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The cytological and molecular role of domains rearranged methyltransferase3 in RNA-dependent DNA methylation of Arabidopsis thaliana.

Costa-Nunes P, Kim JY, Hong E, Pontes O - BMC Res Notes (2014)

Bottom Line: Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases.However, DRM3's regulation of DNA methylation is likely target- or chromatin context-dependent.DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of New Mexico, MSC03 2020, 1 University of New Mexico, Albuquerque, NM 87131, USA. opontes@unm.edu.

ABSTRACT

Background: Plants have evolved a unique epigenetic process to target DNA cytosine methylation: RNA-directed DNA methylation (RdDM). During RdDM, small RNAs (smRNAs) guide methylation of homologous DNA loci. In Arabidopsis thaliana, the de novo DNA methyltransferase that ultimately methylates cytosines guided by smRNAs in all sequence contexts is Domains Rearranged Methyltransferase 2 (DRM2). Recent reports have shown that DRM2 requires the catalytic mutated paralog DRM3 to exert its function through a still largely unknown process. To shed light on how DRM3 affects RdDM, we have further characterized its role at the molecular and cytological levels.

Findings: Although DRM3 is not required for RdDM loci transcriptional silencing, it specifically affects loci's DNA methylation. Interestingly, DRM3 and DRM2 regulate the DNA methylation in a subset of loci differently.Fluorescence In Situ Hybridization and immunolocalization analyses showed that DRM3 is not required for the large-scale nuclear organization of heterochromatin during interphase, with the notable exception of the 45S ribosomal RNA loci. DRM3 localizes exclusively to the nucleus and is enriched in a round-shaped domain located in the nucleolar periphery, in which it colocalizes with components of the RdDM pathway.

Conclusions: Our analyses reinforce the previously proposed chaperone role of DRM3 in RdDM. Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases. However, DRM3's regulation of DNA methylation is likely target- or chromatin context-dependent. DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.

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DRM3 regulates NOR interphase organization. (A) DNA FISH showing decondensation of 45S rRNA loci (red) indrm3anddrm2mutants. Unlike met1, drm3 is not required for chromocenter organization, as observed by WT interphase distribution of (B) CEN repeats (green), 5S rRNA loci (red) and (C) H3K9me2 in a drm3 background. Representative images are shown for each genotype, corresponding to frequencies >91 %. (n > 64). DNA visualized with DAPI (grey). Size bar = 5 μm.
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Fig4: DRM3 regulates NOR interphase organization. (A) DNA FISH showing decondensation of 45S rRNA loci (red) indrm3anddrm2mutants. Unlike met1, drm3 is not required for chromocenter organization, as observed by WT interphase distribution of (B) CEN repeats (green), 5S rRNA loci (red) and (C) H3K9me2 in a drm3 background. Representative images are shown for each genotype, corresponding to frequencies >91 %. (n > 64). DNA visualized with DAPI (grey). Size bar = 5 μm.

Mentions: We evaluated whether DNA methylation regulated by DRM3 is correlated with spatial organization of heterochromatin within the nucleus. To that end, we used DAPI staining and Fluorescence In Situ Hybridization (FISH) to characterize the interphase organization of heterochromatic repeats in the drm3 mutant background, when compared to met1, cmt3 and drm2 loss of function mutants (Figure 4). First, we measured the chromocenter fraction [35] in met1, cmt3, drm2 and drm3. With the exception of met1, all analyzed DNA methyltransferase mutant lines displayed similar heterochromatin content as WT (not shown, see also Figure 3), indicating that DRM3-dependent cytosine methylation is not required for heterochromatin assembly during interphase. These observations are in agreement with previously published results showing that heterochromatin compaction is mainly dependent on MET1 activity [33, 36] and not on DRM2 [19].Figure 4


The cytological and molecular role of domains rearranged methyltransferase3 in RNA-dependent DNA methylation of Arabidopsis thaliana.

Costa-Nunes P, Kim JY, Hong E, Pontes O - BMC Res Notes (2014)

DRM3 regulates NOR interphase organization. (A) DNA FISH showing decondensation of 45S rRNA loci (red) indrm3anddrm2mutants. Unlike met1, drm3 is not required for chromocenter organization, as observed by WT interphase distribution of (B) CEN repeats (green), 5S rRNA loci (red) and (C) H3K9me2 in a drm3 background. Representative images are shown for each genotype, corresponding to frequencies >91 %. (n > 64). DNA visualized with DAPI (grey). Size bar = 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4209038&req=5

Fig4: DRM3 regulates NOR interphase organization. (A) DNA FISH showing decondensation of 45S rRNA loci (red) indrm3anddrm2mutants. Unlike met1, drm3 is not required for chromocenter organization, as observed by WT interphase distribution of (B) CEN repeats (green), 5S rRNA loci (red) and (C) H3K9me2 in a drm3 background. Representative images are shown for each genotype, corresponding to frequencies >91 %. (n > 64). DNA visualized with DAPI (grey). Size bar = 5 μm.
Mentions: We evaluated whether DNA methylation regulated by DRM3 is correlated with spatial organization of heterochromatin within the nucleus. To that end, we used DAPI staining and Fluorescence In Situ Hybridization (FISH) to characterize the interphase organization of heterochromatic repeats in the drm3 mutant background, when compared to met1, cmt3 and drm2 loss of function mutants (Figure 4). First, we measured the chromocenter fraction [35] in met1, cmt3, drm2 and drm3. With the exception of met1, all analyzed DNA methyltransferase mutant lines displayed similar heterochromatin content as WT (not shown, see also Figure 3), indicating that DRM3-dependent cytosine methylation is not required for heterochromatin assembly during interphase. These observations are in agreement with previously published results showing that heterochromatin compaction is mainly dependent on MET1 activity [33, 36] and not on DRM2 [19].Figure 4

Bottom Line: Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases.However, DRM3's regulation of DNA methylation is likely target- or chromatin context-dependent.DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of New Mexico, MSC03 2020, 1 University of New Mexico, Albuquerque, NM 87131, USA. opontes@unm.edu.

ABSTRACT

Background: Plants have evolved a unique epigenetic process to target DNA cytosine methylation: RNA-directed DNA methylation (RdDM). During RdDM, small RNAs (smRNAs) guide methylation of homologous DNA loci. In Arabidopsis thaliana, the de novo DNA methyltransferase that ultimately methylates cytosines guided by smRNAs in all sequence contexts is Domains Rearranged Methyltransferase 2 (DRM2). Recent reports have shown that DRM2 requires the catalytic mutated paralog DRM3 to exert its function through a still largely unknown process. To shed light on how DRM3 affects RdDM, we have further characterized its role at the molecular and cytological levels.

Findings: Although DRM3 is not required for RdDM loci transcriptional silencing, it specifically affects loci's DNA methylation. Interestingly, DRM3 and DRM2 regulate the DNA methylation in a subset of loci differently.Fluorescence In Situ Hybridization and immunolocalization analyses showed that DRM3 is not required for the large-scale nuclear organization of heterochromatin during interphase, with the notable exception of the 45S ribosomal RNA loci. DRM3 localizes exclusively to the nucleus and is enriched in a round-shaped domain located in the nucleolar periphery, in which it colocalizes with components of the RdDM pathway.

Conclusions: Our analyses reinforce the previously proposed chaperone role of DRM3 in RdDM. Overall, our work further demonstrates that DRM3 most likely functions exclusively with DRM2 in RdDM and not with other A. thaliana DNA methyltransferases. However, DRM3's regulation of DNA methylation is likely target- or chromatin context-dependent. DRM3 hypothetically acts in RdDM either upstream of DRM2, or in a parallel step.

Show MeSH