Loss of H3K9 trimethylation alters chromosome compaction and transcription factor retention during mitosis

Recent studies have shown that repressive chromatin machinery, including DNA methyltransferases and polycomb repressor complexes, binds to chromosomes throughout mitosis and their depletion results in increased chromosome size. In the present study, we show that enzymes that catalyze H3K9 methylation, such as Suv39h1, Suv39h2, G9a and Glp, are also retained on mitotic chromosomes. Surprisingly, however, mutants lacking histone 3 lysine 9 trimethylation (H3K9me3) have unusually small and compact mitotic chromosomes associated with increased histone H3 phospho Ser10 (H3S10ph) and H3K27me3 levels. Chromosome size and centromere compaction in these mutants were rescued by providing exogenous first protein lysine methyltransferase Suv39h1 or inhibiting Ezh2 activity. Quantitative proteomic comparisons of native mitotic chromosomes isolated from wild-type versus Suv39h1/Suv39h2 double-null mouse embryonic stem cells revealed that H3K9me3 was essential for the efficient retention of bookmarking factors such as Esrrb. These results highlight an unexpected role for repressive heterochromatin domains in preserving transcription factor binding through mitosis and underscore the importance of H3K9me3 for sustaining chromosome architecture and epigenetic memory during cell division.

bivariate karyotype was assessed by flow cytometry and the gates used to sort all chromosomes, chromosome 19, or the X chromosome are indicated.Proteomic analysis was performed using LC-MS/MS on total mitotic cell lysate pellet, or on flow-purified chromosomes, to identify proteins enriched on metaphase chromosomes.Chromosome 19 and X size measurements were performed using Fiji/imageJ software to estimate chromosome (total DAPI) and centromere (DAPI high) areas, as indicated.(b) Gating strategy for sorting mitotic chromosomes, percentage of each gate is indicated (c) Volcano plots of proteins detected as being significantly enriched (red), depleted (blue) or not significantly enriched (grey) on sorted chromosomes relative to mitotic lysate pellet for WT ESCs (upper plot) or WT MEFs (lower plot).H3K9 KMTs and HP1 proteins are highlighted on the volcano plots.Statistical analysis was performed using unpaired two tailed Student's t-test, permutation-based FDR < 0.05, n = 3 independent experiments each measured in duplicate, see Methods for details).Proteins were plotted as Log2 fold change (LFQ intensity of sorted chromosome pellet /LFQ intensity of mitotic lysate pellet) and significance (-Log10 p) using Perseus software.

Figure S2 :
Figure S2: (a,b) Representative images (right panel) of immunofluorescence labelling of histone H3K9me3 (a) (green) or histone H3K27me3 (b) (pink) on mouse chromosome X isolated from WT or Suv39h dn ESCs where DAPI counterstain is shown in light grey.Scale bars = 5 μm.Plots (left of the images) show H3K9me3 (a) or H3K27me3 (b) mean intensities measured at centromeric regions.Mean ± SD are shown, n = minimum 50 chromosomes over three independent

Figure S4 :
Figure S4: (a) Pairwise correlations (R values from Spearman correlation) between proteomics LFQ values (this study) and polyA RNAseq TPM (Transcripts per million) values in ESCs (dataset from 1 ).In total, 5664 genes were mapped between proteomics and transcriptomics experiments based on gene symbols.(b) Pairwise correlations (R values from Spearman correlation) between log2 fold changes in proteomics LFQ values (this study) and DESeq2 log2 fold changes in polyA RNAseq values (dataset from 1 ) comparing Suv39h dn vs WT ESCs.(c) Western blot of Esrrb in WT and Suv39h dn asynchronous ESCs, representative of three biological replicates.Histone H3 was used as a loading control for the western blot.The uncropped image is provided at the end of this Supplementary information file.(d,e) Average LFQ intensities of Esrrb in WT and Suv39h dn mitotic lysates (d) and sorted chromosomes (e).Mean + SD is shown, n = 3 independent experiments each measured in duplicate.(d,e) P-value of statistically significant change, measured by unpaired two tailed Student's t-tests, is indicated.Source data are provided in Supplemental Data 3. (f) Trend of ATACseq accessibility around Esrrb bookmarked binding sites in WT and Suv39h dn asynchronous ESCs (left), mitotic ESCs (middle) and sorted chromosomes (right).Esrrb peak locations and bookmarking status are taken from 2 .(g,h) Esrrb (g) and Sox2 (h) mean intensities on sorted chromosomes 19 and X from Esrrb-tdTomato ES cell line following treatment with DMSO or 100 nM Chaetocin.Plots show mean ± SD, n = minimum 50 chromosomes over three independent experiments.P-values of statistically significant changes, measured by unpaired two tailed Student's t-tests, are indicated.Source data, including the precise number of chromosomes analysed, are provided in Supplemental Data 3.

Figure S5 :
Figure S5: (a) Volcano plots as in Figure 3d, highlighting H3K9me3-associated factors that are enriched (red), depleted (blue) or not significantly enriched (black) on WT (left) or Suv39h dn (right) ESC mitotic chromosomes versus mitotic lysates.(b) Average LFQ intensity of different histone H1 variants and total histone H1, H2, H3, and H4 in the sorted chromosome samples of WT (grey) and Suv39h dn (blue).Mean + SD is shown, n = 3 independent experiments each measured in duplicate.P-values of statistically significant changes, measured by unpaired two tailed Student's t-tests, are indicated.Source data are provided in Supplemental Data 3.

Figure S6 :
Figure S6: (a) Representative images of Esrrb-tdTomato ESCs (DSG+PFA double fixed) in different phases of the cell cycle.DAPI counterstain in blue.Scale bars = 5 μm, representative of three biological replicates.(b) qRT-PCR expression analysis of LINE-1, SINE-b2 and IAP repeat elements in WT and Suv39h dn asynchronous and mitotic ESCs.Mean + SD is shown, n = 3 independent experiments, P-values of statistically significant changes, measured by unpaired two tailed Student's t-tests, are indicated.(c) Average LFQ intensity of L1ORF1 in mitotic lysates and sorted chromosome samples of WT and Suv39h dn ESCs.Mean + SD is shown, n = 3 independent experiments each measured in duplicate.P-values of statistically significant changes, measured by unpaired two tailed Student's t-tests, are indicated.(b,c) Source data are provided in Supplemental Data 3.