DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis

Abstract

The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, Arabidopsis trithorax-related protein5 (ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs). Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs.

Figure 1. Heterochromatin is specifically re-replicated in atxr5 atxr6 double mutants.

Top panels show enrichment of DNA methylation (CG, CHG, CHH, where H = A,T or C) , transposable element (TE) densities (TE per base-pair) and H3K9me2 over the boundaries of heterochromatic regions of indicated sizes. Values were plotted +/−10 kilobase from the boundary of heterochromatin in 500 bp bins. x = 0 is the heterochromatin boundary, x<0 is outside the region, and x>0 is into the region. Bottom panels show the distribution of DNA contents from atxr5 atxr6 mutants relative to wild-type (log2 ratios) . Heterochromatic regions were defined using previously characterized H3K9me2 regions . Plots in both top and bottom panels were smoothed by taking the moving average over +/−1 bins and +/−3 bins, respectively.

Figure 2. Relationship between ATXR5/6 and DNA methylation in regulating DNA replication in heterochromatin.

A. Chromosomal distribution of transposable element (TE) density, DNA methylation and H3K9me2 data are presented to mark the locations of pericentromeric heterochromatin. B. Chromosomal views of the log2 ratio of genomic DNA reads of atxr5 atxr6 mutants to wild type (WT) are shown in black, and the log2 ratio of ddm1 atxr5 atxr6 mutants to WT are shown in red. C. met1 atxr5 atxr6 mutants, D. cmt3 atxr5 atxr6 mutants, and E. kyp suvh5 suvh6 atxr5 atxr6 mutants. F. Quantitation of reads in heterochromatin in mutants. Fraction of reads falling into previously defined pericentromeric heterochromatin was calculated. *P<10⁻⁵ relative to atxr5 atxr6 mutants.

Figure 3. Relationship between ATXR5/6 and DNA methylation in transcriptionally silencing TEs.

A. Overlap of re-replicated TEs in atxr5 atxr6 mutants, with transcriptionally reactivated TEs in atxr5 atxr6 mutants. B. Overlap of TEs transcriptionally derepressed in atxr5 atxr6 mutants and DNA methylation mutants. C. Overlap of TEs derepressed in indicated mutants. D. TE families of derepressed TEs in indicated mutants. E. Normalized expression levels of key genes in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA repair pathways.