Figure S3. Polycomb-dependent associations between Irx3, 5, and 6 and replicate data for Fig 3 showing reversible disruption of the cluster by 1,6-HD.

(A) Micro-C maps, at 5-kb resolution, of the Irx3, 5, and 6 TAD from WT (Hsieh et al, 2020), CTCF depleted (CTCF-AID) and cohesin-depleted mESCs (RAD21-AID) (Hsieh et al, 2022). Genome co-ordinates (Mb) are from the mm10 assembly of the mouse genome. Genes and RING1B ChIP-seq profiles are shown below. Black arrowheads indicate enriched interactions between Irx3, 5, and 6. (B) Bar plots providing categorical analysis of the spatial location of each of the three polycomb target loci probes shown in Fig 3A relative to each other in untreated, 2,5-HD, 1,6-HD, and recovered WT cells (biological replicate of Fig 3). 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 identified using Fisher’s exact test; ∗ P ≤ 0.05 and > 0.01, ∗∗ P ≤ 0.01. (C, D) Scatter plots depicting the interprobe distances between each of the two fosmid probe pairs with the separation between the third pair indicated by the color in the color bar in WT (C) and Ring1B^−/− cells (D). Dashed red box indicates non-dispersed alleles with at least two sets of interprobe distances <400 nm. Data and statistical evaluation are summarised in Table S3.