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

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          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.

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          Nuclear DNA quantity varies widely between species and is poorly correlated with gene number. Variation in genome size can be explained by differing amounts of repetitive DNA. Repetitive DNA may be mobile, meaning it can increase its copy number within genomes. To prevent this, plants and animals suppress expression of repeats, often by marking the repeated sequence with a methyl group on cytosine bases. DRM2 is an enzyme capable of establishing this methylation, which can be guided to target sequences by short complementary RNA guides. As repeated sequences are prone to generate short RNAs they are efficiently recognized and silenced. We show that DRM2 requires a related inactive DRM3 protein to normally silence repeated sequences. A similar situation exists in mammals, where active and inactive DNA methyltransferases act together to silence repeats. We also demonstrate that non-catalytic regions of the DRM2 enzyme are functionally important, which we speculate to be involved in targeting the enzyme to the genome. Although plant and mammal RNA–directed DNA methylation systems share key similarities, there are important mechanistic differences, meaning that they are likely to have arisen convergently.

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          Most cited references 68

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          Establishing, maintaining and modifying DNA methylation patterns in plants and animals.

          Cytosine DNA methylation is a stable epigenetic mark that is crucial for diverse biological processes, including gene and transposon silencing, imprinting and X chromosome inactivation. Recent findings in plants and animals have greatly increased our understanding of the pathways used to accurately target, maintain and modify patterns of DNA methylation and have revealed unanticipated mechanistic similarities between these organisms. Key roles have emerged for small RNAs, proteins with domains that bind methylated DNA and DNA glycosylases in these processes. Drawing on insights from both plants and animals should deepen our understanding of the regulation and biological significance of DNA methylation.
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            DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

            The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.
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              Highly integrated single-base resolution maps of the epigenome in Arabidopsis.

              Deciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.

                Author and article information

                [1 ]Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America
                [2 ]Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom
                [3 ]New England Biolabs, Ipswich, Massachusetts, United States of America
                [4 ]Howard Hughes Medical Institute, University of California Los Angeles, Los Angeles, California, United States of America
                The Salk Institute for Biological Studies, United States of America
                Author notes

                Conceived and designed the experiments: IRH AD WW XZ HGC GAH SP SEJ. Performed the experiments: IRH AD WW XZ HGC GAH. Analyzed the data: IRH AD WW XZ HGC GAH KAK SP SEJ. Contributed reagents/materials/analysis tools: SP. Wrote the paper: IRH SEJ.

                Role: Editor
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                October 2010
                October 2010
                28 October 2010
                : 6
                : 10
                Henderson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Pages: 11
                Research Article
                Genetics and Genomics/Epigenetics
                Genetics and Genomics/Plant Genetics and Gene Expression
                Plant Biology/Plant Genetics and Gene Expression



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