Limits...
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.

Show MeSH

Related in: MedlinePlus

ROS1 demethylates the ROS1 promoter and reduces expression.(A) Methylation of the 5' region of ROS1 (region 1 in Fig 3) was measured in two ros1 missense mutants by bisulfite PCR. WT strains were segregating Col-0 siblings for ros1-7 and were C24 for ros1-2. N represents the number of independent clones sequenced for each BS-PCR product. (B) Relative ROS1 transcript abundance was measured in seedlings of both mutants using RT-qPCR. Data represented as mean, error bars represent standard deviation. *p = <0.005, two-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4380477&req=5

pgen.1005142.g005: ROS1 demethylates the ROS1 promoter and reduces expression.(A) Methylation of the 5' region of ROS1 (region 1 in Fig 3) was measured in two ros1 missense mutants by bisulfite PCR. WT strains were segregating Col-0 siblings for ros1-7 and were C24 for ros1-2. N represents the number of independent clones sequenced for each BS-PCR product. (B) Relative ROS1 transcript abundance was measured in seedlings of both mutants using RT-qPCR. Data represented as mean, error bars represent standard deviation. *p = <0.005, two-tailed t-test.

Mentions: We noticed that cytosines in the CG context 5’ of ROS1 were intermediately methylated at around 50% in wild-type tissues, with independent variance at every CG position in the sequence (Fig 3, S3 Fig). CG methylation is faithfully copied by MET1 during DNA replication, and so average methylation at symmetric CG sites is usually close to 0 or 100% [20]. The observed intermediate level of CG methylation and the low frequency of bisulfite clones fully methylated in the CG context (S3 Fig) suggested that active DNA demethylation might also be active at the 5’ sequence. We hypothesized that ROS1 might oppose RdDM to remove methylation at its own promoter. We performed bisulfite sequencing of the 5' methylated region in two missense mutants of ROS1, ros1-2 [32] and ros1-7, an allele encoding a protein with an E956K substitution in the ROS1 DNA glycosylase domain [33]. Symmetric CG methylation increased to nearly 100% in both mutants compared to their wild-type siblings, along with increases in non-CG methylation (Fig 5A), indicating that ROS1 actively removes methylation from this region in wild-type plants. At other loci, removal of 5' methylation by ROS1 increases transcription [21]. We examined the effect of DNA demethylation by ROS1 on ROS1 transcription by performing RT-qPCR on ros1-2 and ros1-7 mutants. Because these are missense mutations, nonsense-mediated decay should not be a complicating factor in measuring transcript abundance. ROS1 transcripts were 2 to 4-fold more abundant in ros1 mutants (Fig 5B). This suggests that active demethylation by ROS1 represses transcription of ROS1, counteracting the function of the RdDM pathway, which promotes ROS1 expression. Thus ROS1 regulates the expression of its own gene, forming a negative feedback loop for demethylation activity.


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

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

ROS1 demethylates the ROS1 promoter and reduces expression.(A) Methylation of the 5' region of ROS1 (region 1 in Fig 3) was measured in two ros1 missense mutants by bisulfite PCR. WT strains were segregating Col-0 siblings for ros1-7 and were C24 for ros1-2. N represents the number of independent clones sequenced for each BS-PCR product. (B) Relative ROS1 transcript abundance was measured in seedlings of both mutants using RT-qPCR. Data represented as mean, error bars represent standard deviation. *p = <0.005, two-tailed t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005142.g005: ROS1 demethylates the ROS1 promoter and reduces expression.(A) Methylation of the 5' region of ROS1 (region 1 in Fig 3) was measured in two ros1 missense mutants by bisulfite PCR. WT strains were segregating Col-0 siblings for ros1-7 and were C24 for ros1-2. N represents the number of independent clones sequenced for each BS-PCR product. (B) Relative ROS1 transcript abundance was measured in seedlings of both mutants using RT-qPCR. Data represented as mean, error bars represent standard deviation. *p = <0.005, two-tailed t-test.
Mentions: We noticed that cytosines in the CG context 5’ of ROS1 were intermediately methylated at around 50% in wild-type tissues, with independent variance at every CG position in the sequence (Fig 3, S3 Fig). CG methylation is faithfully copied by MET1 during DNA replication, and so average methylation at symmetric CG sites is usually close to 0 or 100% [20]. The observed intermediate level of CG methylation and the low frequency of bisulfite clones fully methylated in the CG context (S3 Fig) suggested that active DNA demethylation might also be active at the 5’ sequence. We hypothesized that ROS1 might oppose RdDM to remove methylation at its own promoter. We performed bisulfite sequencing of the 5' methylated region in two missense mutants of ROS1, ros1-2 [32] and ros1-7, an allele encoding a protein with an E956K substitution in the ROS1 DNA glycosylase domain [33]. Symmetric CG methylation increased to nearly 100% in both mutants compared to their wild-type siblings, along with increases in non-CG methylation (Fig 5A), indicating that ROS1 actively removes methylation from this region in wild-type plants. At other loci, removal of 5' methylation by ROS1 increases transcription [21]. We examined the effect of DNA demethylation by ROS1 on ROS1 transcription by performing RT-qPCR on ros1-2 and ros1-7 mutants. Because these are missense mutations, nonsense-mediated decay should not be a complicating factor in measuring transcript abundance. ROS1 transcripts were 2 to 4-fold more abundant in ros1 mutants (Fig 5B). This suggests that active demethylation by ROS1 represses transcription of ROS1, counteracting the function of the RdDM pathway, which promotes ROS1 expression. Thus ROS1 regulates the expression of its own gene, forming a negative feedback loop for demethylation activity.

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