Figure 3. Reversible disruption of Irx gene clustering by 1,6-HD.

(A) Hi-C maps of the TAD on chromosome 8 (mm9 co-ordinates in Mb) containing Irx3, 5, and 6 from (upper) WT (Ring1B^+/+) and (lower) Ring1B^−/− mESCs at 5 kb resolution. Genes, fosmid probe–binding locations, H3K27me3, and RING1B ChIP-seq profiles are shown between the two Hi-C maps (data are from the study of Boyle et al [2020]). Black arrowheads indicate enriched interactions between Irx3, 5, and 6 in WT mESCs and the reduced interactions in Ring1B^−/− cells. (B) Representative 3D-FISH images of Irx3, 5, and 6 loci in untreated, 1,6-HD–treated and recovered WT mESCs. Scale bar: 5 μm. (C) Bar plots providing categorical analysis of the spatial relationship of Irx3, 5, and 6 in untreated, 2,5-HD–treated, 1,6-HD–treated and recovered WT cells. Categories are as follows: clustered, at least two of the three loci <200 nm apart; intermediate, at least two of the three loci between 200–399 nm apart; dispersed, all three loci ≥400 nm apart. Differences in clustering and dispersal are identified using Fisher’s exact test; ∗ P ≤ 0.05 and > 0.01, ∗∗ P ≤ 0.01, ∗∗∗ P ≤ 0.001, ∗∗∗∗ P ≤ 0.0001. (D) Scatter plots showing interprobe distances between each of the two probe pairs indicated on x and y axes with the separation between the third pair indicated by the color (in the color bar) in untreated, 2,5-HD–treated, 1,6-HD–treated, and recovered WT mESCs. Dashed red box indicates non-dispersed alleles with at least two sets of interprobe distances <400 nm. (E, F) As for (B, C), but for Ring1B^−/− cells. Scatter plots for those data are in Fig S3, as are data for a biological replicate. All data and statistical evaluation are summarised in Table S3.