Fig. 3: H3K9me3 is required for perinuclear centromere anchoring.
From: Global reorganization of genome architecture at the transition to gametogenesis
a, Representative IF image of H3K9me3 and lamin B1 (n = 3 biological replicates). DNA was stained with DAPI (blue). b, Quantification of total H3K9me3 area and H3K9me3 area within the perinuclear area (Methods). Each dot represents the percentage within each cell (mean ± s.d.). P values were calculated using a two-tailed Mann–Whitney U-test (n = 3 embryos analyzed with 3 independent litters). c, The proportion of cells with perinuclear H3K9me3 (>70% of total H3K9me3 area). d, Representative image of major satellite DNA-FISH combined with IF staining. e, Representative IF image of ATRX. f, Representative image of single molecular RNA-FISH of major satellite combined with IF. Yellow arrowheads indicate signals from PGCs. White arrowheads indicate signals from somatic cells. DNA was stained with DAPI (blue). For the scatter plot, colored dots represent the average percentage of each embryo and gray dots represent the percentage of each tissue section (n = 3 biological replicates; mean ± s.d.). P values were calculated using a two-tailed Mann–Whitney U-test. g, Western blot analysis of lamin B1 and LBR. In total, 10,000 GFP-positive (PGC) and GFP-negative (Soma) cells were sorted using FACS from the gonads of Oct4–GFP mice (GOF 18ΔPE–EGFP) (n = 2 animals, representative of 2 independent experiments). h, Comparative gene expression using published scRNA-seq data27. i, Representative IF image of LBR. Germ cell markers, OCT4 (E9.5) and MVH (E13.5). j, Gonads were cultured with chaetocin for 12 and 24 h. Measurement of the shortest distance of each CENPA focus to its nuclear periphery. The mean value of each nucleus was calculated. n represents the number of cells analyzed. Each cell is represented by a gray dot. Colored dots represent the mean value of each individual biological replicate (mean ± s.d.). P values were calculated using a two-tailed Mann–Whitney U-test. In d, e and i, data are representative of three independent experiments (three embryos from independent litters). Scale bars, 10 µm (a,d–f,i,j). **P < 0.01, ***P < 0.001.
