6930 Nucleic Acids Research, 2011, Vol. 39, No. 16 also tend to be depleted in CG sites, suggesting an import- ant function for these sites in determining DNA methylation density. Thus we can propose a scenario in which TE sequences progressively lose CG sites because of their higher methylation levels compared to CHG and CHH sites and because of the higher mutability of methylcytosines compared to cytosines. This progressive loss would in turn reduce the potential for the affected sequences to perpetuate methylation at CHG and CHH sites, leading ultimately to complete loss of DNA methylation. While our analysis conﬁrms the association of siRNAs with DNA methylation over TE sequences, an unexpect- edly high number of densely methylated TE sequences are characterized by the absence or near absence of matching siRNAs. Such TE sequences are preferentially found in heterochromatin. We have shown that in these cases, DNA methylation most likely results from local spreading (within 500 bp) from ﬂanking, siRNA-targeted sequences (Figure 6c). We have also provided evidence that DNA methylation spread occurs in euchromatin as well, but that the extent of spreading is more limited than in heterochromatin ( 200 bp versus 500 bp; Figure 6c and Supplementary Figure S5). This could reﬂect either a facilitating role of heterochromatin, an inhibitory effect of euchromatin, or, as reported previously (30), a higher DNA demethylation activity in euchromatin. The spreading phenomenon we have uncovered here appears distinct from so-called secondary RdDM, which is caused by the biogenesis of secondary siRNAs from sequences adjoining those initially targeted by RdDM (31–34). Furthermore, the persistence of DNA methylation gradi- ents for CG and CHH sites in the ddc triple mutant back- ground (Figure 7) argues against an important role for RdDM in DNA methylation spreading. However, we cannot rule out that RdDM is involved, notably during the reproductive phase when it is most active (3), and that spreading of DNA methylation is maintained by MET1 and/or other DNA methyltransferases independently of RdDM during plant growth. Finally, our study indicates that TE sequences present in euchromatin are more abundant closer to genes than away from them. This pattern is observed both upstream and downstream of genes, which could reﬂect a preference for TEs to insert in ‘open’ chromatin. Indeed, preferential insertion close to or within genes has been noted for several TE families in maize and rice (35), even though such events are unlikely to be maintained over evolution- ary timescales because of their high potential to be dele- terious. We have also shown that methylated TE sequences are slightly underrepresented compared to their unmethylated counterparts close to the 50-end of genes and that methylated TE sequences with matching siRNAs are least abundant and somewhat more uniformly distributed within the 50-end of genes than methylated se- quences with no matching siRNAs (Supplementary Table S2). Given the known inhibitory effect of DNA methyla- tion on promoter activity, it is therefore reasonable to speculate that DNA methylation spread contributes signiﬁcantly to the negative impact of methylated TE sequences on neighboring gene expression. In support of this view, both in A. thaliana and A. lyrata, genes that are located <500 bp away from TE sequences tend to be ex- pressed at lower levels than genes further away, and this reduction in gene expression is more pronounced when the TE sequences have matching siRNAs (8). SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank David Swarbreck for valuable insights on TE annotation, Eva Huala for helping us to access The Arabidopsis Information Ressource, Franc¸ ois Roudier for critical reading of the manuscript and members of the Colot group for discussions. FUNDING Agence Nationale de la Recherche (ANR) ‘DDB1 project’ (to C.B. and V.C., in part); Centre National de la Recherche Scientiﬁque (CNRS) ‘Groupement de Recherche Elements Transposables’ (to V.C and H.Q., in part). PhD studentships from ANR and CNRS (I.A. and A.S., respectively). Funding for open access charges: CNRS. Conﬂict of interest statement. None declared. REFERENCES 1. Slotkin,R.K. and Martienssen,R. (2007) Transposable elements and the epigenetic regulation of the genome. Nat. Rev. Genet., 8, 272–285. 2. Law,J.A. and Jacobsen,S.E. (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet., 11, 204–220. 3. Teixeira,F.K. and Colot,V. 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