S1 SINE retroposons are methylated at symmetrical and non-symmetrical positions in Brassica napus:: identification of a preferred target site for asymmetrical methylation

DNA methylation has been often proposed to operate as a genome defence system against parasitic mobile elements. To test this possibility, the methylation status of a class of plant mobile elements, the S1(Bn) SINEs, was analysed in detail using the bisulfite modification method. We observed that S1(Bn) SINE retroposons are methylated at symmetrical and asymmetrical positions. Methylated cytosines are not limited to transcriptionally important regions but are well distributed along the sequence. S1(Bn) SINE retroposons are two-fold more methylated than the average methylation level of the Brassica napus nuclear DNA. By in situ hybridization, we showed that this high level of methylation does not result from the association of S1(Bn) elements to genomic regions known to be highly methylated suggesting that S1(Bn) elements were specifically methylated. A detailed analysis of the methylation context showed that S1(Bn) cytosines in symmetrical CpG and CpNpG sites are methylated at a level of 87% and 44% respectively. We observed that 5.3% of S1(Bn) cytosines in non-symmetrical positions were also methylated. Of this asymmetrical methylation, 57% occurred at a precise motif (Cp(A/T)pA) that only represented 12% of the asymmetrical sites in S1(Bn) sequences suggesting that it represents a preferred asymmetrical methylation site. This motif is methylated in S1(Bn) elements at only half the level observed for the Cp(A/T)pG sites. We show that non-S1(Bn) CpTpA sites can also be methylated in DNA from B. napus and from other plant species.