methyltransferase [7,33]. kyp mutations were shown to specifically reduce H3K9m2 in vivo, and KYP is efficient at mono- and di- methylation, but not trimethylation, of H3K9 sites in vitro [34]. The mechanism by which H3K9m2 controls CHG DNA methylation is not yet clear but appears to involve direct recruitment of CMT3 to methylated histones because CMT3 contains a chromodomain that is capable of binding directly to methylated histone peptides [35]. Efficient binding of CMT3 chromodomain to methylated histones in vitro required simulta- neous methylation of both the lysine 9 and lysine 27 positions, suggesting that H3K27 methylation may also be involved in the recruitment of CMT3 [35]. More recently, the SRA domain present within KYP was shown to bind in vitro to oligonucleotides that are methylated at CHG sites, suggesting that KYP is directly recruited to methylated DNA [30]. These results suggest a self- reinforcing feedback loop between CMT3 and KYP that would ensure efficient maintenance of CHG DNA methylation. Recent genome-wide profiling studies of DNA methylation within the Arabidopsis genome utilizing either microarrays or whole genome shotgun bisulfite sequencing have revealed key aspects of DNA methylation patterning [32,36–40]. CG, CHG and CHH methylation are highly correlated with each other and with transposons and other repeat sequences throughout the genome. An interesting exception, however, is in the coding region of genes (gene bodies), where only CG methylation is found [32,39,40]. Gene body methylation occurs on about a third of all genes, and these genes tend to be highly and ubiquitously expressed in different Arabidopsis tissues [39,40]. The function of this methylation is unclear, but it has been proposed to be involved in the suppression of cryptic transcription initiation which would otherwise interfere with transcription from the 59 promoter [36,40]. Multiple studies have also addressed the location of H3K9m2 either by utilizing chromatin immunoprecipitation (ChIP) at individual loci, or by using a ChIP-microarray (ChIP-chip) approach with a microarray consisting of 1-kb probes tiled across Arabidopsis chromosome 4 [41–45]. These studies indicated that H3K9m2 is highly enriched on transposons and pseudogenes and is also associated with repeat elements. In order to gain deeper insight into H3K9m2 methylation patterning, we profiled H3K9m2 by using native nucleosome chromatin immunoprecipitation (native ChIP), coupled with a high resolution Nimblegen array containing ,60 nucleotide probes tiled across the entire Arabidopsis genome and combined these data with recently published DNA methylation profiling data. We found that H3K9m2 was specifically associated with CHG DNA methylation throughout the genome, and not with body methylated genes that only contain CG methylation. Furthermore, we found that pericentromeric heterochromatin and euchromatic chromosome arms show distinct patterns of H3K9m2, suggesting that different targeting mechanisms exist for these different genome compartments. Results and Discussion Genome-wide detection of H3K9m2 We designed a Roche Nimblegen High Density (HD2) tiling array with 1.98 million probes spaced every 60 nucleotides and covering the entire sequenced portion of the Arabidopsis genome. By tiling across the genome, all repeated sequences were included and were represented proportionally to their copy number. We utilized a native chromatin immunoprecipitation (ChIP) method in which the crosslinking/sonication step was eliminated to achieve nucleosome resolution data. Chromatin was subjected to limited digestion with Micrococcal nuclease (MNase) such that the majority of the chromatin was present as mononucleosomes (Figure S1a). Genome-Wide H3K9m2 Profiling Digested chromatin was then immunoprecipitated with a highly specific monoclonal antibody against di-methylated lysine 9 on histone H3 (H3K9m2) (Figure S1b). ChIP with antibody against the unmodified C-terminus of histone H3 was used as a control [46]. DNA fragments extracted from either the H3K9m2 or control immunoprecipitation were amplified, labeled with either cy3 or cy5, and hybridized to the tiling arrays. Three biological replicates experiments were performed and each probe was assigned a Z-score as detailed in Material and Methods. Based on the distribution of Z- scores, we defined a positive probe as having a score higher than 0.2 (Figure S2). In order to minimize false positive signals, we defined a set of ‘‘methylated regions’’ utilizing max gap/min run settings that allow detection of regions not shorter than the size of a mononucleosome (147 bp) (Material and Methods). The level of false positive signals was evaluated by including probes on the array corresponding to the entire chloroplast genome (154,478 bp sequence), which lacks histones and therefore H3K9m2. Only 8 regions (1.19%) were falsely identified as being H3K9m2 positive. Comparing this false positive rate to the 22.5% positive probes for the nuclear genome, suggests that the majority of signals in the nuclear genome represent true signal. The reproduc- ibility of the microarray data was assessed in two ways. First, we performed additional Nimblegen profiling experiments utilizing a conventional, formaldehyde crosslinking ChIP method [46]. The correlation between the two sets of data was very high (Pearson correlation coefficient = 0.9) (Figure S3). Second, we compared our results with a list of H3K9m2-positive regions compiled from previously published studies (Table S1). We confirmed H3K9m2 methylation of 99.6% of previously published regions [41]. These results suggest that the experimental method and the microarray platform allow for efficient detection of H3K9m2. A high correlation between H3K9m2 and CHG DNA methylation We identified 17,099 regions with significant levels of H3K9m2, covering a total of 27 Mb of the sequenced genome (22.5%). Figure 1 shows a distribution of H3K9m2 along each of the five Arabidopsis chromosomes in sliding 100 bp window. Consistent with previous reports and immunofluorescence studies [10,41,47], H3K9m2 is highly enriched in Arabidopsis pericentromeric regions and is correlated with DNA methylation (Figure 1). In order to correlate H3K9m2 with DNA methylation in the different sequence contexts, CG, CHG and CHH, we compared the H3K9m2 profiling data with whole genome shotgun bisulfite sequencing (BS-seq) data, which represents quantitative single nucleotide resolution data for individual cytosines throughout the genome [32]. First, we calculated average DNA methylation percentages within both H3K9m2 highly positive and negative probes (with Z-scores higher than one and lower than zero, respectively) (Figure 2a). All three kinds of DNA methylation were highly enriched within H3K9m2 positive probes, compared to genome average levels. However, H3K9m2 negative probes were virtually devoid of CHG and CHH methylation, while CG methylation was still present. Second, we asked what fraction of methylated cytosines (defined with the thresholds of .50% for CG, .20% for CHG and .0% for CHH) fall within H3K9m2 positive regions (as defined as regions with Z-scores.0.2). Figure 2b shows that only a very small fraction of CHG methylation does not overlap with these H3K9m2 positive regions (also see examples in Figure S4), while CG and CHH methylation shows a higher level of non-overlap. The relatively high fraction of CG methylated regions that do not overlap with H3K9m2 positive regions corresponds to genes (Figure S5), consistent with previous observations of CG-only methylation in the transcribed region PLoS ONE | www.plosone.org 2 September 2008 | Volume 3 | Issue 9 | e3156