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Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat.

Williams BP, Pignatta D, Henikoff S, Gehring M - PLoS Genet. (2015)

Bottom Line: We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant.Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive.We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America.

ABSTRACT
Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.

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Methylation-sensitive expression of ROS1 is evolutionarily conserved.(A) ROS1 is locally duplicated in the closely related species Arabidopsis lyrata. Percentage DNA sequence identity between each paralog and A. thaliana ROS1 is shown for upstream, coding and downstream regions. (B) Bayesian reconstruction of the phylogeny of ROS1 homologs within Brassicales shows AlROS1a and AlROS1b diverged prior to the divergence of A. thaliana and A. lyrata. Support values are Bayesian posterior probabilities. (C) Methylation of the sequence 5’ of AlROS1a was examined by BS-PCR. N represents the number of independent clones sequenced for each BS-PCR product. (D) Annotation of methylated cytosines in the AtROS1 and AlROS1a 5’ region. Blue lines annotate sequences matching sRNAs from their respective species. (E) Relative AlROS1a transcripts are significantly decreased when plants are grown on 5-azaC. (F) Relative AlROS1b transcripts levels are unaffected by growth on 5-azaC. Data represented as mean, error bars represent standard deviation. *p = <0.05, **p = <0.005, two-tailed t-test.
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pgen.1005142.g006: Methylation-sensitive expression of ROS1 is evolutionarily conserved.(A) ROS1 is locally duplicated in the closely related species Arabidopsis lyrata. Percentage DNA sequence identity between each paralog and A. thaliana ROS1 is shown for upstream, coding and downstream regions. (B) Bayesian reconstruction of the phylogeny of ROS1 homologs within Brassicales shows AlROS1a and AlROS1b diverged prior to the divergence of A. thaliana and A. lyrata. Support values are Bayesian posterior probabilities. (C) Methylation of the sequence 5’ of AlROS1a was examined by BS-PCR. N represents the number of independent clones sequenced for each BS-PCR product. (D) Annotation of methylated cytosines in the AtROS1 and AlROS1a 5’ region. Blue lines annotate sequences matching sRNAs from their respective species. (E) Relative AlROS1a transcripts are significantly decreased when plants are grown on 5-azaC. (F) Relative AlROS1b transcripts levels are unaffected by growth on 5-azaC. Data represented as mean, error bars represent standard deviation. *p = <0.05, **p = <0.005, two-tailed t-test.

Mentions: To determine if regulation of ROS1 by methylation might be adaptive, we assessed whether methylation-sensitive expression of ROS1 is conserved in other species. Arabidopsis lyrata, which diverged from A. thaliana approximately 10 million years ago, has two highly conserved paralogs of ROS1 in tandem in the genome, which we termed AlROS1a and AlROS1b (Fig 6A). We performed a Bayesian reconstruction of the phylogeny of ROS1 homologs within all sequenced Brassicales (Fig 6B) and found that the duplication giving rise to the two ROS1 paralogs in A. lyrata occurred prior to the divergence of A. lyrata from A. thaliana. AtROS1 belongs to the same clade as AlROS1a, and no true homologs to AlROS1b exist in A. thaliana (Fig 6B). The homolog to AlROS1b was likely lost in the lineage that gave rise to A. thaliana. AlROS1a and AlROS1b share a high degree of sequence similarity in their coding region, but no significant similarity in their upstream sequences. Only AlROS1a has an upstream region conserved with AtROS1 (Fig 6A), including the presence of the same 5’ TE. The 5’ sequences are 78% identical over the first 1.4 kb.


Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat.

Williams BP, Pignatta D, Henikoff S, Gehring M - PLoS Genet. (2015)

Methylation-sensitive expression of ROS1 is evolutionarily conserved.(A) ROS1 is locally duplicated in the closely related species Arabidopsis lyrata. Percentage DNA sequence identity between each paralog and A. thaliana ROS1 is shown for upstream, coding and downstream regions. (B) Bayesian reconstruction of the phylogeny of ROS1 homologs within Brassicales shows AlROS1a and AlROS1b diverged prior to the divergence of A. thaliana and A. lyrata. Support values are Bayesian posterior probabilities. (C) Methylation of the sequence 5’ of AlROS1a was examined by BS-PCR. N represents the number of independent clones sequenced for each BS-PCR product. (D) Annotation of methylated cytosines in the AtROS1 and AlROS1a 5’ region. Blue lines annotate sequences matching sRNAs from their respective species. (E) Relative AlROS1a transcripts are significantly decreased when plants are grown on 5-azaC. (F) Relative AlROS1b transcripts levels are unaffected by growth on 5-azaC. Data represented as mean, error bars represent standard deviation. *p = <0.05, **p = <0.005, two-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4380477&req=5

pgen.1005142.g006: Methylation-sensitive expression of ROS1 is evolutionarily conserved.(A) ROS1 is locally duplicated in the closely related species Arabidopsis lyrata. Percentage DNA sequence identity between each paralog and A. thaliana ROS1 is shown for upstream, coding and downstream regions. (B) Bayesian reconstruction of the phylogeny of ROS1 homologs within Brassicales shows AlROS1a and AlROS1b diverged prior to the divergence of A. thaliana and A. lyrata. Support values are Bayesian posterior probabilities. (C) Methylation of the sequence 5’ of AlROS1a was examined by BS-PCR. N represents the number of independent clones sequenced for each BS-PCR product. (D) Annotation of methylated cytosines in the AtROS1 and AlROS1a 5’ region. Blue lines annotate sequences matching sRNAs from their respective species. (E) Relative AlROS1a transcripts are significantly decreased when plants are grown on 5-azaC. (F) Relative AlROS1b transcripts levels are unaffected by growth on 5-azaC. Data represented as mean, error bars represent standard deviation. *p = <0.05, **p = <0.005, two-tailed t-test.
Mentions: To determine if regulation of ROS1 by methylation might be adaptive, we assessed whether methylation-sensitive expression of ROS1 is conserved in other species. Arabidopsis lyrata, which diverged from A. thaliana approximately 10 million years ago, has two highly conserved paralogs of ROS1 in tandem in the genome, which we termed AlROS1a and AlROS1b (Fig 6A). We performed a Bayesian reconstruction of the phylogeny of ROS1 homologs within all sequenced Brassicales (Fig 6B) and found that the duplication giving rise to the two ROS1 paralogs in A. lyrata occurred prior to the divergence of A. lyrata from A. thaliana. AtROS1 belongs to the same clade as AlROS1a, and no true homologs to AlROS1b exist in A. thaliana (Fig 6B). The homolog to AlROS1b was likely lost in the lineage that gave rise to A. thaliana. AlROS1a and AlROS1b share a high degree of sequence similarity in their coding region, but no significant similarity in their upstream sequences. Only AlROS1a has an upstream region conserved with AtROS1 (Fig 6A), including the presence of the same 5’ TE. The 5’ sequences are 78% identical over the first 1.4 kb.

Bottom Line: We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant.Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive.We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.

View Article: PubMed Central - PubMed

Affiliation: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America.

ABSTRACT
Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.

Show MeSH
Related in: MedlinePlus