Active Demethylation of the Foxp3 Locus Leads to the Generation of Stable Regulatory T Cells within the Thymus

Abstract

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 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 guarantees full functionality and long-term lineage stability of Tregs.

Vitamin C Facilitates Demethylation of the Foxp3 Enhancer in a Tet-Dependent Manner

“…7A). These results led us to hypothesize that a lack of sensitivity of tTregs to vitamin C might be caused by the high levels of Tet expression (30). Indeed, Foxp3 CNS2 in Tet2 2/2 tTregs was methylated after sulfinpyrazone treatment.…”

“…6 in Ref. 30) and vice versa. Next, we tested the role of Dnmt1 in CNS2 demethylation using RNA interference.…”

“…Furthermore, CNS2 is demethylated in tTregs expressing Foxp3 stably, but it is fully methylated in activated T cells or iTregs expressing Foxp3 transiently (18,29). CNS2 demethylation has been reported to occur within the thymus during the developmental stages and achieved through an active DNA oxidation process including ten-eleven-translocation (Tet) family (30,31). However, the underlying mechanism of the epigenetic regulation of CNS2 is still elusive.…”

Unravelling the molecular basis for regulatory T‐cell plasticity and loss of function in disease

“…The modifiable mediators of this regulation include methylation,37, 38, 39, 40, 41, 42, 43, 44 acetylation45, 46, 47 and microRNA‐mediated control of transcript translation 28, 48, 49, 50, 51, 52, 53, 54, 55. The global regulation of FOXP3 is influenced by chromatin organises, including SATB1 acts both during development and in mature cells 48, 58.…”

Central Role of CD45RA− Foxp3hi Memory Regulatory T Cells in Clinical Kidney Transplantation Tolerance

“…20,21 Demethylation of the Foxp3 region is not only important for the induction and stabilization of Foxp3 expression but also ensures the differentiation and suppressive function of Tregs. 22,23 In this article, we characterize the profile of Tregs in circulating blood from operationally tolerant patients and investigate the Foxp3 demethylation level of these cells in conjunction with their suppressive properties. To our knowledge, this study is the first report of an enhanced frequency of CD45RA 2 Foxp3 hi memory Tregs (mTregs) expressing CD39 and GITR and exhibiting increased Foxp3 TSDR demethylation and stronger suppressive properties in operationally tolerant patients.…”