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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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Association of Asr1 with a repressive histone epigenetic mark. ChIP was performed using Dynabeads protein A (Invitrogen) and anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam). Quantitative Real-Time PCR was carried out as indicated in the Methods section. Comparison between non-stress vs. stress yielded p < 0.05. Actin was included as a housekeeping gene control.
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Figure 7: Association of Asr1 with a repressive histone epigenetic mark. ChIP was performed using Dynabeads protein A (Invitrogen) and anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam). Quantitative Real-Time PCR was carried out as indicated in the Methods section. Comparison between non-stress vs. stress yielded p < 0.05. Actin was included as a housekeeping gene control.

Mentions: As gene expression could be regulated also by post-translational histone modifications, which, in turn, may interact with the methylation of cytosines, we decided to explore H3K27me3 and H3K4me3, abundant histone marks in Arabidopsis [24]. We found the expression level of gene Asr1 tightly associated with H3K27me3, a major repressive mark for gene expression. Such a covalent modification quantitatively appeared to decrease with water stress (p < 0.05) (Figure 7). In contrast, H3K4me3, a mark distinctive of gene activation, was not significantly detected under any condition in the context of Asr1 (Figure 7).


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)

Association of Asr1 with a repressive histone epigenetic mark. ChIP was performed using Dynabeads protein A (Invitrogen) and anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam). Quantitative Real-Time PCR was carried out as indicated in the Methods section. Comparison between non-stress vs. stress yielded p < 0.05. Actin was included as a housekeeping gene control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Association of Asr1 with a repressive histone epigenetic mark. ChIP was performed using Dynabeads protein A (Invitrogen) and anti-H3K4me3 or anti-H3K27me3 antibodies (Abcam). Quantitative Real-Time PCR was carried out as indicated in the Methods section. Comparison between non-stress vs. stress yielded p < 0.05. Actin was included as a housekeeping gene control.
Mentions: As gene expression could be regulated also by post-translational histone modifications, which, in turn, may interact with the methylation of cytosines, we decided to explore H3K27me3 and H3K4me3, abundant histone marks in Arabidopsis [24]. We found the expression level of gene Asr1 tightly associated with H3K27me3, a major repressive mark for gene expression. Such a covalent modification quantitatively appeared to decrease with water stress (p < 0.05) (Figure 7). In contrast, H3K4me3, a mark distinctive of gene activation, was not significantly detected under any condition in the context of Asr1 (Figure 7).

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