Fig. 3: Formation of chromatin loops by nonconvergently oriented CBSs.

a, Schematic of a convergent loop that indirectly generates a nonconvergent loop in the opposite direction. b, Percentage of loop anchors establishing bidirectional loops (n = 12,635 loops from mESCs from ref. ²⁶). Anchor categories: convergent-only (only CBSs oriented in the same direction as their anchored loops, n = 7,769), nonconvergent (anchor loops in a direction for which they lack a directional CBS, n = 960) and no-CTCF (no CBS, n = 3,906). c, Loop strengths in pairs of convergent/nonconvergent loops classified into Non-conv.-associated (nonconvergent loop sharing the nonconvergent anchor with a convergent loop in the opposite direction, n = 322) and Conv.-associated (convergent loop sharing one anchor with a nonconvergent loop in the opposite direction, n = 496). Boxplots defined as in Fig. 1c. Two-sided Benjamini–Hochberg-corrected Mann–Whitney U-test P = 6.2 × 10⁻⁶. d, Aggregated loop signal for categories in c. Arrows represent CBS orientation. e, Pax3 WISH in E11.5 forelimbs from CBS mutants. Arrowheads represent reverse- (blue) and forward- (orange) oriented CBSs. Crosses indicate deleted CBSs. Note the positive correlation between expanded Pax3 misexpression and increased number of deleted CBSs. Scale bar, 250 μm. f, Pax3 qPCR analysis in E11.5 limbs from CBS mutants. Bars represent mean and dots individual replicates. Values were normalized against DelBs mutant (ΔΔCt). Note the positive correlation of Pax3 misexpression with the increase in deleted CBSs (Pearson correlation significantly > 0; ***P ≤ 0,001). g, cHi-C maps from E11.5 mutant distal limbs at 10-kb resolution (top). Data were mapped on a custom genome containing the DelBs deletion (n = 1 with an internal control comparing 6 different experiments; Methods). Insets represent a magnification (5-kb resolution) of the centromeric (left) and telomeric (right) loops highlighted by brackets on the map. Gained or lost chromatin loops are represented by full or empty dots. Subtraction maps (bottom) showing gain (red) or loss (blue) of interactions in mutants compared with DelBs. h, Insulation score values. Dots represent the local minima of the insulation score at the EP boundary for each mutant. i, Virtual 4C profiles for the region in g (viewpoint in Pax3). The gray rectangle highlights Epha4 enhancers. Note increased interactions between the Pax3 promoter and the Epha4 enhancers in ΔR-all compared with DelBs.