Extended Data Fig. 4: SCR can activate Sox2 across CTCF-mediated insulation.

a Top left scheme depicts targeting strategy for generation of the transgenic lines carrying insertion of CTCF motifs. Arcs represent CTCF-mediated loops generated in each insertion line as predicted by the loop-extrusion model. We assumed that the sites where cassettes were targeted did not contain regulatory elements based on conservation and analysis of ENCODE datasets. Top right browser shows CTCF ChIP-seq enrichment and insertion sites of CTCF transgenes. Left bottom panel shows the targeting of the C57Bl6 genome to generate CTCFi3×⁻ mice. Right bottom panel shows targeting to generate CTCFi3×⁺, CTCFi3×⁻;3×⁺, and CTCFi18×⁺ mice. Nucleotides shown in red represent the protospacer sequence of gRNAs used for injection while underlined nucleotides highlight location of the Cas9 PAM in the mouse mm10 genome. gRNA mm10 coordinates are shown in red. The central sequence of the repair templates is shown, with parts of the protospacer sequence used in the gRNA shown in red and mutated PAM nucleotides underlined. The complete sequence of homology arms on both sides is omitted. Each colored rectangle represents a different region from the mouse genome containing a CTCF motif and adjacent regions. Complete sequences of the CTCF motifs and adjacent regions are shown in different color with the central CTCF motif in blue representing targeting to negative strand in the CTCFi3×⁻ line. The first and last two nucleotides of each of the three CTCF regions are shown in the schema of the repair template. The same three CTCF-carrying regions were used in CTCFi3×⁻ and CTCFi3×⁺ lines but targeted to different strands and locations. Therefore, the central region of repair template is the same but in different strands and containing different repair templates. Retargeting of the CTCFi3×⁺ transgene on a homozygous CTCFi3×⁻ background generated the CTCFi3×⁻;3×⁺ line. The CTCFi18×⁺ line was obtained as a consequence of the CTCFi3×⁺ injection because of concatemerization of the repair template. The resulting allele is shown in the bottom right. b Differential CHi-C interaction frequency heatmap. Red signal represents interactions occurring at higher frequency in mutant cell lines compared to control. Dotted lines represent insertion sites of CTCF transgenes.