Extended Data Fig. 6: Extended Data Figure. 6 related to Fig. 4. Chromatin landscape of active replication forks remains unaltered in absence of G9a activity.
From: Dynamic de novo heterochromatin assembly and disassembly at replication forks ensures fork stability
(a) Schematic representation of the iPOND-SILAC-MS experiment comparing the protein present at replication fork when G9a is active (DMSO) vs when G9a is inactive (+UNC0642). This comparison was done at stalled replication fork (Fig.4a) or ongoing replication fork (Extended Data Fig. 6b). (b) Diagram showing a selection of the protein that are enriched (shades of green) or depleted (shades of red) at ongoing replication fork in the absence of G9a activity. Proteins considered enriched when log2 (ratio of UT + G9a inhibition/UT) ≥ 0.2. Proteins considered depleted when log2 (ratio of UT+G9a inhibition/UT) ≤ - 0.2. (c) Distribution of H2AK15Ub-EdU total PLA spot intensity per nucleus assessing the level of H2AK15Ub at replication sites for the indicated conditions. (nWT-UT = 1337, nWT-HU = 1312, nWT-REL = 1370, nUNC0642-UT = 308, nUNC0642-HU = 1408, nUNC0642-REL = 1426 cells analyzed; blue dashed line = mean of the distribution, **** = P ≤ 0.0001, ** = P ≤ 0.01, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). (d) Distribution of H4K20me0-EdU total PLA spot intensity per nucleus assessing the level of H4K20me0 at replication sites for the indicated conditions. (nWT-UT = 1504, nWT-HU = 1400, nWT-REL = 1374, nUNC0642-UT = 1358, nUNC0642-HU = 1096, nUNC0642-REL = 1210 cells analyzed; blue dashed line = mean of the distribution, **** = P ≤ 0.0001, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). (e) Fold change in transcript levels of dysregulated genes (red) and DNA damage repair (DDR) genes (blue) in wild type cells treated with UNC0642 normalized to untreated wild type cells. Left: In the absence of replication stress. Right: In the presence of replication stress (1 mM HU 1 hr), N = number of genes, unpaired two-sided t-test. (f) Distribution of PCNA-EdU total PLA spot intensity per nucleus assessing the level of PCNA at replication sites for the indicated conditions. (nWT-UT = 732, nWT-HU = 628, nWT-REL = 391, nUNC0642-UT = 723, nUNC0642-HU = 763, nUNC0642-REL = 688 cells analyzed; blue dashed line = mean of the distribution, **** = P ≤ 0.0001, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). (g) Distribution of RPA-EdU total PLA spot intensity per nucleus assessing the level of RPA at replication sites for the indicated conditions. (nWT-UT = 1344, nWT-HU = 1437, nWT-REL = 1352, nUNC0642-UT = 965, nUNC0642-HU = 793, nUNC0642-REL = 514 cells analyzed; blue dashed line = mean of the distribution, **** = P ≤ 0.0001, * = P ≤ 0.05, ns = non-significant, Kruskal-Wallis test followed by Dunn’s test). Source numerical data are available in source data.
