Fig. 7: Mcm2-2A, Pole3 KO, and Pole4 KO mutants defective in parental histone transfer show abnormal chromosome segregation.
From: Stable inheritance of H3.3-containing nucleosomes during mitotic cell divisions
a Deletion of either H3f3a or H3f3b results in defects in mitosis. Representative live-cell images of H3.1/H3.3-SNAP at the indicated time points (20 min before (−) and after (+) anaphase) in WT, H3f3a KO and H3f3b KO mES cells (n > 160). Nuclear abnormalities marked by arrows including chromosome bridges, misaligned chromosomes and lagging chromosomes observed in anaphase was counted in H3f3a KO/H3f3b KO mES cells. Scale bar, 10 µm. b Percentages of abnormal mitotic cells in WT, H3f3a KO and H3f3b KO mES cells. Images at anaphase were used for quantification. c Representative live-cell images of H3.3-SNAP signals collected at indicated time (20 min before (−) and after (+) anaphase) in WT, Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, Mcm2-2A + Pole4 KO, Hira KO, and Daxx KO mES cells (n > 170). The nuclear abnormalities mentioned in (a) were observed in Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, Mcm2-2A + Pole4 KO, and Daxx KO mES cells. Defects are indicated by arrows. Scale bar, 10 µm. d Percentages of abnormal mitotic cells in WT, Pole3 KO, Pole4 KO, Mcm2-2A, Mcm2-2A + Pole3 KO, Mcm2-2A + Pole4 KO, Hira KO, and Daxx KO cell lines. Images at anaphase were used for quantification. b, d Data are presented as means ± SD. Statistical analysis was performed by two-tailed unpaired Student t test with the P values shown on the graphs (ns, no significant difference). a–d n > 160 cells from three independent experiments were analyzed. e The percentage of G2/M phase cells increases in WT, Pole3 KO, Pole4 KO, Mcm2-2A, single, Mcm2-2A + Pole3 KO, and Mcm2-2A Pole4 KO double mutant cell lines. Cells were collected for flow cytometry analysis of DNA content, and the percentage of cells at G2/M was calculated. Data are presented as means ± SD. Statistical analysis was performed by two-tailed unpaired Student t test, and the P values were marked on the graphs (ns no significant difference). Four independent replicates were conducted for each cell line.
