Processive and stochastic CHH methylation by plant DRM2 and CMT2 revealed by single-read methylome analysis

Abstract

Cytosine methylome data is commonly generated through next-generation sequencing (NGS). Analyses of this data average methylation states of individual reads. We propose an alternate method of analysing single-read methylome data. Using this method, we identified patterns that relate to the mechanism of two plant non-CG methylating enzymes, DRM2 and CMT2: DRM2 has higher processivity than CMT2, and DRM2-methylated regions have higher variation among cells. Based on these patterns, we developed a classifier that predicts enzyme activity in different species and tissues. To facilitate further single-read analyses, we developed a genome browser optimised for visualising and analysing NGS data at single-read resolution.Background DNA methylation is a conserved epigenetic mechanism that regulates genome stability and expression in diverse eukaryotes [1][2][3][4]. This regulation is based on a dynamic addition or removal of a methyl group to/from the fifth carbon of a cytosine residue. DNA methylation appears in distinct genomic features, such as genes and transposable elements (TEs), and in different chromatin states, such as heterochromatin and euchromatin [2, 5-9]. In plants, DNA methylation occurs in three contexts: CG, CHG and CHH (where H is any base except G). These contexts are differentially regulated by four DNA methyltransferase (DNMT) families that share conserved methyl-transferase domain (MTD). METHYLTRANSFERASE1 (MET1) recognises hemi-methylated CG following DNA replication, and methylates the naked cytosine in the daughter strand [10,11]. CHROMOMETHYLASEs (CMTs), which are plant-specific DNMTs, bind histone H3 lysine 9 (H3K9me2) heterochromatin via a chromodomain (CD) to methylate non-CG contexts [12]. In flowering plants, CMT3 methylate mostly CHG sites, whereas CMT2 methylates mostly CHH sites [13,14]. The CHH methylation state is additionally regulated by plant DNMT3 orthologues or homologs, i.e. the DOMAINS REARRANGED METHYLASEs (DRMs) [15,16]. Similar to animal DNMT3, plant DNMT3 and DRMs function as de novo methylases, establishing methylation on unmethylated sites.

Contiguous and stochastic CHH methylation patterns of plant DRM2 and CMT2 revealed by single-read methylome analysis

“…The source code of the software presented is available on GitHub [58]. The version of the code used for the analyses in this paper is available on Zenodo [59].…”

DNA methylation mutants in Physcomitrella patens elucidate individual roles of CG and non-CG methylation in genome regulation

“…In flowering plants, DNMT3s become extinct, and their non-CG methylation is mediated by CMTs and DRMs (14,15,30). The chromatin targeting mechanism of plant DNMT3 orthologs is still unknown; however, similarly to CMTs, moss DNMT3 preferentially methylates heterochromatic TEs enriched by the histone mark H3K9me2 (30,36). In contrast, DRMs are generally targeted to euchromatic TEs via the RNA-directed DNA methylation pathway (RdDM) (14,15,30).…”