Extended Data Fig. 4: Extended Data Figure. 4 related to Fig. 2. Transient accumulation of H3K9me and G9a at stalled replication forks is dependent upon checkpoint activation.
From: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability
(a–f) ChromStretch analysis assessing: (a) Dynamics of H3K9me3 at replication sites at ongoing (UT) and stalled (HU) replication forks. Mean ± SD percentage of colocalization of H3K9me3 and EdU is shown as a bar plot. (n = 3 independent experiments). (b) Dynamics of H3K9me2 at replication sites upon replication stress. For experimental design see Fig. 2c. Quantification of H3K9me2 at EdU sites for the indicated conditions. (nUT= 132, nHU10 = 74, nHU20 = 140, nHU30 = 75, nHU60 = 86; **** = P ≤ 0.0001, ** = P ≤ 0.01, Kruskal-Wallis test followed by Dunn’s test). (c) Dynamics of H3K9me2 at replication sites after release from replication stress. For experimental design see Fig. 2d. Quantification of H3K9me2 at EdU sites for the indicated conditions. (nUT= 100, nHU = 92, nrel30 = 92, nrel60 = 93; **** = P ≤ 0.0001, Kruskal-Wallis test followed by Dunn’s test). (d–f) Dynamics of H3K9 PTM at replication sites in the presence (UNC0642-) or in the absence of G9a activity (UNC0642+) at ongoing (UT) and stalled (HU) replication forks. Bar plot of the mean of the percentages of H3K9me1 (d), H3K9me2 (e), H3K9me3 (f) colocalization with EdU. (n = 2 independent experiments). (g) Top: Schematic representation of the G9a isoform A. Exon1 was targeted using CRISPR/Cas9 to generate a G9a knock-out. UNC0642 binds G9a SET domain preventing G9a catalytic activity. Bottom: Immunoblot showing G9a levels in wild type cells (WT), wild type where G9a activity was inhibited with UNC0642 (WT + UNC0642) for 2hrs and a G9a knockout clone (G9a-/-). Tubulin is used as a loading control. (h) Top: Cell cycle profile of the indicated cells. Bottom: Mean percentage of cells in various phases of the cell cycle ± standard deviation from 3 independent experiments. (ns = non-significant, One-way ANOVA). (i) Immunoblot showing pCHK1 levels in wild type cells (WT), wild type where G9a activity was inhibited with UNC0642 (WT + UNC0642) and a G9a knock out clone (G9a-/-), in the presence or in the absence of replication stress. CHK1 is used as a loading control. This experiment was reproduced independently three times with similar results. Source numerical data and unprocessed blots are available in source data.
