Analysis of Chromatin Regulators Reveals Specific Features of Rice DNA Methylation Pathways

Abstract

Plant DNA methylation that occurs at CG, CHG, and CHH sites (H = A, C, or T) is a hallmark of the repression of repetitive sequences and transposable elements (TEs). The rice (Oryza sativa) genome contains about 40% repetitive sequence and TEs and displays specific patterns of genome-wide DNA methylation. The mechanism responsible for the specific methylation patterns is unclear. Here, we analyzed the function of OsDDM1 (Deficient in DNA Methylation 1) and OsDRM2 (Deficient in DNA Methylation 1) in genome-wide DNA methylation, TE repression, small RNA accumulation, and gene expression. We show that OsDDM1 is essential for high levels of methylation at CHG and, to a lesser extent, CG sites in heterochromatic regions and also is required for CHH methylation that mainly locates in the genic regions of the genome. In addition to a large member of TEs, loss of OsDDM1 leads to hypomethylation and up-regulation of many protein-coding genes, producing very severe growth phenotypes at the initial generation. Importantly, we show that OsDRM2 mutation results in a nearly complete loss of CHH methylation and derepression of mainly small TE-associated genes and that OsDDM1 is involved in facilitating OsDRM2-mediated CHH methylation. Thus, the function of OsDDM1 and OsDRM2 defines distinct DNA methylation pathways in the bulk of DNA methylation of the genome, which is possibly related to the dispersed heterochromatin across chromosomes in rice and suggests that DNA methylation mechanisms may vary among different plant species.DNA methylation in flowering plants occurs in three sequence contexts: the so-called symmetric CG and CHG and the asymmetric CHH, where H is any nucleotide except G. Methylation in each context is believed to be catalyzed primarily by a specific family of DNA methyltransferases: MET1 (homologous to animal Dnmt1) for CG, plant-specific chromomethylases (CMT3 and CMT2) for CHG, and DRM2 (homologous to animal Dnmt3) for CHH (Law and Jacobsen, 2010).Recently, it was shown that CMT2 is a major CHH methyltransferase in Arabidopsis (Arabidopsis thaliana; Zemach et al., 2013;Stroud et al., 2014). The majority of plant methylation is found in transposable elements (TEs) and transcribed TE genes (known as transposable element-related genes [TEGs]) and is crucial for the repression of TE expression and transposition (Law and Jacobsen, 2010). Substantial methylation also is found in the bodies of active genes, where methylation is generally restricted to the CG context (Law and Jacobsen, 2010;Zemach et al., 2010). Methylation in all sequence contexts also is found in the promoter regions of many genes, which represses gene expression.In Arabidopsis, the maintenance of CHH methylation is mediated by RNA-directed DNA methylation (RdDM) involving the methyltransferase activity of DRM2 (Law and Jacobsen, 2010). DNA methylation also is influenced by chromatin factors. For instance, the Arabidopsis Snf2 family nucleosome remodeler DDM1, which can shift nucleosomes in vitro (Brzeski and Jerzmanowski, 2003), is essential fo...

Decrease in DNA methylation 1 (DDM1) is required for the formation of mCHH islands in maize

“…In contrast, in maize the levels of m CHG and m CHH were affected more strongly than those of m CG. Our results are similar to the data from the rice ddm1 mutation, which mostly affects m CHG levels (Tan et al ). These observations suggest that DDM1 has different effects on DNA methylation patterns in diverse species.…”

“…Previously, it was thought that DDM1 facilitates CMT2 ‐mediated CHH methylation independently of the RdDM pathway (Zemach et al ). However, a recent study in rice has indicated that OsDDM1 may be involved in RdDM‐mediated m CHH methylation at genic regions (Tan et al ). Thus, whether DDM1 participate in RdDM pathway may vary among plant species.…”

DNA Methylation in Rice and Relevance for Breeding

“…The genome-wide DNA methylation level is reduced to 17% in Osdrm2 compared to the WT and the RdDM pathway is deficient. In contrast to Arabidopsis, OsDRM2 is the major CHH MET in rice [46]. Heterologous expression of OsDRM2 in yeast confirmed that OsDRM2 could methylate DNA de novo [48].…”

“…These two genes share 93% similarity and their expression patterns are also similar. Interestingly, only the double mutants Osddm1a and Osddm1b show severe developmental defaults and a complete sterility, suggesting functional redundancy [46,47]. The global DNA methylation level is reduced by 54% in the double mutant Osddm1a Osddm1b compared to the WT.…”

“…The global DNA methylation level is reduced by 54% in the double mutant Osddm1a Osddm1b compared to the WT. Genome-wide analyses of DNA methylation have shown that OsDDM1 is involved in both CG and CHG methylation at euchromatic and heterochromatic regions but is also involved in CHH methylation of small TEs such as MITEs mainly located in euchromatic regions [46]. Transcriptomic analyses in Arabidopsis ddm1 mutants have shown an important deregulation of gene transcription [39,66] suggesting that OsDDM1 could also play a role in gene regulation and ensure normal plant development.…”