6928 Nucleic Acids Research, 2011, Vol. 39, No. 16 that are simultaneously defective for the de novo DNA methyltransferases DRM1 and DRM2 and the CHG- speciﬁc DNA methyltransferase CMT3 [drm1, drm2 and cmt3 (ddc); Supplementary Figure S3a and b]. Nonetheless, the gradient in the ddc triple mutant is less steep than in wild type. Finally, plants defective for three of the four known Arabidopsis DNA demethylases [ros1dml2dml3 or rdd triple mutant; (6)] display DNA methylation gradients similar to wild type (Supplementary Figure S3c). Taken together, these results rule out any signiﬁcant contribution of active DNA demethyl- ation to the gradients observed and suggest a complex set of interactions between different DNA methyl- transferases in promoting or limiting DNA methylation spread. To analyze further the local spreading of DNA methyl- ation from siRNA-targeted TE sequences, methylation densities were plotted separately for CG, CHG and CHH sites (Figure 7). Although gradients are observed in wild type for the three types of sites, slopes are maximal for CHG, suggesting that CHG methylation spreads over shorter distances than CG and CHH methyl- ation. Furthermore, CHG methylation is completely abol- ished both in the ﬂanks and within TE sequences in the ddc triple mutant (Figure 7), suggesting that at least in this background the residual CG and CHH methylation gra- dients are contributed by DNA methyltransferases other than DRM1, DRM2 and CMT3. These results, together with the absence of any discernible methylation gradient for CHG and CHH in met1 (Supplementary Figure S4), provide additional evidence that the extent of DNA methylation spread results from complex interactions between different DNA methyltransferases. Whereas most MMM TE sequences with no matching siRNAs show decreasing DNA methylation towards their middle, uniform DNA methylation across the entire length is observed for some large TE sequences. This suggests either a more extensive spreading of DNA methylation in these cases or the existence of DNA methylation mechanisms not associated, directly or indirectly, with siRNAs. In agreement with the latter hypothesis, the few euchromatic MMM TE sequences Figure 7. Analysis of methylation spreading for CG, CHG and CHH sites. The ﬁgures in the ﬁrst and second columns correspond to wild type (Col0) and the drm1, drm2, cmt3 triple mutant (ddc), respectively.