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In plants, epigenetic reprogramming occurs at key developmental stages and in response to environmental stimuli (Feng et al., 2010b). These epigenomic events play important roles in genome protection, control of gene expression and inheritance via chromatin structural reworking (Teixeira & Colot, 2010). Among epigenetic marks, DNA methylation shows high stability through mitosis and meiosis and has been thoroughly investigated. In plants, methylated cytosines are found mainly in CG dinucleotides and to a lesser extent in CHG and asymmetric CHH contexts (where H is A, T, or C). Non-CG methylation is specific to plants and fungi, with the exception of mammal embryonic stem cells (Suzuki & Bird, 2008; Lister et al., 2009). The enzymatic machinery of methylation (Goll & Bestor, 2005; Teixeira & Colot, 2010) and demethylation (Penterman et al., 2007), driven by both developmental and environmental stimuli, shapes a methylome that is the set of nucleic acid methylation modifications in an organism's genome or in a particular cell.
The first methylome characterization was performed in Arabidopsis (Zhang et al., 2006) and revealed that DNA methylation is concentrated in heterochromatin and repeats but is also present on 30% of genes. Several other approaches have confirmed this distribution across the genomes of other species, including animals, and raised the problem of repeated sequence data processing, as a consequence of both their repetitive nature and their high DNA methylation (Beck & Rakyan, 2008; Cokus et al., 2008). Indeed, repeats are affected by high CG and non-CG methylation, while the gene body is only methylated in CG (Cokus et al., 2008). Despite its high conservation in many organisms, the functional role of gene-body methylation needs to be clarified.
DNase I is a pancreatic enzyme that preferentially digests with no sequence-based bias (Crawford et al., 2006) nucleosome-depleted DNA, while tightly packaged chromatin is more resistant to cleavage (Crawford et al., 2004). In plants, genome-scale assays revealed tissue-specific DNase hypersensitive sites in promoters and potential regulatory elements, but also within genes, allowing the preferential isolation of genes and the elimination of repeated regions (Zhang et al., 2012a,b). In addition, while DNA methylation and nucleosomes co-localize on exons, some nucleosome depleted regions are also methylated (Chodavarapu et al., 2010; Pecinka et al., 2010).
Draft sequencing of the poplar (Populus trichocarpa; western poplar) genome has led to the construction of 19 scaffolds which contain 370 Mb of sequence (out of 403 Mb) and the identification of > 40 000 protein-coding transcripts (http://www.phytozome.net/poplar; Tuskan et al., 2006). As a consequence of its genome sequence becoming available, along with various molecular tools, and its substantial genetic and phenotypic variation, Populus spp. has become widely used as a model tree (Tuskan et al., 2006; Jansson & Douglas, 2007). As perennial plants with long life-spans and generation times, trees have to face and acclimate to changing environments and are therefore models of interest for epigenetic studies (Hamanishi & Campbell, 2011). Interestingly, genetic variability of global DNA methylation, ranging from 4% to 12%, has been observed in Populus × euramericana hybrid shoot apices (Gourcilleau et al., 2010) and has been positively correlated with biomass production. Furthermore, differences in global DNA methylation paralleled transcriptome level trends in leaves of poplar genotypes during drought, suggesting an epigenomic basis for the clone history-dependent transcriptome divergence (Raj et al., 2011). Recently, drafts of genome-wide P. trichocarpa methylome revealed several particularities of this model, such as a high CHG methylation (Feng et al., 2010a), as well as a negative correlation between promoter and, particularly, gene-body DNA methylation and gene expression level (Vining et al., 2012). However, the relationships between gene-body DNA methylation and tissue-specific gene expression remain to be clarified.
In this context, our aim was to characterize the methylome of noncondensed chromatin to investigate gene-body DNA methylation characteristics in an open chromatin state. Moreover, the elimination of heterochromatic repeated loci would reduce the complexity of the whole-genome analysis. For this purpose, the chromatin fraction hypersensitive to DNase I was isolated from P. trichocarpa shoot apical meristem (SAM) cells and was used in MethylDNA Immunoprecipitation followed by Illumina/Solexa (Fasteris, Plan-les-Ouates, Switzerland) sequencing (MeDIP-SEQ). The mapping on the P. trichocarpa genome was confirmed by bisulfite sequencing of candidate mapped loci to assess the cytosine contexts and the corresponding methylation levels. Taken together, our results showed that methylated DNase I hypersensitive sites covered 2% of the genome but were regularly distributed along poplar scaffolds and strongly enriched in genes, particularly in exons. Furthermore, gene-body DNA methylation in the noncondensed chromatin fraction was dependent on structural gene characteristics, redundancy in the genome, tissue-specific pattern of expression and among poplar genotypes.