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Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene.

González RM, Ricardi MM, Iusem ND - BMC Plant Biol. (2011)

Bottom Line: In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites.We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires e IFIByNE-CONICET, Buenos Aires, Argentina.

ABSTRACT

Background: Eukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.

Results: We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region.

Conclusions: These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants.

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Analysis of methylation at a particular site. A pair of isoschizomers (HpaII and MspI) with different methylation specificities was used as described in the Methods section to discriminate between CG and CNG contexts in the leaves of both normal and stressed plants. For site 32 (indicative of CNG methylation), **p < 0.0001; for site 33 (indicative of CG methylation), *p < 0.01)
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Figure 6: Analysis of methylation at a particular site. A pair of isoschizomers (HpaII and MspI) with different methylation specificities was used as described in the Methods section to discriminate between CG and CNG contexts in the leaves of both normal and stressed plants. For site 32 (indicative of CNG methylation), **p < 0.0001; for site 33 (indicative of CG methylation), *p < 0.01)

Mentions: With the intention of further validating the bisulphite methodology, we measured the extent of methylation at a single CCGG site (which obviously contains both CG and CNG contexts) by methylation-sensitive and -insensitive restriction enzymes; the chosen site was C32C33GG, belonging to exon 1. The result (Figure 6) is in agreement with that obtained with bisulphite for those particular cytosine residues for both basal and stress conditions (Figure 2 and 4).


Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene.

González RM, Ricardi MM, Iusem ND - BMC Plant Biol. (2011)

Analysis of methylation at a particular site. A pair of isoschizomers (HpaII and MspI) with different methylation specificities was used as described in the Methods section to discriminate between CG and CNG contexts in the leaves of both normal and stressed plants. For site 32 (indicative of CNG methylation), **p < 0.0001; for site 33 (indicative of CG methylation), *p < 0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117769&req=5

Figure 6: Analysis of methylation at a particular site. A pair of isoschizomers (HpaII and MspI) with different methylation specificities was used as described in the Methods section to discriminate between CG and CNG contexts in the leaves of both normal and stressed plants. For site 32 (indicative of CNG methylation), **p < 0.0001; for site 33 (indicative of CG methylation), *p < 0.01)
Mentions: With the intention of further validating the bisulphite methodology, we measured the extent of methylation at a single CCGG site (which obviously contains both CG and CNG contexts) by methylation-sensitive and -insensitive restriction enzymes; the chosen site was C32C33GG, belonging to exon 1. The result (Figure 6) is in agreement with that obtained with bisulphite for those particular cytosine residues for both basal and stress conditions (Figure 2 and 4).

Bottom Line: In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites.We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires e IFIByNE-CONICET, Buenos Aires, Argentina.

ABSTRACT

Background: Eukaryotic DNA methylation is one of the most studied epigenetic processes, as it results in a direct and heritable covalent modification triggered by external stimuli. In contrast to mammals, plant DNA methylation, which is stimulated by external cues exemplified by various abiotic types of stress, is often found not only at CG sites but also at CNG (N denoting A, C or T) and CNN (asymmetric) sites. A genome-wide analysis of DNA methylation in Arabidopsis has shown that CNN methylation is preferentially concentrated in transposon genes and non-coding repetitive elements. We are particularly interested in investigating the epigenetics of plant species with larger and more complex genomes than Arabidopsis, particularly with regards to the associated alterations elicited by abiotic stress.

Results: We describe the existence of CNN-methylated epialleles that span Asr1, a non-transposon, protein-coding gene from tomato plants that lacks an orthologous counterpart in Arabidopsis. In addition, to test the hypothesis of a link between epigenetics modifications and the adaptation of crop plants to abiotic stress, we exhaustively explored the cytosine methylation status in leaf Asr1 DNA, a model gene in our system, resulting from water-deficit stress conditions imposed on tomato plants. We found that drought conditions brought about removal of methyl marks at approximately 75 of the 110 asymmetric (CNN) sites analysed, concomitantly with a decrease of the repressive H3K27me3 epigenetic mark and a large induction of expression at the RNA level. When pinpointing those sites, we observed that demethylation occurred mostly in the intronic region.

Conclusions: These results demonstrate a novel genomic distribution of CNN methylation, namely in the transcribed region of a protein-coding, non-repetitive gene, and the changes in those epigenetic marks that are caused by water stress. These findings may represent a general mechanism for the acquisition of new epialleles in somatic cells, which are pivotal for regulating gene expression in plants.

Show MeSH
Related in: MedlinePlus