Demethylation at the Foxp3 intronic TSDR region in WT versus Dnmt1^−/− Tregs and Tcon. Demethylation was determined by (A) bisulphite conversion, cloning, and sequencing and was (B) confirmed by pyrosequencing. (C) Assessment of demethylation of the Foxp3 promoter region in WT versus Dnmt1^−/− Tregs and Tcon was determined by bisulphite conversion. Open circles indicate demethylated CpG and closed circles indicate methylated CpG. (D) Measurement of total DNA methylation in WT versus Dnmt1^−/− Treg cells (mean ± SD, n = 3 per group).