Limits...
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.

Show MeSH

Related in: MedlinePlus

DRM2 catalytic and UBA domains are required for maintenance of non-CG methylation.(A) A Myc-epitope tagged DRM2 transgene was mutated to introduce amino acid substitutions in the UBA domains or the methyltransferase domain, depicted graphically. (B) Micrographs of intact nuclei stained with DAPI and immunostained using α-Myc antibodies. (C) Southern blots hybridized for the MEA-ISR repeat using DNA digested with the methyl-sensitive restriction enzyme MspI. (D) Graphical representation of bisulfite sequencing of MEA-ISR with % cytosine methylation shown for each genotype. Error bars represent 95% confidence limits (Wilson score interval). CG sequence contexts are shaded dark grey, CHG are shaded light grey and white represents CHH.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2965745&req=5

pgen-1001182-g004: DRM2 catalytic and UBA domains are required for maintenance of non-CG methylation.(A) A Myc-epitope tagged DRM2 transgene was mutated to introduce amino acid substitutions in the UBA domains or the methyltransferase domain, depicted graphically. (B) Micrographs of intact nuclei stained with DAPI and immunostained using α-Myc antibodies. (C) Southern blots hybridized for the MEA-ISR repeat using DNA digested with the methyl-sensitive restriction enzyme MspI. (D) Graphical representation of bisulfite sequencing of MEA-ISR with % cytosine methylation shown for each genotype. Error bars represent 95% confidence limits (Wilson score interval). CG sequence contexts are shaded dark grey, CHG are shaded light grey and white represents CHH.

Mentions: DRM2 and DRM3 show a similar domain organization of N-terminal UBA domains and a C-terminal cytosine methyltransferase domain (Figure 1A). To further understand DRM2 function we performed site-directed mutagenesis of these domains and tested the consequence on RNA–directed DNA methylation. Cytosine methyltransferases require a set of 10 conserved motifs, including motif IV which contains the catalytic cysteine involved in transfer of the methyl group to cytosine (Figure 1B and 1C) [13]. We substituted the DRM2 motif IV catalytic cysteine C587 for an alanine, within an otherwise complementing DRM2 transgene containing a Myc-epitope translational fusion (hereafter termed DRM2cat-Myc) (Figure 4A) [58]. DRM proteins also possess N-terminal UBA domains, which consist of three helices connected by two conserved loop regions forming an ubiquitin interaction surface [63], [64]. The first loop contains a highly conserved MGF/MGY motif, which is required for correct folding and maintenance of UBA domain structure [64]. To test the functional importance of the DRM2 UBA domains we substituting conserved phenylalanine residues (F73, F123, F206) in each loop-I region to alanines (hereafter termed DRM2uba-Myc) (Figure 4A). The wild type DRM2-Myc and mutant DRM2cat-Myc and DRM2uba-Myc transgenes were transformed into drm1 drm2 and tested for their ability to complement mutant RNA–directed DNA methylation phenotypes.


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)

DRM2 catalytic and UBA domains are required for maintenance of non-CG methylation.(A) A Myc-epitope tagged DRM2 transgene was mutated to introduce amino acid substitutions in the UBA domains or the methyltransferase domain, depicted graphically. (B) Micrographs of intact nuclei stained with DAPI and immunostained using α-Myc antibodies. (C) Southern blots hybridized for the MEA-ISR repeat using DNA digested with the methyl-sensitive restriction enzyme MspI. (D) Graphical representation of bisulfite sequencing of MEA-ISR with % cytosine methylation shown for each genotype. Error bars represent 95% confidence limits (Wilson score interval). CG sequence contexts are shaded dark grey, CHG are shaded light grey and white represents CHH.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965745&req=5

pgen-1001182-g004: DRM2 catalytic and UBA domains are required for maintenance of non-CG methylation.(A) A Myc-epitope tagged DRM2 transgene was mutated to introduce amino acid substitutions in the UBA domains or the methyltransferase domain, depicted graphically. (B) Micrographs of intact nuclei stained with DAPI and immunostained using α-Myc antibodies. (C) Southern blots hybridized for the MEA-ISR repeat using DNA digested with the methyl-sensitive restriction enzyme MspI. (D) Graphical representation of bisulfite sequencing of MEA-ISR with % cytosine methylation shown for each genotype. Error bars represent 95% confidence limits (Wilson score interval). CG sequence contexts are shaded dark grey, CHG are shaded light grey and white represents CHH.
Mentions: DRM2 and DRM3 show a similar domain organization of N-terminal UBA domains and a C-terminal cytosine methyltransferase domain (Figure 1A). To further understand DRM2 function we performed site-directed mutagenesis of these domains and tested the consequence on RNA–directed DNA methylation. Cytosine methyltransferases require a set of 10 conserved motifs, including motif IV which contains the catalytic cysteine involved in transfer of the methyl group to cytosine (Figure 1B and 1C) [13]. We substituted the DRM2 motif IV catalytic cysteine C587 for an alanine, within an otherwise complementing DRM2 transgene containing a Myc-epitope translational fusion (hereafter termed DRM2cat-Myc) (Figure 4A) [58]. DRM proteins also possess N-terminal UBA domains, which consist of three helices connected by two conserved loop regions forming an ubiquitin interaction surface [63], [64]. The first loop contains a highly conserved MGF/MGY motif, which is required for correct folding and maintenance of UBA domain structure [64]. To test the functional importance of the DRM2 UBA domains we substituting conserved phenylalanine residues (F73, F123, F206) in each loop-I region to alanines (hereafter termed DRM2uba-Myc) (Figure 4A). The wild type DRM2-Myc and mutant DRM2cat-Myc and DRM2uba-Myc transgenes were transformed into drm1 drm2 and tested for their ability to complement mutant RNA–directed DNA methylation phenotypes.

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