Fig. 4: Runx1 promoter-proximal CTCF sites play a role in establishing Runx1 chromatin architecture.

a Schematic of Runx1 TAD showing CTCF binding in mESCs⁴² and the orientation of CTCF motifs underlying peaks. P1 and P2 promoter-proximal CTCF sites are indicated with CRISPR/Cas9 strategies to delete them. Distance to Runx1 transcription start sites is indicated. Vertebrate conservation (phastCons, cons), CTCF occupancy in 416B HPCs, core motif sequence, single guide (sg)RNA, and deletion alleles (dels) are indicated. b, c Subtraction of Tiled-C matrices between P2-CTCF-KO (b) P1-CTCF-KO (c) and wild-type hematopoietic cells is shown at 2 kb resolution with threshold at +/−97th percentile of subtracted normalized interactions (subtracted norm. ints.) (n = 4). Locations of CTCF site deletions are indicated by a pink and green cross. RPKM-normalized ATAC-seq in wild-type HPCs and CTCF occupancy in 416B cells is shown. The locations of the main Runx1 TAD and sub-TADs are indicated. d, e Tiled-C matrix from P2-CTCF-KO (d) and P2-CTCF-KO (e) (2 kb resolution, threshold at 94th percentile, n = 4). f Insulation scores (intra-TAD interaction ratio) for main Runx1 TAD and sub-TADs in wild type, P1-CTCF-KO, and P2-CTCF-KO HPCs (*, Kruskal–Wallis and Dunn’s test, two-sided adjusted p-values: main TAD WT and P1-CTCF-KO p = 4.8⁻⁴, WT and P2-CTCF-KO p = 0.03, P1-P2 sub-TAD WT and P1-CTCF-KO p = 0.003). Boxplot centre shows median, bounds of the box indicate 25th and 75th percentiles, and maxima and minima show the largest point above or below 1.5 * interquartile range. Outlying points are not shown. Data were analyzed from the total number of bins indicated above each boxplot from four biologically independent experiments.