Fig. 2: Suv39h dn mitotic chromosomes show elevated levels of H3K27me3 and H3S10ph and can be resized by inhibiting PRC2 activity.
a,b, Representative images (right) of immunofluorescence labeling of histone H3K9me3 (a) (green) or histone H3K27me3 (b) (pink) on chromosome 19 isolated from WT or Suv39h dn ESCs, where DAPI counterstain is shown in light gray. Scale bar, 5 μm. Plots (left of the images) show H3K9me3 (a) or H3K27me3 (b) mean intensities, measured at centromeric regions. c, Representative images of immunofluorescence labeling of histone H3S10ph (yellow) on WT and Suv39h dn metaphase-arrested ESCs, where DAPI counterstain is shown in blue. Scale bar, 4 μm. H3S10ph mean intensities were measured on mitotic chromosomes for each condition (n = minimum 40 chromosomes analyzed for each cell line over 3 independent experiments (a–c)). P values of statistically significant changes, measured by unpaired, two-tailed Student’s t-tests, are indicated. d, Experimental strategy used to measure mitotic chromosome size of Suv39h dn ESCs after treatment with DNA methylation or PRC2 inhibitors (5-Aza or GSK343, respectively). e,f, Representative images of immunofluorescence labeling of histone H3K27me3 (red) on mouse chromosome 19 (e) and chromosome X (f) isolated from Suv39h dn ESCs pretreated with DMSO, 5-Aza or GSK343. DAPI counterstain is shown in light gray. Scale bar, 5 μm. H3K27me3 mean intensities were measured at centromeric regions and whole chromosomes; the mean ± s.d. is shown (n = minimum 50 chromosomes analyzed over 3 independent experiments). P values of statistically significant decreases compared with DMSO treatment, measured by unpaired, two-tailed Student’s t-tests, are indicated. Boxplots show area measurements of individual chromosomes and centromeres for each condition. Minimum, lower quartile, median, upper quartile and maximum values are indicated (n = minimum 100 chromosomes analyzed for each condition over 3 independent experiments). P values of statistically significant increases compared with DMSO treatment, measured by unpaired, two-tailed Student’s t-tests, are indicated. g, Chromatin accessibility profile across chromosome 19 for WT and Suv39h dn asynchronous (Asynch.) and mitotic ESCs and flow-sorted mitotic chromosomes, shown as log2(enrichment of ATAC-seq signal). a–c,e,f, Source data, including the precise numbers of chromosomes analyzed, are provided.
