughout numerous cell divisions [29]. Therefore, once lineage-specific epigenetic modifications are installed, they can “lock” at least part of the gene expression profile and consequently facilitate lineage commitment. A key question to be addressed then is whether epigenetic modifications are prerequisites or consequences of cell lineage specification. As an example of the former, during brain development in mice, astrocyte differentiation has been shown to be dependent on the DNA hypomethylation of the STAT3 recognition site in Gfap (glial fibrillary acidic protein) promoter and on STAT3 signaling [30]. STAT3 cannot recognize the methylated STAT3-binding sequence in neuroepithelial cells on embryonic day 11.5, but demethylation occurs by embryonic day 14.5, making the cells epigenetically poised to receive differentiation-inducing signals [30]. In contrast, DNA demethylation at Il4 gene locus has been shown to occur after the initiation of Il4 transcription during murine Th2 differentiation, likely facilitated by the master regulator Gata3, suggesting that DNA demethylation in this case is a consequence of a cell fate decision and contributes to the reinforcement of lineage commitment (reviewed in [31]). Thus, although the roles of epigenetic modifications are not yet fully understood in many cell types, some cases of lineage specification may require prior modulation of the chromatin landscape in order for the master transcription factors to be able to initiate gene regulation, while other cell differentiation programs may solely depend on master transcription factors, which modulate the chromatin landscape to regulate gene expression. In cases where epigenetic modifications need to occur prior to the expression of master transcription factors, one of the determinants of lineage specification is the inducer of such epigenetic modifications. Among molecules involved in epigenetic modifications, the most downstream ones are enzymes that catalyze the modifying processes. For example, there are DNA methyltransferases for DNA methylation, Tet family members for DNA demethylation, and histone acetyltransferases and histone deacetylases for histone modifications [25, 26]. Furthermore, there are groups of enzymes known as chromatin remodelers, which utilize ATP to alter nucleosome positioning. These epigenetic modifying enzymes often act in concert to modulate the overall chromatin architecture (reviewed in [32]). However, these enzymes are ubiquitously expressed and it is likely that their activity and specificity during lineage specification is regulated by their recruitment to particular genomic regions. Therefore, where these enzymes are recruited to determines lineage specificity, and the way in which they are recruited to particular genomic regions holds the key for understanding the mechanism of lineage specification. One possible model to position general epigenetic modifying enzymes at specific loci is the use of “pioneer” factors that specify target genes. In order to change the chromatin architecture of a locus from a condensed to a loosened structure, such pioneer factors can bind either to nearby accessible sites which are already epigenetically open, or directly to epigenetically closed sites, and
www.eji-journal.eu
Eur. J. Immunol. 2015. 45: 11–16
HIGHLIGHTS
Figure 1. Possible model of lineage specification programs initiated by pioneer factors. Some lineage specification programs may involve gene regulation by modulation of the chromatin landscape, which is initiated by the binding of pioneer factors, followed by the recruitment of epigenetic modifying enzymes such as Tet and histone acetyltransferases to demethylate DNA and modify histones, respectively, resulting in a more open chromatin structure. These chromatin remodeling and permissive epigenetic modifications allow the binding of transcription factors (TF), which activate gene transcription.
then modify the epigenetic features at these sites. In both scenarios, the pioneer factors need to be able to induce epigenetic changes by themselves, or to recruit factors with such capabilities, so that the target loci become accessible to general transcription factors as their consensus sequences become revealed. In fact, FoxA and Gata4 can be considered as pioneer factors for embryonic hepatocyte differentiation; these factors have been shown to bind to their target regions in compacted chromatin in liver precursor cells and open up the compacted chromatin [33]. FoxA has also been shown to bind to methylated cell type specific target sites during neural cell differentiation and subsequently induce DNA demethylation, which is essential for the transcription of target genes [34]. Taken together, these examples suggest that, at least in some cases of lineage specification, chromatin remodeling and other epigenetic modifications by pioneer factors that recruit the modifying enzymes allow the appropriate positioning of transcription factors, which then initiate lineage-specific gene regulation (Fig. 1).
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Epigenetic regulation in Treg-cell differentiation T cells originate from multipotent HSCs, which differentiate into common lymphoid progenitors, and then into T cells. The path of differentiation is accompanied by progressive lineage restriction, consequent loss of the potential to differentiate into other lineages, and acquisition of specific lineage identity with specific transcriptional programs and functions (reviewed in [35, 36]). Similarly to the cases of aforementioned hepatocyte and neural cell differentiation, lineage commitment is accompanied by permissive epigenetic modifications in functionally relevant genes and repressive modifications of the genes associated with alternative lineages [2, 3, 37]. For example, during differentiation of the lymphoid lineage from HSCs, lymphoid lineage-associated genes such as Lck have been shown to undergo DNA demethylation, whereas myeloid lineageassociated genes become methylated [2, 3]. Epigenetic modifications continue further along the road of specification, from that of
www.eji-journal.eu
13
14
Yohko Kitagawa et al.
lymphocytes into T and B cells, and that of T cells into CD4+ and CD8+ T cells [38–40]. These findings demonstrate that there is an accumulation of epigenetic modifications during the sequential differentiation of HSCs to mature T-cell subpopulations, including Treg cells. Treg cells, as a subset of CD4+ T cells, possess specific epigenetic features in addition to those in conventional CD4+ T cells. For example, DNA hypomethylation is specifically observed at Treg signature gene loci, such as Foxp3, Ctla4, Ikzf4, and Ikzf2 [4] and permissive histone marks are specifically present in Treg cells at the Foxp3 promoter region [20]. Particularly, DNA hypomethylation at Foxp3 CNS2 (conserved noncoding region 2), an enhancer region, is important for Treg-cell lineage specification, as it enhances Foxp3 transcription by allowing the binding of transcription factors, which otherwise would not recognize the sequence [41]. Unlike the T cells that express Foxp3 without possessing Treg-cell-specific DNA hypomethylation, such as TGFβ-induced Foxp3+ T cells, Treg cells with these open chromatin structures at Treg signature genes are able to maintain their protein expression as they divide in vivo, suggesting a pivotal contribution of epigenetic modifications to Treg-cell lineage stability [4, 42]. Furthermore, STAT5 and NFAT activated by IL-2 and T cell receptor (TCR) signaling, respectively, were found to interact with the hypomethylated Foxp3 CNS2 to counteract the destabilizing effects of pro-inflammatory cytokines on Treg-cell identity [43, 44]. These results demonstrate the roles of Treg-cell-specific epigenetic modifications in the maintenance of Treg-cell lineage commitment. In addition, there are several findings that suggest their contribution to thymic Treg-cell development. First, Treg-cell-specific DNA demethylation occurs as Treg cells develop in the thymus, independently of Foxp3 expression; moreover, Foxp3-null Treg cells with hypomethylated Treg signature genes still express a substantial proportion of the Treg-cell-type gene profile [4]. Second, it has been shown that thymic Treg precursors are already primed such that general TCR stimulation and IL-2 signaling are sufficient to induce DNA demethylation and Foxp3 expression, which is a phenomenon observed specifically in thymic Treg precursors but not in conventional CD4SP thymocytes [45]. This suggests that some forms of epigenetic modulation occur to the chromatin landscape prior to Foxp3 induction in Treg-cell precursors, making them poised to differentiate into Treg cells. Third, genomewide analyses of gene expression, Foxp3-binding sites, and DNA methylation status in steady-state and activated Treg cells have revealed that DNA hypomethylated sites are associated with gene activation in steady-state Treg cells, whereas Foxp3 is involved in gene repression in activated Treg cells [21]. Consistently, deletion of DNA methylation maintenance enzyme, Dnmt1, in T cells has been shown to induce Foxp3 expression even in CD8+ T cells upon TCR stimulation, suggesting a role for DNA demethylation in Foxp3 induction [46]. Taken together, these findings suggest that thymic Treg-cell development involves preconditioning of the chromatin landscape in precursor cells, followed by the induction of Foxp3 and DNA demethylation. It is speculated that a pioneer factor may bind to Treg signature genes and alter some epigenetic C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2015. 45: 11–16
features in precursor cells, so that upon further TCR and IL-2 signaling, Tet enzymes recruited by the pioneer factor may actively demethylate Treg signature gene loci, and transcription factors, such as cRel and STAT5, downstream of these signaling cascades, bind to Treg signature genes for transcription initiation. It is likely that Foxp3 then acts as a transcriptional repressor at a late stage of the Treg-cell differentiation program to complete Treg lineage specification. In contrast to the idea that a Treg-cell-specific chromatin architecture needs to be generated to induce and maintain the expression of Treg signature genes, including Foxp3, recent studies have demonstrated that the chromatin landscape required for Foxp3-mediated gene regulation is commonly observed in peripheral conventional CD4+ T cells and Treg cells. Analysis of DNase hypersensitive regions in conventional CD4+ T cells and Treg cells showed that Foxp3 does not actively modulate the chromatin landscape to bind to its cognate motifs, but instead binds to sites that are accessible in both conventional CD4+ T cells and Treg cells and are prebound by its co-factors or structurally similar factors [47]. Furthermore, overexpression of Foxp3 with any one of its five co-factors (Eos, Irf4, Satb1, Lef1, and Gata1) in peripheral conventional CD4+ T cells was found to induce expression of a large proportion of Treg-cell-specific genes, far more than that achieved by Foxp3 alone [15]. These findings suggest that preestablishment of the chromatin landscape prior to Foxp3 induction and the expression of co-factors that interact with Foxp3 are critical components for Foxp3-dependent gene regulation. Since Foxp3binding sites are accessible in conventional CD4+ T cells, it is likely that a part of the chromatin landscape required for Tregcell development is established during CD4+ T-cell development or perhaps even earlier, allowing cells to be poised to receive signals for thymic Treg-cell differentiation. Then, upon receiving Treg-cell differentiation-inducing signals, such as relatively strong TCR stimulation by recognition of self-peptide or MHC ligands in the thymus, further modification of the chromatin landscape and induction of Foxp3 are thought to complete the Treg-cell lineage commitment by allowing stable expression of both Foxp3dependent and Foxp3-independent genes (Fig. 2).
Future perspectives With consideration to the pivotal roles of Treg cells in the control of immunological homeostasis, understanding how Treg-cell stability is generated during their development is critical. Foxp3 function in Treg cells has been comprehensively studied in the last decade, and it has revealed that there is more to Treg-cell development than just Foxp3 expression. Since the establishment of a Treg-cellspecific DNA hypomethylation pattern occurs independently of Foxp3 expression, studying the molecular mechanisms underlying its installation could pave the way for the identification of novel factors involved in Treg-cell development. It could also help us elucidate the signals and progenitor conditions required for Tregcell differentiation, which may ultimately lead to the generation of Treg cells in vitro from conventional T cells for use in cell therapies, www.eji-journal.eu
HIGHLIGHTS
Eur. J. Immunol. 2015. 45: 11–16
Figure 2. Distinct roles of epigenetic modification and Foxp3 expression in thymic Treg-cell development. Thymic Treg-cell development involves gene regulation by two mechanisms; gene activation by Tregcell-specific epigenetic modification, which facilitates the binding of transcription factors, including Foxp3 (left), and gene repression by Foxp3, which binds to a preestablished chromatin landscape in an activationdependent manner (right).
and to the discovery of new therapeutic targets for autoimmune and other immunological diseases.
5 Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M. and Toda, M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases. J. Immunol. 1995. 155: 1151–1164. 6 Bennett, C. L., Christie, J., Ramsdell, F., Brunkow, M. E., Ferguson, P. J., Whitesell, L., Kelly, T. E. et al., The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by muta-
Acknowledgments: We thank J. B. Wing for critical reading of the manuscript and the members of our laboratory for discussion. This work is supported by Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency to S.S., research support program for combined research fields to N.O., and grants-in-aid for JSPS Fellows 261560 from Japanese Society for the Promotion of Science to Y.K.
tions of FOXP3. Nat. Genet. 2001. 27: 20–21. 7 Brunkow, M. E., Jeffery, E. W., Hjerrild, K. A., Paeper, B., Clark, L. B., Yasayko, S. A., Wilkinson, J. E. et al., Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat. Genet. 2001. 27: 68–73. 8 Fontenot, J. D., Gavin, M. A. and Rudensky, A. Y., Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 2003. 4: 330–336. 9 Sakaguchi, S., Naturally arising CD4+ regulatory t cells for immuno-
Conflict of interest: The authors declare no financial or commercial conflict of interest.
logic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 2004. 22: 531–562. 10 Kim, J. M., Rasmussen, J. P. and Rudensky, A. Y., Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice. Nat.
References
Immunol. 2007. 8: 191–197. 11 Hori, S., Nomura, T. and Sakaguchi, S., Control of regulatory T cell
1 Jaenisch, R. and Bird, A., Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 2003. 33(Suppl): 245–254.
development by the transcription factor Foxp3. Science 2003. 299: 1057– 1061. 12 Sugimoto, N., Oida, T., Hirota, K., Nakamura, K., Nomura, T., Uchiyama,
2 Bock, C., Beerman, I., Lien, W. H., Smith, Z. D., Gu, H., Boyle, P., Gnirke, A.
T. and Sakaguchi, S., Foxp3-dependent and -independent molecules spe-
et al., DNA methylation dynamics during in vivo differentiation of blood
cific for CD25+CD4+ natural regulatory T cells revealed by DNA microar-
and skin stem cells. Mol. Cell 2012. 47: 633–647.
ray analysis. Int. Immunol. 2006. 18: 1197–1209.
3 Ji, H., Ehrlich, L. I., Seita, J., Murakami, P., Doi, A., Lindau, P., Lee, H.
13 Hill, J. A., Feuerer, M., Tash, K., Haxhinasto, S., Perez, J., Melamed, R.,
et al., Comprehensive methylome map of lineage commitment from
Mathis, D. and Benoist, C., Foxp3 transcription-factor-dependent and -
haematopoietic progenitors. Nature 2010. 467: 338–342.
independent regulation of the regulatory T cell transcriptional signature.
4 Ohkura, N., Hamaguchi, M., Morikawa, H., Sugimura, K., Tanaka, A.,
Immunity 2007. 27: 786–800.
Ito, Y., Osaki, M. et al., T cell receptor stimulation-induced epigenetic
14 Ono, M., Yaguchi, H., Ohkura, N., Kitabayashi, I., Nagamura, Y., Nomura,
changes and Foxp3 expression are independent and complementary
T., Miyachi, Y. et al., Foxp3 controls regulatory T-cell function by inter-
events required for Treg cell development. Immunity 2012. 37: 785–799.
acting with AML1/Runx1. Nature 2007. 446: 685–689.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eji-journal.eu
15
16
Yohko Kitagawa et al.
Eur. J. Immunol. 2015. 45: 11–16
15 Fu, W., Ergun, A., Lu, T., Hill, J. A., Haxhinasto, S., Fassett, M. S., Gazit, R.
34 Serandour, A. A., Avner, S., Percevault, F., Demay, F., Bizot, M., Lucchetti-
et al., A multiply redundant genetic switch ’locks in’ the transcriptional
Miganeh, C., Barloy-Hubler, F. et al., Epigenetic switch involved in acti-
signature of regulatory T cells. Nat. Immunol. 2012. 13: 972–980. 16 Rudra, D., deRoos, P., Chaudhry, A., Niec, R. E., Arvey, A., Samstein, R. M., Leslie, C. et al., Transcription factor Foxp3 and its protein partners form a complex regulatory network. Nat. Immunol. 2012. 13: 1010–1019. 17 Bacchetta, R., Passerini, L., Gambineri, E., Dai, M., Allan, S. E., Perroni, L., Dagna-Bricarelli, F. et al., Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J. Clin. Invest. 2006. 116: 1713–1722.
vation of pioneer factor FOXA1-dependent enhancers. Genome Res. 2011. 21: 555–565. 35 Rothenberg, E. V., Moore, J. E. and Yui, M. A., Launching the Tcell-lineage developmental programme. Nat. Rev. Immunol. 2008. 8: 9– 21. 36 Koch, U. and Radtke, F., Mechanisms of T cell development and transformation. Annu. Rev. Cell Dev. Biol. 2011. 27: 539–562. 37 Vigano, M. A., Ivanek, R., Balwierz, P., Berninger, P., van Nimwegen, E.,
18 Aarts-Riemens, T., Emmelot, M. E., Verdonck, L. F. and Mutis, T., Forced
Karjalainen, K. and Rolink, A., An epigenetic profile of early T-cell devel-
overexpression of either of the two common human Foxp3 isoforms can
opment from multipotent progenitors to committed T-cell descendants.
induce regulatory T cells from CD4(+)CD25(-) cells. Eur. J. Immunol. 2008.
Eur. J. Immunol. 2014. 44: 1181–1193.
38: 1381–1390. 19 Floess, S., Freyer, J., Siewert, C., Baron, U., Olek, S., Polansky, J., Schlawe, K. et al., Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol. 2007. 5: e38. 20 Schmidl, C., Klug, M., Boeld, T. J., Andreesen, R., Hoffmann, P., Edinger, M. and Rehli, M., Lineage-specific DNA methylation in T cells correlates with histone methylation and enhancer activity. Genome Res. 2009. 19: 1165–1174. 21 Morikawa, H., Ohkura, N., Vandenbon, A., Itoh, M., Nagao-Sato, S., Kawaji, H., Lassmann, T. et al., Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation. Proc. Natl. Acad. Sci. USA 2014. 111: 5289–5294. 22 Xie, W., Schultz, M. D., Lister, R., Hou, Z., Rajagopal, N., Ray, P., Whitaker, J. W. et al., Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 2013. 153: 1134–1148. 23 Chen, T. and Dent, S. Y., Chromatin modifiers and remodellers: regulators of cellular differentiation. Nat. Rev. Genet. 2014. 15: 93–106. 24 Mohn, F., Weber, M., Rebhan, M., Roloff, T. C., Richter, J., Stadler, M. B., Bibel, M. et al., Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 2008. 30: 755–766. 25 Bird, A., DNA methylation patterns and epigenetic memory. Genes Dev. 2002. 16: 6–21. 26 Williams, K., Christensen, J. and Helin, K., DNA methylation: TET proteins-guardians of CpG islands? EMBO Rep. 2012. 13: 28–35.
38 Taniuchi, I. and Ellmeier, W., Transcriptional and epigenetic regulation of CD4/CD8 lineage choice. Adv. Immunol. 2011. 110: 71–110. 39 Barneda-Zahonero, B., Roman-Gonzalez, L., Collazo, O., Mahmoudi, T. and Parra, M., Epigenetic regulation of B lymphocyte differentiation, transdifferentiation, and reprogramming. Comp. Funct. Genomics 2012. 2012: 564381. 40 Wilson, C. B., Makar, K. W., Shnyreva, M. and Fitzpatrick, D. R., DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin. Immunol. 2005. 17: 105–119. 41 Polansky, J. K., Schreiber, L., Thelemann, C., Ludwig, L., Kruger, M., Baumgrass, R., Cording, S. et al., Methylation matters: binding of Ets-1 to the demethylated Foxp3 gene contributes to the stabilization of Foxp3 expression in regulatory T cells. J. Mol. Med. 2010. 88: 1029–1040. 42 Ohkura, N., Kitagawa, Y. and Sakaguchi, S., Development and maintenance of regulatory T cells. Immunity 2013. 38: 414–423. 43 Li, X., Liang, Y., LeBlanc, M., Benner, C. and Zheng, Y., Function of a Foxp3 cis-element in protecting regulatory T cell identity. Cell 2014. 158: 734–748. 44 Feng, Y., Arvey, A., Chinen, T., van der Veeken, J., Gasteiger, G. and Rudensky, A. Y., Control of the inheritance of regulatory T cell identity by a cis element in the Foxp3 locus. Cell 2014. 158: 749–763. 45 Toker, A., Engelbert, D., Garg, G., Polansky, J. K., Floess, S., Miyao, T., Baron, U. et al., Active demethylation of the Foxp3 locus leads to the generation of stable regulatory T cells within the thymus. J. Immunol. 2013. 190: 3180–3188.
27 Pina, B., Bruggemeier, U. and Beato, M., Nucleosome positioning modu-
46 Josefowicz, S. Z., Wilson, C. B. and Rudensky, A. Y., Cutting edge: TCR
lates accessibility of regulatory proteins to the mouse mammary tumor
stimulation is sufficient for induction of Foxp3 expression in the absence
virus promoter. Cell 1990. 60: 719–731. 28 Boyes, J. and Bird, A., DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell 1991. 64: 1123–1134. 29 Probst, A. V., Dunleavy, E. and Almouzni, G., Epigenetic inheritance dur-
of DNA methyltransferase 1. J. Immunol. 2009. 182: 6648–6652. 47 Samstein, R. M., Arvey, A., Josefowicz, S. Z., Peng, X., Reynolds, A., Sandstrom, R., Neph, S. et al., Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification. Cell 2012. 151: 153–166.
ing the cell cycle. Nat. Rev. Mol. Cell Biol. 2009. 10: 192–206. 30 Takizawa, T., Nakashima, K., Namihira, M., Ochiai, W., Uemura, A., Yanagisawa, M., Fujita, N. et al., DNA methylation is a critical cellintrinsic determinant of astrocyte differentiation in the fetal brain. Dev. Cell 2001. 1: 749–758. 31 Ansel, K. M., Djuretic, I., Tanasa, B. and Rao, A., Regulation of Th2 differ-
Full correspondence: Prof. Shimon Sakaguchi, Department of Experimental Immunology, World Premier International Immunology Frontier Research Center, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan Fax: +81-6-6879-4464 e-mail: [email protected]
entiation and Il4 locus accessibility. Annu. Rev. Immunol. 2006. 24: 607–656. 32 Cedar, H. and Bergman, Y., Linking DNA methylation and histone modification: patterns and paradigms. Nat. Rev. Genet. 2009. 10: 295–304. 33 Cirillo, L. A., Lin, F. R., Cuesta, I., Friedman, D., Jarnik, M. and Zaret, K. S., Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol. Cell 2002. 9: 279–289.
C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Received: 17/9/2014 Revised: 17/10/2014 Accepted: 22/10/2014 Accepted article online: 28/10/2014
www.eji-journal.eu
