Figure 5(A) K-means clustering of gene expression data for Th0 cells and iTreg cells differentiated in vitro under the conditions of TGFβ alone, TGFβ + VitC, TGFβ + RA; TGFβ + RA + VitC using genes differentially expressed between the pairwise comparisons indicated by the arrows on top of the heatmap. The number of genes in each cluster is shown on the left. (B) Gene set enrichment analysis (GSEA) of expression changes induced by Vitamin C treatment, for genes upregulated in Treg cells vs conventional T cells (GSE7460, left panel), genes upregulated in WT Treg cells vs CD4+ naïve T cells (our data, middle panel) and genes upregulated in WT Treg cells vs Tet2/3 DKO Treg cells (our data, right panel), presented as enrichment score (Y-axis) and overlap of genes from each set (X-axis, shown on the bottom) with genes expressed higher in TGFβ + VitC and TGFβ + RA + VitC compared to TGFβ alone and TGFβ + RA, respectively. Data are from two biological replicates. (C) K-means clustering of chromatin accessibility data (ATAC-seq) for Th0 cells and in vitro differentiated iTregs using regions differentially accessible between the pairwise comparisons indicated by the arrows on top of the heatmap. The number of regions is shown on the left and data are represented as normalized log2 (RPM+1). (D) Scatter plot of ATAC-seq data for TGFβ + VitC vs TGFβ iTregs (left), TGFβ + RA + VitC vs TGFβ + RA iTregs (middle) and TGFβ + RA + VitC vs TGFβ iTregs (right). Differentially-accessible regions (DARs) are shown in color and the total number of DARs (N) are shown on each graph. (E) Genome browser view of ATAC-seq data showing the accessibility profile at the Foxp3 locus; the CNS2 region is highlighted (gray shading). DARs are denoted above the cell type tracks with regions of more accessibility identified by color. Scale is reads per million (RPM) and conservation track (Cons) is shown at the bottom. (F) Beeswarm plots of mean chromatin accessibility measured as log2 (RPM+1) for regions more accessible in TGFβ + VitC vs TGFβ (left) and TGFβ + RA + VitC vs TGFβ + RA (right). DARs denote the regions in the respective cell types used for the comparison, and are indicated by the gray shading. P-values are calculated using an analysis of variances with Tukey's post-hoc test and DARs are determined using a negative binomial test with Benjamini-Hochberg correction FDR ≤ 0.05 (see methods). Data are from two biological replicates. (G) Scatter plots show the comparisons of whole genome bisulfite sequencing (WGBS) data between TGFβ + RA + VitC iTregs vs TGFβ iTregs. The differentially-methylated CpGs (DM-CpGs) are shown in color and the numbers are shown on top of each graph. (H-J) Box-and-whisker plots of DNA methylation levels for DM-CpGs less methylated in TGFβ + RA + VitC iTregs vs TGFβ iTregs (H), DM-CpGs less methylated in WT Treg cells vs CD4+ naïve T cells (I) and DM-CpGs less methylated in WT Treg cells vs Tet2/3 DKO Treg cells (J). DM-CpGs denotes the CpGs in the respective cell types used for the comparison. Boxplots show the median and quartiles with the whiskers extending to the most extreme data point within 1.5 times the interquartile range. P-values are shown above relevant comparisons and are calculated using an analysis of variances with Tukey's post-hoc test. Data are from two biological replicates.