are characterized by low median frequencies of methylated CHH sites (12–13%). In contrast, the M pattern (20% of total) is characterized by a much higher median frequency of methylated sites (33%), close to that of the MMM pattern (36%). Similarly, the other four patterns (-MM, -MU, M-M, M-U, 30% of total) have median frequencies of methylated sites comparable to those of the MMM pattern. Thus, whereas half of the 3418 TE sequences with no CG or CHG sites are densely methylated, the other half have no or very low methylation, which is twice the fraction of TE sequences with no or very low methylation among those containing all three types of sites. This latter result indicates therefore a critical role for CG and CHG sites in dictating methylation of TE sequences. Arabidopsis TE sequences can be classiﬁed into 13 superfamilies, four corresponding to retroelements (Copia, Gyspy, LINE and SINE), ﬁve to well-deﬁned DNA transposons (En-Spm, Harbinger, HAT, MuDR and Pogo) and one each to Helitrons, TEs of a composite nature, Tc1/mariner and other DNA transposons. As shown in Figure 4, the Gypsy and /En-Spm superfamilies have the highest proportion ( 90%) of methylated TE sequences, and RC/Helitrons and Tc1/mariner superfamilies the lowest such fraction (40–50%). Genomic distribution of TE sequences The 119 Mb of available Arabidopsis genome sequence can be divided into gene-rich/TE-poor and gene-poor/ TE-rich regions that form the euchromatic arms of chromosomes and pericentromeric heterochromatin plus interstitial heterochromatic knobs, respectively (4,21,27). Our analysis indicates that more than two thirds of densely methylated TE sequences (MMM, -MM, M-M and M) and a similar proportion of unmethylated or poorly methylated TE sequences are located within pericentromeric heterochromatin and euchromatin, re- spectively (Table 1 and Supplementary Table S1). Furthermore, the last two categories of TE sequences Figure 4. DNA methylation patterns within TE superfamilies. Unmethylated TE sequences are found across all classes but >90% of the sequences for LTR/Gypsy and DNA/En-Spm superfamilies are methylated. The RC/Helitron and Tc1/mariner superfamilies comprise the largest fraction (50–60%) of unmethylated TE sequences. Nucleic Acids Research, 2011, Vol. 39, No. 16 6925 correspond mainly to TE relics depleted in CpGs, as indicated by their shorter size compared to their densely methylated counterparts and their higher divergence from the cognate reference TE sequence (Figure 5). Thus, the dense DNA methylation characteristic of heterochromatin results not only from the much higher density of TE se- quences compared to euchromatin, but also from the larger ratio of methylated to unmethylated TE sequences within heterochromatin and the longer length of methylated TE sequences on average. Although the vast majority of TE sequences are located outside of genes and cluster within heterochromatin, 17% of euchromatic TE sequences intersect with gene annota- tions (Table 2). Furthermore, most of these TE sequences overlay with exons, suggesting a high incidence of ‘exonization’ of TE sequences in Arabidopsis. Finally, whereas 53% of these exonic TE sequences are unmethylated (UUU+UUM), 24% are highly methylated (MMM), suggesting a recent origin. TE sequences and siRNAs Deep sequencing of small RNAs has revealed that a large fraction of methylated repeat elements present in the Arabidopsis genome are characterized by an abundance of matching 24-nt siRNAs throughout development (6). To investigate more precisely the association of different DNA methylation patterns of TE sequences with en- dogenous 24-nt-long siRNAs, small RNA deep sequenc- ing data obtained from Arabidopsis whole-aerial tissues were downloaded from GEO (accession: GSE14696) (24) and used to calculate the 24-nt siRNA density for all TE sequences with deﬁned DNA-methylation patterns (see ‘Materials and Methods’ section). As expected, almost all (95%) unmethylated TE sequences and many (48– 58%) of those poorly methylated are devoid of matching 24-nt siRNAs, irrespective of their location in euchroma- tin or heterochromatin. On the other hand, 89 and 82% of densely methylated TE sequences located respectively in euchromatin and heterochromatin have matching 24-nt siRNAs (Figure 6a and b). This result conﬁrms that most densely methylated TE sequences are associated not only with siRNAs, but also indicates a lower propor- tion of such sequences in heterochromatin. Indeed, differences between euchromatic and heterochromatic methylated TE sequences were even more pronounced when considering the proportion of those having an abun- dance of matching siRNAs (density >0.25 reads/kb/105 library reads), which is 61% in euchromatin but only 21% in heterochromatin. Given the high density of TE sequences in heterochro- matin, we explored the possibility that methylation of heterochromatic TE sequences with no matching 24-nt siRNAs could occur through local spreading from ﬂanking siRNA-targeted sequences. To this end we con- sidered the set of 749 heterochromatic MMM TE se- quences longer than 200 bp and with no matching 24-nt siRNAs but ﬂanked within 1 kb on one or both sides by sequences associated with siRNAs. Each TE sequence was split in equal halves and DNA methylation densities were calculated in non-overlapping 100-bp windows for