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

BMC Plant Biology Atypical epigenetic mark in an atypical location: cytosine methylation at asymmetric (CNN) sites within the body of a non-repetitive tomato gene Rodrigo M Gonzlez 0 Martiniano M Ricardi 0 Norberto D Iusem 0 0 Departamento de Fisiologia, Biologia Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires e IFIByNE- CONICET , Buenos Aires , Argentina 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. epigenetics asymmetric methylation; Asr1; water stress; tomato - Background Epigenetics refers to mitotically and meiotically heritable variation in gene regulation and function that cannot be accounted for by changes in DNA sequence but rather results from enzyme-mediated chemical modifications to DNA and its associated chromatin proteins [1]. Over the last decade, epigenetic research has focused mainly on mammals, whereas plants have received less attention, although there is a fair amount of information on certain plant models such as Arabidopsis [2,3], rice [4] and maize [5]. Whereas methylation in animal genomes occurs mostly in regulatory regions, methylation in Arabidopsis is found in transcribed sequences, not only at canonical CG sites but also at CNG (N denotes A, C or T) and CNN (asymmetric) sites. The latter sites are preferentially methylated in repetitive elements and transposons [6,7]. It has been well established through chemical analyses on mutants that MET1, the orthologous enzyme to mammalian DNMT1 (DNA methyltransferase 1), maintains DNA methylation at CG sites [8]. On the other hand, the plant-specific methyltransferase CMT3 maintains DNA methylation at CNG sites [7] while at the same time cross-talking with the histone H3 methyltransferase KYP [9]. Finally, the third type of plant cytosine methylation (CNN, called asymmetric) was demonstrated by pioneer mutant analysis to arise due to the methylase DRM2 [10], a homologue of the mammalian de novo methyltransferase DNMT3. DRM2, together with endogenous small interfering RNAs, also maintains DNA methylation at CNN sites [11], a less-studied epigenetic modification. Our studies focused on the tomato plant (Solanum lycopersicum), an edible plant crop (http://mips.helmholtz-muenchen.de/plant/tomato/index.jsp) of great economic importance with a genome that is almost 10 times larger than that of Arabidopsis and of which there have been few epigenetics studies [12]. Using this model system, we investigated cytosine methylation status in different contexts and the intragenic distribution of cytosine methylation in Asr1, a non-transposon, proteincoding, water stress-inducible gene of the LEA superfamily [13] that is conserved in the plant kingdom but lacks an orthologous counterpart in Arabidopsis. This gene has been extensively studied by us and other groups at the DNA [14], RNA [15] and protein [16,17] levels and in terms of physiological function [18] and evolution [14]. This 1,199-bp gene has a very simple organisation, consisting of exon 1 and exon 2 of 153 and 358 nt, respectively, separated by an intron of 688 nt. We chose the leaf as the source of genomic DNA because it is the organ in which Asr1 expression is the greatest upon water stress [15]. A second aspect of our work dealt with the intriguing link between epigenetics and stress in plants [19-21]. Stress-induced physiological responses in Arabidopsis are thought to depend on altered DNA methylation [22]. To test this hypothesis experimentally, we examined the gain and loss of cytosine methylation marks on our model gene as a consequence of imposing water stress conditions on tomato plants. Results Overall non-CG methylation in the tomato genome To explore the general features of methylation in tomato leaf DNA, we first observed a panoramic view of both CG and CNG methylation using several restriction enzymes. Comparisons between methylation-sensitive and -insensitive enzymes provided an evaluation of the overall CG methylation. This low-resolution but illustrative analysis (Figure 1) displayed a pronounced level of typical CG methylation and a noticeable degree of overall CNG methylation (Figure 1, Msp I treatment), a Figure 1 Panoramic view of CG and CNG methylation in the tomato plant. Total leaf genomic DNA was treated with the indicated restriction enzymes (right). Recognition sites are listed in the Methods section. As a control for enzymatic cutting efficiency and specificity, pBluescript plasmid was similarly treated (left). modification that is typically, though not exclusively, associated with repeated and/or transposable elements. Non-CG methylation in the Asr1 gene body Motivated by the results described above, we wanted to gain insight into methylation events in cytosine contexts other than the well-known CpG. For that purpose, we performed a closer inspection of Asr1 in the leaf. For this analysis, we used the bisulphite procedure [23], which allows a higher resolution as it is able to d (...truncated)