Fig. 7: CTCF orchestrates the inter-chromosomal association between Gm26793 and Cubn loci.

a Schematic diagram showing the strategy for GKO fragment knockin. b Bar plot showing the partial rescue of primitive endoderm markers upon GKO region knockin on XEN-D7. c Genome browser view demonstrating the binding of CTCF and RAD21 at Gm26793 and Cubn loci in WT and GKO mESCs. Grey boxes represent the co-binding sites of CTCF and RAD21, which were subjected to genomic knockout in WT cells and following experiments. The light yellow box represents the CCCTC motif within GKO region. d Schematic illustration depicting the specific knockout of CTCF-binding site within the Gm26793 (CBS-KO) and Cubn (CKO) loci. e Relative expression changes of primitive endoderm-related genes on XEN-D7 upon CTCF binding site knockout. f Morphologies of differentiated cells at bright field in the indicated cell groups. g Quantification of Cubn expression by RNA-seq analysis on XEN-D7. h Heatmap showing the DEGs upon CTCF-binding site knockout or knockin on XEN-D7. The average expression levels of up- and down-regulated genes are listed on the right panel. i Quantification of the contact frequency with neighboring regions and Cubn locus by using Gm26793 as a viewpoint on ESC-D0 and XEN-D7 in the indicated cell groups. j Representative images recording the spatial distribution of the Gm26793 and Cubn loci in the indicated cells on ESC-D0 and XEN-D7. Scale bars, 5 μm. (ESC-D0: WT, n = 43 cells; GKO, n = 46 cells; GKO + KI, n = 62 cells; CKO, n = 3 6 cells; CBS-KO, n = 36 cells. XEN-D7: WT, n = 84 cells; GKO, n = 105 cells; GKO + KI, n = 95 cells; CKO, n = 7 0 cells; CBS-KO, n = 52 cells). k Scatter plots showing the statistical changes of 3D-distance between the Gm26793 and Cubn loci in the indicated cell groups on ESC-D0 (top) and XEN-D7 (bottom). qPCR data are shown as means ± SEM. 3D-distance statistics are shown as means ± SD. One-way ANOVA analysis with Tukey’s test is used in b, e, h, k; *P < 0.05, **P < 0.01, ***P < 0.001.