PLoS BIOLOGY

Epigenetic Control of the foxp3 Locus in Regulatory T Cells
Stefan Floess1[, Jennifer Freyer1[, Christiane Siewert1[, Udo Baron2, Sven Olek2, Julia Polansky1, Kerstin Schlawe1,
Hyun-Dong Chang3, Tobias Bopp4, Edgar Schmitt4, Stefan Klein-Hessling5, Edgar Serfling5, Alf Hamann1,
Jochen Huehn1*
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1 Experimentelle Rheumatologie, Charite Universitaetsmedizin Berlin, Berlin, Germany, 2 Epiontis, Berlin, Germany, 3 Deutsches Rheuma-Forschungszentrum, Berlin,
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Germany, 4 Institut fur Immunologie, Johannes-Gutenberg-Universitat, Mainz, Germany, 5 Abteilung fur Molekulare Pathologie, Pathologisches Institut, Wuerzburg,
Germany

Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4þ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity.
Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as
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histone modifications within the conserved region in ex vivo isolated Foxp3þCD25þCD4þ Tregs, but not in naıve
CD25ÀCD4þ T cells. Partial DNA demethylation is already found within developing Foxp3þ thymocytes; however, Tregs induced by TGF-b in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural
Tregs, these TGF-b–induced Foxp3þ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-b. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.
Citation: Floess S, Freyer J, Siewert C, Baron U, Olek S, et al. (2007) Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 5(2): e38. doi:10.1371/journal.pbio.
0050038

application in vivo [9–11] or upon activation in the presence of TGF-b in vitro [12–17]. To what extent these induced populations of Tregs acquire a stable phenotype corresponding to that of natural, thymus-derived Tregs is, however, unclear. An emerging paradigm in understanding the development of stable cellular lineages emphasizes the role of epigenetic mechanisms for the permanent, heritable fixation of distinct gene expression patterns. Molecular mechanisms of epigenetic imprinting include selective demethylation of CpG motifs and histone modifications as shown for cytokine genes
[18–20]. Whether Treg differentiation also involves elements of epigenetic regulation has not been studied thus far. We therefore investigated whether epigenetic alterations such as
DNA methylation and histone modifications of the foxp3 locus correlate with Foxp3 expression. The selective association of chromatin remodeling with a stable Treg phenotype suggests

Introduction
Regulatory T cells (Tregs), which have been shown to play a pivotal role in the maintenance of self-tolerance within the immune system, were described originally as CD4þ T cells constitutively expressing CD25 [1]. More recently, the forkhead transcription factor Foxp3 has been shown to be specifically expressed in Tregs and to be a central control element in Treg development and function [2]. Mutation or deletion of the gene encoding Foxp3 causes severe autoimmune disease in mice and humans, due to a failure to generate CD25þCD4þ Tregs [3,4], whereas ectopic expression of Foxp3 in conventional T cells confers suppressive activity
[4,5]. These findings provided compelling evidence that
Foxp3 acts as a master switch controlling the development and function of