Fig. 5
From: Live-cell single-molecule dynamics of PcG proteins imposed by the DIPG H3.3K27M mutation
The DIPG H3.3K27M mutation prolongs the search processes of Ezh2 and Cbx7, but has different effects on their stability on chromatin. a Survival probability distribution of the dwell times for HaloTag-Ezh2 in HEK293T cells expressing H3.3-FLAG (N = 211 cells, n = 3825 trajectories) or H3.3K27M-FLAG (N = 160 cells, n = 3137 trajectories), and for HaloTag-Cbx7 in HEK293T cells expressing H3.3-FLAG (N = 89 cells, n = 8024 trajectories) or H3.3K27M-FLAG (N = 45 cells, n = 6866 trajectories). The distributions were fitted with a two-component exponential decay model. Fitted data are shown in Supplementary Table 6. b Survival probability distribution of the dwell times for HaloTag-Ezh2 in 9427 (N = 90 cells, n = 6078 trajectories) and SF8628 (N = 54 cells, n = 4485 trajectories) cells, and for HaloTag-Cbx7 in 9427 (N = 110 cells, n = 4641 trajectories) and SF8628 (N = 67 cells, n = 5376 trajectories) cells. The distributions were fitted with a two-component exponential decay model. Fitted parameters are shown in Supplementary Table 6. c–e Residence time (c), fraction (d), and search time (e) of the long-lived population (F1sb) for HaloTag-Ezh2 and HaloTag-Cbx7. Results are means ± SD from three biological replicates. f A proposed model for the effects of H3.3K27M on the binding and search mechanisms of PcG proteins. The H3.3K27M mutation prolongs the residence time and search time of Ezh2, but has no effect on the bound level of Ezh2 (right, top panel). Given that the target search time of Ezh2 is ∼20-fold longer than its residence time, we propose that the reduced sampling frequency of Ezh2 for the target sites by prolonging its target search time reprograms the genomic distributions of H3K27me3 and PRC2 (right, top panel). The H3.3K27M mutation has no effect on the residence time of Cbx7 on chromatin; however, increases the target search time of Cbx7, indicating that the prolonged search time by H3K27M reduces the bound level of Cbx7 (right, bottom panel). The thicker arrowhead represents a longer search time and the bigger font depicts a prolonged residence time
