High occupancy APBSs delineate TADs and associate with robust enhancer-blocking activity. (a) Heatmap representing Hi-C interaction frequencies at single fragment resolution for a 1 Mb region across Drosophila chromosome 3 L in Kc167 cells. White lines demarcate previously defined TAD boundaries . A high occupancy APBS (left) is present at a single fragment topological domain border strongly separating two TADs (white arrowhead). Colorbar represents (log2) interaction frequencies observed between restriction fragments, ranging from low (blue) to high (red). (b) Percentage of TADs defined in Kc167 cells delineated by a high, medium, or low occupancy APBSs ± one restriction cut site (TAD borders n = 1,110, high occupancy APBSs n = 1,638, P < 0.00001, permutation test). (c) Topological border strength defined by the ratio of intra- versus inter-TAD interaction frequencies scales with the occupancy (number of bound proteins) at APBSs. (d) Architectural protein occupancy and DNase I hypersensitivity at DNA fragments previously tested for enhancer-blocking activity in transgenic reporter assays [13, 51, 52]. Sequences shown to possess robust activity (red) correlate with both the highest occupancy and DNase I activity, whereas sites incapable of insulator activity are marked by low occupancy (P < 0.01, Wilcoxon rank sum test, two-sided). RPM, reads per million. (e) Quantification of topological domain border strengths at sequences tested for insulator function within their endogenous context. Robust insulator sequences are characterized by significantly greater topological border strength than non-enhancer-blocking sequences (P < 0.05, Wilcoxon rank sum test, two-sided). (f) Tag density plots of rank-order normalized DNase-seq profiles throughout embryonic stages of development at APBSs , and at transcription factor binding sites shown to function as developmental enhancers during early embryogenesis. The progressive loss of DNase accessibility at highly bound transcription factor binding sites (right) contrasts with the combinatorially bound APBSs (left), which are marked by strong DNase I hypersensitivity throughout each stage of development.