Genome-Wide H3K9m2 Profiling Figure 1. Distribution of DNA methylation and K9H3m2 methylation along chromosomes. Top panels show average DNA methylation levels in a sliding 100 kb window [32]. Bottom panels show the number of H3K9m2 positive probes (z-score.0.2) in a sliding 100 kb window. doi:10.1371/journal.pone.0003156.g001 (body) of genes. Thus, H3K9m2 is tightly linked to CHG methylation throughout the genome, and is not present at CG- only methylated genes. CHG methylation at individual sites shows a broad distribution of methylation levels from 20% to 100%. To test if H3K9m2 levels correlate with different CHG methylation levels, we computed the average H3K9m2 level within each decile of CHG methylation level (Figure 2c). Regions with CHG methylation lower than 10% were mostly devoid of H3K9m2. In contrast, CHG sites methylated at levels between 10% and 50% showed a monoton- ically increasing level of H3K9m2. This quantitative correlation between H3K9m2 and CHG DNA methylation is consistent with the molecular genetic characterization of KYP and CMT3 [7,30,33–35] and suggests that the linkage between CHG methylation and H3K9m2 is a genome-wide phenomenon. Interestingly, CHG sites methylated at a level between 50% and 100% showed a relatively uniform level of H3K9m2, possibly because this is the maximum level of H3K9m2 attainable. Distinct patterns of H3K9m2 in pericentromeric heterochromatin and euchromatic arms Inspection of the H3K9m2 profiling data in the UCSC genome browser revealed dramatic differences in the patterns of H3K9m2 in pericentromeric heterochromatin and the euchromatic arms (Figure 3a). Pericentromeric regions contained long, uninterrupted regions of high levels of H3K9m2, while euchromatic arms showed smaller, isolated patches of H3K9m2 with overall lower levels. To quantitate this phenomenon, we assigned regions of the five Arabidopsis chromosomes to either pericentromeric regions or regions corresponding to the euchromatic arms of the chromo- somes, based on the overall abundance of repeats, genes and DNA methylation (Figure S6), and then analyzed these two genome compartments separately. We observed that overall H3K9m2 levels, as measured by the distribution of Z-scores of individual probes, were higher in pericentromeric regions (median score of 1.78) than in the arms (median score of 0.74). The majority of H3K9m2 positive probes (those with Z-scores.0.2) in chromo- some arms had a Z-score lower than 1.2, while the majority of probes in pericentromeric regions had scores higher than 1.2 (Figure 3b). To avoid the potential ambiguity introduced by cross- hybridization of repetitive sequences, we performed the same test using only probes with unique sequences (probes that match only once to the genome as defined by BLAT analysis) [48]. Consistent with the observations for all probes, the distribution of Z-scores in chromosome arms was generally lower than the distribution in pericentromeric regions (Figure 3b). To eliminate the possibility that this difference is caused by insensitivity of pericentromeric regions to MNase digestion, we made similar comparisons utilizing data obtained from formaldehyde crosslinking ChIP experiments (Figure S7). We observed the same general trend in this dataset, with a higher proportion of pericentromeric probes showing high Z-scores. Another distinction between H3K9m2 patterns in pericentromeric regions and chromosome arms is the length of H3K9m2 positive regions (Figure 3a and 3c). H3K9m2 in pericentromeric regions tended to occupy large domains, showing an average length of 3.4 kilobases and a maximum length of 350 kilobases (Figure 3c). Conversely, the average length of H3K9m2 positive regions in chromosome arms was only 0.6 kilobases. Because of the strong correlation between CHG methylation and H3K9m2, and because H3K9m2 levels in pericentromeric heterochromatin are generally higher than in the euchromatic arms, we expected that CHG methylation would also be higher in pericentromeric regions than in the arms. Surprisingly however, when we analyzed the average CHG levels from whole genome shotgun sequencing data [32], we found that H3K9m2 positive regions in pericentromeric heterochromatin showed lower overall levels of CHG methylation (19.9%) than in H3K9m2 positive regions in the arms (35.9%). We also analyzed the relationship between CHG methylation levels and the length of the H3K9m2 positive regions in either pericentromeric heterochromatin or the euchromatic arms. As previously seen for specific elements of the genome [32], we found a positive correlation between the length of a region and the percentage of CHG methylation (Figure 4). This analysis again showed that at any given length of H3K9m2- positive region, average levels CHG methylation are lower in pericentromeric heterochromatin than in the arms. Our results indicate that H3K9m2 tends to be distributed in large blocks in pericentromeric regions, while in chromosome arms H3K9m2 forms smaller regions that have lower levels of H3K9m2 with higher levels of CHG methylation. While it is not clear why these differences exist, the data suggest that H3K9m2 PLoS ONE | www.plosone.org 3 September 2008 | Volume 3 | Issue 9 | e3156