Epigenetic regulation of Foxp3 expression in regulatory T cells by DNA methylation

FOXP3, a winged-helix family transcription factor, serves as the master switch for CD4(+) regulatory T cells (Treg). We identified a unique and evolutionarily conserved CpG-rich island of the FOXP3 nonintronic upstream enhancer and discovered that a specific site within it was unmethylated in natural Treg (nTreg) but heavily methylated in naive CD4(+) T cells, activated CD4(+) T cells, and peripheral TGFbeta-induced Treg in which it was bound by DNMT1, DNMT3b, MeCP2, and MBD2. Demethylation of this CpG site using the DNA Methyltransferase Inhibitor 5-aza-2'-deoxycytidine (Aza) induced acetylation of histone 3, interaction with TIEG1 and Sp1, and resulted in strong and stable induction of FOXP3. Conversely, IL-6 resulted in methylation of this site and repression of FOXP3 expression. Aza plus TGFbeta-induced Treg resembled nTreg, expressing similar receptors, cytokines, and stable suppressive activity. Strong FOXP3 expression and suppressor activity could be induced in a variety of T cells, including human CD4(+)CD25(-) T cells. Epigenetic regulation of FOXP3 can be predictably controlled with DNMT inhibitors to generate functional, stable, and specific Treg.