Fig. 6

DNA replication hindrance provokes CHG hypermethylation and release of silencing. A, B Metaplots showing TE methylation rates in CHG context in the indicated conditions. Annotations were aligned to their 5′ or 3′ end and average methylation was calculated for each 100-bp bin from 3 kb upstream to 3 kb downstream. For B, we used published datasets for rpa2a (Stroud et al. [22]) and pold2 (Zhang et al. [16, 17]). C, D Average methylation rates in CHG context in L5 and pol2a-12 in the absence (0 mm) or presence (2 mM) of hydroxyurea (HU) calculated at all TEs (C) or LTR/Gypsy TEs (D). E CHG methylation rates over non-overlapping 100 kb bins on chromosome 4. F Proportion of γ-H2A.X labeled nuclei in L5 and pol2a-12 plants treated or not with HU. A two-way ANOVA showed the significant effect (P < 2e−16) of HU treatment. Lowercase letters indicate significant differences between groups using Tukey’s post hoc tests (P < 0.05). Error bars represent standard error of the mean across three or four biological replicates, as indicated above bars. G Number of γ-H2A.X foci per nucleus in L5 and pol2a-12 plants treated or not with HU, excluding nuclei without γ-H2A.X signal. P-values from a two-sided unpaired Wilcoxon rank-sum test are indicated. H Transcript accumulation at three silent loci analyzed by RT-qPCR in L5 and pol2a-12 seedlings treated with various concentrations of HU, normalized to the ACTIN2 gene with L5 0mM HU set to 1. Asterisks mark statistically significant differences (two-sided unpaired Student’s t test, *P < 0.05, **P < 0.005). Error bars represent standard error of the mean across three biological replicates