Fig. 6: Loss of KDM3A rescues fork degradation, ssDNA gap accumulation and drug sensitivity of cells lacking G9a activity.
From: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability
a, Top: schematics of ssDNA gap accumulation. Bottom: IdU track length (µm) distribution for the indicated conditions. n = 100 replication forks analysed per condition (nsiCTL_S1− = 206, nsiPRIMPOL_S1− = 91, nsiKDM3_S1− = 206, nsiCTL+UNC0642_S1− = 201, nsiPRIMPOL+UNC0642_S1− = 202, nsiKDM3+UNC0642_S1− = 201, nsiCTL_S1+ = 206, nsiPRIMPOL_S1+ = 206, nsiKDM3_S1+ = 209, nsiCTL+UNC0642_S1+ = 209, nsiPRIMPOL+UNC0642_S1+ = 203, nsiKDM3+UNC0642_S1+ = 205, replication tracks analysed; ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, NS, non-significant, Kruskal–Wallis test followed by Dunn’s test). b, Top: schematics of the Fork restart assay. Bottom: IdU track length (µm) distribution (nsiCTL = 650, nsiPRIMPOL = 376, nsiKDM3 = 316, nsiCTL+UNC0642 = 502, nsiPRIMPOL+UNC0642 = 369, nsiKDM3+UNC0642 = 302 replication tracks analysed; ****P ≤ 0.0001, ***P ≤ 0.001,**P ≤ 0.01, *P ≤ 0.05, NS, non-significant, Kruskal–Wallis test followed by Dunn’s test). c, Fork degradation performed as Fig. 5a. Ratio of IdU to CldU tract length was plotted for the indicated conditions (nsiCTL = 161, nsiBRCA1 = 164, nsiKDM3 = 161, nsiCTL+UNC0642 = 162, nsiBRCA1+UNC0642 = 163, nsiKDM3+UNC0642 = 176 replication tracks analysed; ****P ≤ 0.0001, NS, non-significant, Kruskal–Wallis test followed by Dunn’s test). d, Dynamics of H3K9me3 at replication sites in the presence (DMSO) or in the absence of G9a activity (UNC0642) and in the presence (siCTL) or absence of KDM3A (siKDM3). Distribution of H3K9me3-EdU PLA spots intensity per nucleus upon unperturbed (UT), stressed (HU) and restarted (Rel) replication. Cells were labelled with EdU for 20 min and were either left untreated (UT) or treated with 1 mM HU for 1 h or treated with 1 mM HU for 1 h and released for 25 min before labelling with EdU for 20 min (Rel). Blue dashed indicates mean of the distribution, nsiCTL-UT = 1,509, nsiCTL-HU = 1,509, nsiCTL-REL = 1,506, nsiCTL+UNC0642-UT = 2,003, nsiCTL+UNC0642-HU = 1,529, nsiCTL+UNC0642-REL = 1,543, nsiKDM3-UT = 1,516, nsiKDM3-HU = 1,504, nsiKDM3-REL = 1,524, nsiKDM3+UNC0642-UT = 1,505, nsiKDM3+UNC0642-HU = 1,536, nsiKDM3+UNC0642-REL = 1,514 cells analysed; ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, NS, non-significant, Kruskal–Wallis test followed by Dunn’s test is used for all statistical analysis. e,f, Colony survival assay. Mean survival in wild type (WT) and cells lacking G9a (G9a−/−), in the presence (siCTL) or absence of KDM3A (siKDM3) and treated with different concentrations of olaparib (PARPi, e) or cisplatin (f). Data are normalized to the 0 dose of the corresponding condition. Error bars represent ± standard deviation (n = 3 independent experiment) (****P ≤ 0.0001, **P ≤ 0.01, NS, non-significant, ordinary two-way analysis of variance was used for multiple comparisons). Source numerical data are available in Source Data.
