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Exploring the role of DNA methylation in epigenetic variation in higher plants

Final Activity Report Summary - VARIOMETH (Exploring the role of DNA methylation in epigenetic variation in higher plants)

In plants and animals, DNA methylation has two essential roles: defending the genome against transposons and regulating gene expression. The first aim of our work was to determine if oil palm (Elaeis guineensis Jacq.) contains the full complement of the DNA-methyltransferase types that are found in model plants. In oil palm, an average 5 % of somatic embryo-derived clonal plants were found to be affected by somaclonal variation, giving rise to an aberrant floral structure (Rival and Parveez, 2005). Both genome-wide and sequence-specific DNA hypomethylation have been demonstrated in variant tissues (Jaligot et al., 2000, 2002 and 2004). It is thus of paramount importance to investigate relationship between changes in global methylation rates in 'mantled' oil palm variants and expression profiles of members of the three families of methyltansferases.

Oil palm encodes three families of plant DNA methyltransferases.
- MET1: MET1 has been identified very early as the main maintenance methyltransferase targeting symmetric sites (CG), although marginal de novo activity has been recently demonstrated on silenced transgene sequences. The EgMET1 coding sequence is approx. 5 kb in length and is mostly similar to rice and maize sequences.
- CMT3: The CMT (chromomethylase) family is unique to the plant kingdom (Henikoff and Comai, 1998); it is in charge of the maintenance of methylation at symmetrical sequence CpNpG. CMTs are characterized by the presence of a chromatin-association domain (or chromodomain) embedded in the catalytic part of the protein. CMT plays a major role in the long-term inactivation of repeated sequences such as retrotransposons. The EgCMT sequence (circa 3 kb) displays extensive homologies with rice, maize and barley sequences.
DRM: The de novo methylation activity is controlled by the DRM (domain-rearranged) family, which shows an inversion in the arrangement of motifs in the catalytic domain, compared to the Dnmt3 de novo family found in mammalians (Finnegan and Kovac, 2000). This activity is required to establish methylation at previously unmethylated sites and to maintain methylation present at asymmetric (CNN) sites throughout mitotic divisions (Cao and Jacobsen, 2002). The EgDRM sequence (2.4 kb) is mostly similar to DRM-like sequences found in tobacco, rice and maize.
- Global DNA hypomethylation in variant calluses is not consistent with changes in METases expression.

Differential expression analyses by real-time quantitative PCR (rtQ-PCR) have been performed on oil palm embryogenic calli. Repeatable overexpression of both EgMET1 and EgCMT3 genes was measured in Fast-growing calli (FGC, generating 100 % of 'mantled' palms) when compared to Nodular Compact Calli (which yield on average 5 % of variant palms). The fate of EgDRM was found to be more variable. We found a consistent over expression of both MET1 and CMT3 in FGC while the genome of FGC shows a 4.5 % decrease in global methylation rate compared to that of NCC (Jaligot et al., 2000).

Our results parallel the major unresolved paradox in DNA methylation and cancer research (Laird and Jaenisch, 1994; Eads et al., 1999). Indeed, there is a net loss of m5C in many tumour cell genomes which is inconsistent with increased levels of transcripts from both de novo and maintenance DNA methyltransferases (James et al., 2003; Goll and Bestor, 2005).

Full lengths cDNAs coding for three different DNA (cytosine-5)-methyltransferases families (namely, MET, CMT and DRM) were isolated from oil palm (Elaeis guineensis L) and the corresponding genes designated as EgMET, EgCMT and EgDRM. Global DNA hypomethylation which was previously measured in variant calluses is not related with decrease in expression of any of the three isolated METases.