Constitutive CTCF binding forms insulated neighborhoods encompassing T _MP genes. (A) Heatmaps showing ChrInt among +/−500 kb regions centered on the incongruous (left) and congruous sites (right), based on pile-up analysis of Hi-C data in T_N cells. (B) Heatmaps showing ChrInt pile-up profile centered on ChrAcc⁺ (top) and ChrAcc⁻ motif⁺ (bottom) constitutive CTCF binding sites that are located at 100–200 kb upstream or downstream of the incongruous sites in T_N cells.. (C) Heatmap showing the interaction strength between upstream and downstream ChrAcc⁻ constitutive CTCF binding sites flanking the incongruous sites in T_N cells.based on aggregate peak analysis. In A–C, color scale denotes ChrInt strength. (D) Boxplots showing insulation scores at the upstream (left) and downstream (right) ChrAcc⁻ motif⁺ constitutive CTCF binding sites flanking the incongruous sites in T_N and T_EFF cells. The P values are determined with one-sided Mann–Whitney U test. (E) Heatmaps showing ChrInt strength at the Tcf7, Sell, and Id3 gene loci and associated incongruous sites in T_N and T_EFF cells. The green-edged triangle marks an insulated neighborhood, and that with dotted lines represents extended regions where additional ChrAcc⁻ constitutive CTCF binding sites may contribute to enforcing the boundary. The yellow circle represents the insulating knots formed through interactions between the boundary anchors. The end of red arrow represents TSS of genes of interest, and the length is proportional to the gene length. CTCF binding tracks in WT T_N and T_EFF cells are the same as Fig. 7 A (for Tcf7 and Id3) or Fig. S5 A (for Sell), shown as location reference for the constitutive (green) and dynamic (blue) CTCF binding sites. Insulation (Ins) scores of each 10 kb bin across each gene locus are displayed, with lower scores in orange representing stronger insulation effects.