mTECany2-T-DMRs correlate with gene expression in mTECs and associate with marker genes of mTECs. A. Hierarchical clustering of D-REAM scores corresponding to mTECany2-T-DMRs identified by comparison of Aire−/− mTECs with the brain, thymus, liver, and ES cells by Pearson’s correlations. Among mTECany2-T-DMRs, those hypomethylated in Aire−/− mTECs in comparison with all of the reference cells and tissues were defined as mTECu-T-DMRs (indicated by asterisk). Br, brain; Thy, thymus; Lv, liver. B. Relative expression levels of genes associated with mTECu-T-DMRs in proximal regions (−6 to +2.5 kb) from TSS. The horizontal axis of the boxplot represents the log ratios of gene expression levels relative to Aire−/− mTECs. The numbers in round brackets indicate independent gene expression profiles for each type of cell or tissue (Additional file 5: Table S2). The p-values of t-tests showing significant differences in relative expression levels are shown. C. Bisulfite sequencing of regions around the TSS of Aire. The arrowhead indicates the position of the mTECu-T-DMR. The regions upstream (−674 to −191 bp) and downstream (+1672 to +2082 bp) of the TSS were analyzed independently using bisulfite sequencing. Open and closed circles represent unmethylated and methylated CpGs, respectively. Positions of CpGs are shown as vertical bars above the genomic structure (shown at the bottom of the panel), and the boxes indicate exons. D. COBRA analysis of mTECu-T-DMRs associated with Cd80 and Trp63. Positions of HpyCH4IV sites and mTECu-T-DMRs are shown above the genomic sequences within −1 to +2.5 kb of the TSS. Electropherograms represent the bisulfite PCR products after digestion with HpyCH4IV. Hypomethylated fragments were resistant to HpyCH4IV digestion (+). The levels of DNA methylation (%) are shown under each lane of the electropherograms.