The De Novo Cytosine Methyltransferase DRM2 Requires Intact UBA Domains and a Catalytically Mutated Paralog DRM3 during RNA–Directed DNA Methylation in Arabidopsis thaliana

Abstract

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA–directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA–directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA–directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA–directed DNA methylation systems consist of a combination of ancestral and convergent features.

DOMAINS REARRANGED METHYLTRANSFERASE3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

“…Similar to DRM2, DRM3 has N-terminal ubiquitin-associated (UBA) domains and a C-terminal methyltransferase domain (24). Sequence alignments of the DRM3 methyltransferase domains from many plant species revealed the absence of highly Significance DNA methylation, a chemical mark on chromatin, while not affecting DNA's primary sequence, plays important roles in silencing "bad DNA" that would become deleterious to cells if abnormally expressed.…”

“…However, DRM3 is required for full levels of DRM2-mediated DNA methylation (24,25). Similarly, mammalian genomes encode a catalytically inactive methyltransferase termed Dnmt3L that also lacks catalytically critical residues but is required for de novo DNA methylation in vivo (26)(27)(28)(29).…”

“…Dnmt3L has an N-terminal ADD domain that specifically recognizes histone 3 unmethylated at lysine 4 and targets Dnmt3 to chromatin (30,31). In contrast, DRM3 has N-terminal UBA domains of unknown function (23,24). Despite the importance of DRM3 for DRM2-mediated DNA methylation, it is unclear whether DRM3 acts similarly to mammalian Dnmt3L by interacting with DRM2 and stimulating DRM2 activity.…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…The UBA domains of DRM2 MTase in A. thaliana were experimentally shown to be required for normal RNA-directed DNA methylation. Perhaps, these domains are essential for proper localization of MTases in the cell [138].…”

An atypical component of RNA-directed DNA methylation machinery has both DNA methylation-dependent and -independent roles in locus-specific transcriptional gene silencing

“…The rdm4 mutation affects Pol V-dependent transcripts and plant development [21,22]. DRM2 and the recently characterized DRM3 are the de novo DNA methytransferases in the Arabidopsis RdDM pathway [23][24][25][26].…”