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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.

Henderson IR, Deleris A, Wong W, Zhong X, Chin HG, Horwitz GA, Kelly KA, Pradhan S, Jacobsen SE - PLoS Genet. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

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.

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A conserved clade of catalytically mutated DRM cytosine methyltransferase paralogs in angiosperms.(A) Phylogenetic relationships between DRM and Dnmt3 proteins (tree constructed using Unweighted Pair Group Method with Arithmetic Means (UPGMA)). Beneath are graphical representations of DRM2 and DRM3 (UBA domains are shaded black and cytosine methyltransferase domains are shaded grey). (B) Sequence alignment of DRM methyltransferase domains. Conserved catalytic residues are highlighted in red and numbered according to established nomenclature. (C) Sequence alignment of Dnmt3 methyltransferase domains, labeled as in (B). NCBI accession numbers or gene numbers for the proteins analyzed are (Arabidopsis thaliana) AtDRM1 At5g15380, AtDRM2 At5g14620, AtDRM3 At3g17310, (Populus trichocarpa) PtDRM2 DMT905, PtDRM3 DMT907, (Oryzae sativa) OsDRM2 Os3g02010, OsDRM3 Os5g04330, (Homo sapiens) HsDnmt3a NP_783328, HsDnmt3b CAB53071, HsDnm3L BAA95556, (Mus musculus) MmDnmt3a O88508, MmDnmt3b CAM27225, MmDnmt3L AAH83147 and (Haemophilus parahaemolyticus) HhaI P05102.
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pgen-1001182-g001: A conserved clade of catalytically mutated DRM cytosine methyltransferase paralogs in angiosperms.(A) Phylogenetic relationships between DRM and Dnmt3 proteins (tree constructed using Unweighted Pair Group Method with Arithmetic Means (UPGMA)). Beneath are graphical representations of DRM2 and DRM3 (UBA domains are shaded black and cytosine methyltransferase domains are shaded grey). (B) Sequence alignment of DRM methyltransferase domains. Conserved catalytic residues are highlighted in red and numbered according to established nomenclature. (C) Sequence alignment of Dnmt3 methyltransferase domains, labeled as in (B). NCBI accession numbers or gene numbers for the proteins analyzed are (Arabidopsis thaliana) AtDRM1 At5g15380, AtDRM2 At5g14620, AtDRM3 At3g17310, (Populus trichocarpa) PtDRM2 DMT905, PtDRM3 DMT907, (Oryzae sativa) OsDRM2 Os3g02010, OsDRM3 Os5g04330, (Homo sapiens) HsDnmt3a NP_783328, HsDnmt3b CAB53071, HsDnm3L BAA95556, (Mus musculus) MmDnmt3a O88508, MmDnmt3b CAM27225, MmDnmt3L AAH83147 and (Haemophilus parahaemolyticus) HhaI P05102.

Mentions: The A.thaliana genome encodes a previously uncharacterized protein with high amino acid identity to DRM2, which we term DRM3 (At3g17310) (Figure 1A and 1B). Like DRM2, DRM3 contains N-terminal ubiquitin associated (UBA) domains and a C-terminal region which shares identity with the Dnmt3 cytosine methyltransferase domain, but whose catalytic motifs are rearranged such that motifs VI–X precede I–V (Figure 1A and 1B) [54]. During catalysis cytosine methyltransferases form a covalent bond between a conserved cysteine in motif IV and carbon-6 of the cytosine base [14]. The catalytic cysteine is preceded by an invariant proline, which hydrogen bonds to exo-cyclic NH2 groups of cytosine to promote specific recognition and stabilize interaction between the base and catalytic site [14]. Close inspection of the DRM3 methyltransferase domain reveals the absence of highly conserved residues and notably the invariant proline-cysteine sequence is absent from motif IV (Figure 1B). Additionally, DRM3 lacks a conserved glutamic acid within motif IX and glycine within motif X (Figure 1B). It is likely that the absence of these residues inactivate DRM3 cytosine methyltransferase activity, because a drm2 mutation alone is sufficient to block de novo DNA methylation [15]–[17].


The de novo cytosine methyltransferase DRM2 requires intact UBA domains and a catalytically mutated paralog DRM3 during RNA-directed DNA methylation in Arabidopsis thaliana.

Henderson IR, Deleris A, Wong W, Zhong X, Chin HG, Horwitz GA, Kelly KA, Pradhan S, Jacobsen SE - PLoS Genet. (2010)

A conserved clade of catalytically mutated DRM cytosine methyltransferase paralogs in angiosperms.(A) Phylogenetic relationships between DRM and Dnmt3 proteins (tree constructed using Unweighted Pair Group Method with Arithmetic Means (UPGMA)). Beneath are graphical representations of DRM2 and DRM3 (UBA domains are shaded black and cytosine methyltransferase domains are shaded grey). (B) Sequence alignment of DRM methyltransferase domains. Conserved catalytic residues are highlighted in red and numbered according to established nomenclature. (C) Sequence alignment of Dnmt3 methyltransferase domains, labeled as in (B). NCBI accession numbers or gene numbers for the proteins analyzed are (Arabidopsis thaliana) AtDRM1 At5g15380, AtDRM2 At5g14620, AtDRM3 At3g17310, (Populus trichocarpa) PtDRM2 DMT905, PtDRM3 DMT907, (Oryzae sativa) OsDRM2 Os3g02010, OsDRM3 Os5g04330, (Homo sapiens) HsDnmt3a NP_783328, HsDnmt3b CAB53071, HsDnm3L BAA95556, (Mus musculus) MmDnmt3a O88508, MmDnmt3b CAM27225, MmDnmt3L AAH83147 and (Haemophilus parahaemolyticus) HhaI P05102.
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Related In: Results  -  Collection

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pgen-1001182-g001: A conserved clade of catalytically mutated DRM cytosine methyltransferase paralogs in angiosperms.(A) Phylogenetic relationships between DRM and Dnmt3 proteins (tree constructed using Unweighted Pair Group Method with Arithmetic Means (UPGMA)). Beneath are graphical representations of DRM2 and DRM3 (UBA domains are shaded black and cytosine methyltransferase domains are shaded grey). (B) Sequence alignment of DRM methyltransferase domains. Conserved catalytic residues are highlighted in red and numbered according to established nomenclature. (C) Sequence alignment of Dnmt3 methyltransferase domains, labeled as in (B). NCBI accession numbers or gene numbers for the proteins analyzed are (Arabidopsis thaliana) AtDRM1 At5g15380, AtDRM2 At5g14620, AtDRM3 At3g17310, (Populus trichocarpa) PtDRM2 DMT905, PtDRM3 DMT907, (Oryzae sativa) OsDRM2 Os3g02010, OsDRM3 Os5g04330, (Homo sapiens) HsDnmt3a NP_783328, HsDnmt3b CAB53071, HsDnm3L BAA95556, (Mus musculus) MmDnmt3a O88508, MmDnmt3b CAM27225, MmDnmt3L AAH83147 and (Haemophilus parahaemolyticus) HhaI P05102.
Mentions: The A.thaliana genome encodes a previously uncharacterized protein with high amino acid identity to DRM2, which we term DRM3 (At3g17310) (Figure 1A and 1B). Like DRM2, DRM3 contains N-terminal ubiquitin associated (UBA) domains and a C-terminal region which shares identity with the Dnmt3 cytosine methyltransferase domain, but whose catalytic motifs are rearranged such that motifs VI–X precede I–V (Figure 1A and 1B) [54]. During catalysis cytosine methyltransferases form a covalent bond between a conserved cysteine in motif IV and carbon-6 of the cytosine base [14]. The catalytic cysteine is preceded by an invariant proline, which hydrogen bonds to exo-cyclic NH2 groups of cytosine to promote specific recognition and stabilize interaction between the base and catalytic site [14]. Close inspection of the DRM3 methyltransferase domain reveals the absence of highly conserved residues and notably the invariant proline-cysteine sequence is absent from motif IV (Figure 1B). Additionally, DRM3 lacks a conserved glutamic acid within motif IX and glycine within motif X (Figure 1B). It is likely that the absence of these residues inactivate DRM3 cytosine methyltransferase activity, because a drm2 mutation alone is sufficient to block de novo DNA methylation [15]–[17].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus