Fig. 5: The large CTCF cluster separating the Six3 and Six2 TADs prevents promoter competition between these two genes. | Nature Communications

Fig. 5: The large CTCF cluster separating the Six3 and Six2 TADs prevents promoter competition between these two genes.

From: Cooperative insulation of regulatory domains by CTCF-dependent physical insulation and promoter competition

Fig. 5

A NPC Hi-C19 data shows that Six3 and Six2 are located in neighbouring TADs separated by a cluster of seven CTCF sites122. The orientation of key CTCF sites is illustrated with red (sense) and blue (antisense) triangles. B Graphical overview of genomic rearrangements generated in mESC within the Six3/Six2 locus: Δ6XCTCF; 156KbINV; Δ6XCTCF:156KbINV. C, D The expression of Six3 and Six2 was measured by RT–qPCR in NPC that were WT or homozygous for the genomic re-arrangements described in (B). For each cell line, Six3 and Six2 expression was measured in the following number of biological replicates: WT -16 replicates; Δ6XCTCF -19 replicates using a previously characterized clonal line63; 156KbINV −10 replicates for Six3 and 14 replicates for Six2 using two different clonal lines; Δ6XCTCF:156KbINV −18 replicates using three different clonal lines. E Graphical overview of the 226KbINV mESC line. F Six3 and Six2 expression was measured by RT–qPCR in NPC that were WT or homozygous for the 226KbINV. For each cell line, Six3 and Six2 expression was measured in the following number of biological replicates: WT -4 replicates; 226KbINV -4 replicates using two different clonal lines. G Graphical overview of the Δ6XCTCF:Six2−/− mESC line. H Six3 expression was measured by RT–qPCR in NPC that were either WT or homozygous for the Δ6XCTCF and Δ6XCTCF:Six2−/− deletions. For each cell line, Six3 was measured in the following number of biological replicates: WT −16 replicates (same as in C); Δ6XCTCF −19 replicates (same as in C); Δ6XCTCF:Six2−/− -11 replicates using three different clonal lines. Expression differences among cell lines were calculated using two-sided unpaired t-tests (NS (not significant): fold-change < 2 or p > 0.05). In (C, D, F, H) box plots, the upper and lower parts of the box are the upper and lower quartiles, respectively, the horizontal line that splits the box in two is the median and the upper and lower whiskers indicate the maximum and minimum, respectively. Source RT-qPCR data are provided as a Source Data file.

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