Dot1L licenses DNA demethylation to establish regulatory T cell identity

Foxp3+ regulatory T (Treg) cells rely on DNA demethylation to establish and maintain the gene expression program that defines their identity and suppressive function. Although sustained transcription of Treg-specific genes is known to drive this demethylation, the precise mechanism has remained unclear. Here, we show that Dot1L-catalyzed methylation of histone H3 at lysine 79 (H3K79me) is essential for Treg-specific DNA demethylation in both thymic and induced Treg lineages. H3K79me promotes chromatin activation and recruits TET family DNA demethylases to key regulatory loci. Treg-restricted deletion of Dot1L disrupts locus-specific DNA demethylation, diminishes expression of core Treg genes, and precipitates a fatal, early-onset autoimmune syndrome. Conversely, pharmacologic enhancement of TET activity during differentiation rescues DNA demethylation, restores Treg-gene expression, and reinstates suppressive function even in the presence of Dot1L inhibition. Together, these findings identify a critical epigenetic axis--Dot1L-mediated H3K79 methylation driving TET-dependent DNA demethylation--that safeguards Treg cellular identity, function, and immune homeostasis.