Fig. 4: Loss of transiently accumulated H3K9me drastically alters the chromatin landscape of stalled forks.
From: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability
a, Colour-coded diagram showing a selection of proteins enriched (shades of green) or depleted (shades of red) at stalled replication fork in the absence of G9a activity. Proteins were considered enriched when the log2 ratio of HU + G9a inhibition/HU was greater than 0.2 and depleted when the log2 ratio of HU + G9a inhibition/HU was lower than −0.2. b–d, Dynamics of BARD1 (b), BRCA1 (c) and RAD51 (d), at replication sites in the presence (WT) and in the absence of G9a activity (UNC0642). The plots are showing the distribution of PLA spots intensity per nucleus in either unperturbed (UT), stalled (HU) and restarted (Rel) replication. BARD1-EdU (nWT-UT = 1,648, nWT-HU = 2,008, nWT-REL = 2,022, nUNC0642-UT = 2,004, nUNC0642-HU = 2,008, nUNC0642-REL = 2,002 cells analysed) (b), BRCA1-EdU (nWT-UT = 1,502, nWT-HU = 1,508, nWT-REL = 1,510, nUNC0642-UT = 1,511, nUNC0642-HU = 1,521, nUNC0642-REL = 1,505 cells analysed) (c) or RAD51-EdU (nWT-UT = 1,511, nWT-HU = 1,510, nWT-REL = 1,503, nUNC0642-UT = 1,521, nUNC0642-HU = 1,502, nUNC0642-REL = 1,505 cells analysed) (d). Cells were labelled with EdU for 20 min and were either left untreated (UT) or treated with 1 mM HU for 1 h (HU) or treated with 1 mM HU for 1 h and released from HU for 25 min and labelled with EdU for 20 min (Rel). e, Dynamics of H3K9me3 at replication sites in the presence (DMSO) and in the absence of G9a activity (UNC0642) as well as in the presence (siCTL) or absence of SMARCAL1 (siSMARCAL1). Plots showing distribution of H3K9me3-EdU PLA spots intensity per nucleus in either unperturbed (UT), stalled (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 (HU) or treated with 1 mM HU for 1 h and released from HU for 25 min and labelled with EdU for 20 min (Rel). It is interesting to note that transient accumulation of H3K9me3 at replication sites upon replication stress is independent of fork reversal activity. nsiCTL-UT = 1,338, nsiCTL-HU = 1,337, nsiCTL-REL = 1,339, nsiCTL+UNC0642-UT = 1,341, nsiCTL+UNC0642-HU = 1,138, nsiCTL+UNC0642-REL = 1,343, nsiSMARCAL1-UT = 1,339, nsiSMARCAL1-HU = 1,342, nsiSMARCAL1-REL = 747, nsiSMARCAL1+UNC0642-UT = 1,341, nsiSMARCAL1+UNC0642-HU = 1,340, nsiSMARCAL1+UNC0642-REL = 1,338 cells analysed; blue dashed line represents the mean of the distribution, ****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. Source numerical data are available in Source Data.
