Fig. 6
From: LSD1 is required for euchromatic origin firing and replication timing
LSD1 promotes origin firing through H3K4me2 demethylation-dependent loading of TICRR. a LSD1-deficient or overexpressing U2OS cells were synchronized by double-thymidine block followed by release for 0 and 3 (S), 9 (M), and 16 (G1) h. Chromatin fractions were analyzed by western blotting for MCM6, CDC45, and PCNA. The bands were quantified using ImageJ software. b Genome-wide transcriptome analysis by RNA sequencing in LSD1-deficient U2OS cells. The differentially expressed genes are shown in volcano graphs (left) and analyzed by KEGG database for the significant pathway (right). c Western blotting analysis of MCM6, TICRR, CDC45, and PCNA in U2OS cells depleted of LSD1. Cadherin-5 and NAGS were used as positive or negative control. d PLA assays for the proximity of TICRR, CDC45, LSD1, or H3K4me2 relative to EdU-biotin-labeled nascent DNAs in U2OS cells following immunostaining for EdU-biotin (green). PCNA was used as a positive control. Left, cells pulse-labeled with EdU. Right, cells pulse-labeled with EdU and chased with thymidine. Scale bar, 10 μm. e Quantification of PLA spots (n = 8 cells) (left). Western blot of H3K4me2 following the thymidine chase (right). f PLA assays for the proximity of ORC1 or TICRR relative to H3K4me2 followed by immunostaining for ORC1 or TICRR (green). Quantification of the PLA signals is shown right (n = 12 cells). Scale bar, 10 μm. g, h PLA assays in LSD1-deficient U2OS cells for the proximity of TICRR, CDC45, or MCM6 relative to biotin-labeled nascent DNAs followed by immunostaining for biotin (green). Scale bar, 10 μm. Quantification of PLA spots from three independent experiments is shown below (n = 22 cells). P-values were determined by two-tailed student’s t-test. i qChIP assays for TICRR, CDC45, LSD1, and MCM6 at early origins (Ori#1 and 2) and late origins (Ori#3 and 4). Results are presented as fold of change over input with IgG as a negative control. Each bar represents mean ± SD for three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001; t-test). j The proposed model of LSD1-regulated DNA replication initiation in human cells
