Fig. 4: KRAS^G12V-induced transcription-dependent chromatin remodeling causes RS.

a Dot plot and mean of fork speed in Control cells treated with 0.1 μM of 4OHT for 3 days. 100 μM of DRB or 40 μM of Chloroquine (CQ) was added 30 min prior to IdU/CldU labeling. Representative result of three independent reproducible experiments are shown. b Quantification of EU intensity of Control and ATR-1 cells treated with 0.1 μM of 4OHT for 3 days. 100 μM of DRB or 40 μM of CQ was added 30 min prior to 1 mM of EU labeling for final 60 min. The results represent the means ± SEM of five independent experiments. one-way ANOVA Tukey’s test. arbitrary units, a. u. c Quantification of total RNA of Control cells treated with 0.1 μM of 4OHT for 3 days. 1 mM of EU was added 23 h prior to 100 μM of DRB treatment for final 60 min. The results represent the means ± SEM of three independent experiments. one-way ANOVA Tukey’s test. arbitrary units, a. u. d, e After 0.1 μM of 4OHT treatment for 3 days, Control and ATR-1 cells were treated with 100 μM of DRB or 2.5 μM of GSK126 for 90 min, followed by staining with anti-H3K27me3 antibody with pre-extraction method. d Representative image of chromatin-bound H3K27me3 staining. Scale bar = 20 μm. e Quantification of the H3K27me3 intensity shown in (d). Representative result of three independent reproducible experiments are shown. Black lines indicate the mean; arbitrary units, a. u.; n = 1000; one-way ANOVA Tukey’s test. f Dot plot and mean of fork speed in Control cells treated with 0.1 μM of 4OHT for 3 days with or without 2.5 μM of GSK126. GSK126 was added 30 min prior to IdU/CldU labeling. Representative result of four independent reproducible experiments are shown. g Left, representative result of MNase sensitivity assay. After 0.1 μM of 4OHT treatment for 3 days, Control cells treated with 100 μM of DRB, 2.5 μM of GSK126 or 40 μM of CQ for 90 min, followed by permeabilization and MNase digestion for 5, 10, 20 min respectively. Right, quantification of digested DNA intensity. Representative result of two independent reproducible experiments are shown. arbitrary units, a. u. h The model of KRAS^G12V-induced RS and its response. KRAS^G12V induces the transcription of nascent RNA enriched in polycomb repressive complex 2 (PRC2)-regulated genes leading to PRC2 recruitment and trimethylation of H3K27, generating locally compacted heterochromatin region, resulting in a cause of RS. a, f Black lines indicate the mean; n = 200; one-way ANOVA Tukey’s test. All source data are provided as a Source Data file.