Dnmt1 affects genome-wide methylation, leading to loss of the Treg phenotype. (A) Genome-wide overview of fold change in scaled log₂ ratios and peak scores of methylated genomic DNA from Dnmt1^−/− and WT Treg. Heatmap scale optimized for visualization of −2 to +2.5–fold changes (>96.6% of data; range, −5.68 to +9.28). Hypomethylation shown indicated in blue, and hypermethylation is indicated in red. Results pooled from 3 independent experiments and visualized with the Broad Institute’s Integrative Genomics Viewer. (B) Methylation assessment of the IFN-γ enhancer region, as in Figure 5, confirmed greater than 4× increase in demethylation in Dnmt1^−/− Treg. (C) Real-time PCR analysis (mean ± SD, n = 4 per group) of the effects of Dnmt1 loss on Treg expression of various cytokines, and signature Treg genes showed increased IFN-γ expression, consistent with enhancer demethylation. (D-E) Selection transcripts with greater than 2× differential expression; microarray data are shown after z-score transformation (n = 3 per group), data shown after z-score transformation. (D) Transcripts identified as differentially methylated in (A). (E) Treg-relevant genes. (F) Functional annotation enrichment of probes with greater than 5 higher raw scores in Dnmt1^−/− versus WT Treg (P < .05). Gene ontology (GO), cellular component (CC), biological process (BP), molecular function (MF). Functional Annotation Clustering calculated using DAVID.⁵⁰  (G) Loss of Dnmt1 disrupted the normal expression of genes associated with adenosine production (real-time PCR, mean ± SD, n = 4 per group).