Genome-Wide H3K9m2 Profiling Figure 4. The percentage of DNA methylation increases as the length of H3K9m2 positive regions increases. Average percent DNA methylation of CG (red), CHG (green) and CHH (blue) was calculated for H3K9m2 positive regions of different lengths. doi:10.1371/journal.pone.0003156.g004 the targeting or maintenance of H3K9m2 in different genome compartments. Association of H3K9m2 with genes We analyzed the distribution of average levels of H3K9m2 within protein coding genes found in the euchromatic arms of chromosomes and found that H3K9m2 levels are very low in the vast majority of genes (Figure 5a). These results are consistent with the nature of H3K9m2 as a gene silencing mark, and with whole genome bisulfite sequencing results showing that the majority of genes are devoid of DNA methylation in their promoters, and lacking in CHG and CHH methylation in their coding regions [32,37]. It is also consistent with our finding that H3K9m2 is not associated with CG-only gene body DNA methylation (Figure S4). We also analyzed levels of H3K9m2 within the 3518 genes present in pericentromeric heterochromatin and found that 31% had significant levels of H3K9m2 methylation (average score is higher than 0.5), while the majority of genes (75%) were devoid of H3K9m2 (Figure 5a). These results are consistent with earlier findings that genes present in heterochromatic regions ‘‘escape’’ targeting by H3K9m2 and DNA methylation and thereby avoid gene silencing [47], but also show that a significant minority of the genes are indeed associated with H3K9m2. We utilized publicly available microarray expression data averaged over 79 tissues and conditions [49] to analyze expression levels of 2049 of the 3518 pericentromeric genes which were present on the ATH1 array used for expression analysis. We found that the 547 pericentromeric genes with high H3K9m2 levels (27%) were expressed at very low levels compared to the unmethylated pericentromeric genes (Figure 5b). In contrast, genes lacking in H3K9m2 showed a distribution of expression levels (Figure 5b) similar to those previously reported for all protein coding genes in the genome (see Figure 3 in [39]). These results are consistent with previous observations that H3K9m2 methylation is usually associated with gene silencing and show that genes that escape H3K9m2 silencing in pericentromeric regions show normal levels of expression. Association of H3K9m2 with transposons, repeat elements and endogenous siRNAs Transposons (which are often annotated as pseudogenes in the Arabidopsis genome) are frequently DNA methylated and silenced, and associated with H3K9m2 [7,32,33,37,39,41– 43,47]. We examined H3K9m2 levels of transposons/pseudogenes located in pericentromeric regions (77% of all elements) and in the euchromatic arms (23%). Figure 6 shows that transposons/ pseudogenes located in pericentromeric regions are much more frequently associated with high levels of H3K9m2 than are transposons/pseudogenes in the euchromatic arms. We also analyzed three classes of DNA repeats for H3K9m2 methylation (tandem repeats, inverted repeats, and interspersed repeats), and found a much higher frequency of H3K9m2 on the repeats present in pericentromeric regions as compared to those found in the euchromatic arms (Figure 7a). The majority of unmethylated repeats (more than 80%) were found in the euchromatic arms. The fact that many repeats remain unmethy- lated in the euchromatic arms (for instance 4420 out of 7778 tandem repeats and 2428 out of 3471 inverted repeats), suggests that repeat character alone may not be sufficient for recognition and silencing. These findings also underscore the unique nature of silencing in pericentromeric regions, which encompasses large swaths of silent chromatin and encompasses many repeat elements. We examined the distribution of H3K9m2 levels of repeats found in either pericentromeric heterochromatin or euchromatic PLoS ONE | www.plosone.org 6 September 2008 | Volume 3 | Issue 9 | e3156