Active Demethylation of the <i>Foxp3</i> Locus Leads to the Generation of Stable Regulatory T Cells within the Thymus

TLDR

Stable Foxp3 expression in regulatory T cells requires demethylation of the Treg‑specific demethylated region (TSDR) within the Foxp3 locus, but the developmental stage at which this demethylation occurs is unknown. This study investigates whether stable lineage commitment of thymic Tregs is established during early thymic development. The authors demonstrate that active demethylation of the TSDR is mediated by ten‑eleven‑translocation enzymes and hydroxylation of methylated cytosines, initiating a mitosis‑independent DNA demethylation pathway. Early TSDR demethylation in thymic Tregs stabilizes Foxp3 expression, ensuring full functionality and long‑term lineage stability.

Abstract

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.