Fig. 4

H4K20me1 depletion in CIZ1-null PEFs. A Frequency of H4K20me1, H3K27me3, and CIZ1 assemblies at the Xi in female WT (green) and CIZ1-null (blue) PEFs, in the cycling state (darker shades) and after a short or long SW (paler shades), with ± SEM. n denotes the total number of cells evaluated. B Example images of H4K20me1 (green) at H3K27me3-marked (red) Xi and H4K20me1 at CIZ1-marked (red) Xi in WT cells. C Nuclear area for female WT PEFs demonstrating a gradual drop in size over a short and long SW window. D Western blot illustrating H4K20me1 levels over a 24-h SW time course for WT and CIZ1-null female PEFs and histone H3. E Strategy describing GFP-CIZ1 doxycycline (dox) induction in CIZ1-null PEFs [14]. Right, effect on frequency of H4K20me1 assemblies in a dox-induced CIZ1-null female cycling population (yellow) after 48 h, categorised by whether GFP-CIZ1 expressing cells had formed a distinct CIZ1 assembly. Below, representative image illustrating colocalisation of GFP-CIZ1 (green) and H4K20me1(red) assemblies (white arrow). F Mean H4K20me1 immunofluorescence signal in nuclei from CIZ1-null cycling PEFs with (yellow) or without (blue) induction of GFP-CIZ1 (+ dox). Right, nuclear area in the same cells following a long SW, showing restoration of condensation capability. G WT cycling PEFs before (green) and after a 2-h incubation with 10 μM UNC0379 (purple), showing reduced H4K20me1 assemblies (histogram) and fluorescence intensity (dot plots), but no effect on H3K27me3. Right, histogram shows no effect on the proportion of replicating cells (red), detected after 30 min of EdU labelling. H Effect of UNC0379 on nuclear area before and after a short SW, showing loss of function in female WT PEFs. Results are compared by t-test (E, F (left), G), one-way ANOVA (C), or two-way ANOVA (A, B, F (right), H) where ns denotes no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001. DNA is stained with DAPI (blue) and scale bars represent 10 μm