First High Resolution Rat Methylome Figure 1. Chromosomal distribution of DNA methylation. Graphical representation of chromosome wide distribution of methylation peaks of chromosome 1, 2 and 3 along with their GC percentage (dark black color), Refseq genes (blue color), CpG Islands (green color), and chromosome band in UCSC Genome Browser. doi:10.1371/journal.pone.0031621.g001 existence of a differential pattern of promoter methylation (proximal vis a` vis distal), which may in turn play a crucial role in regulating gene expression. Inside the gene body, we observed that exons were more methylated than the introns. At the intron-exon (6200 bases from exon start site)/exon-intron (6200 bases from exon end site) junctions for all RefSeq gene exons, the exon start site was found to be more methylated than the exon end site (intron start site) and there was a sharp transition at the exon boundaries (Figure 5A, B). To check if the methylation patterns in TSS and exon-intron and intron-exon boundary that we observed in rats are also similar in human and mice, we downloaded the MeDIP-Seq data from the Sequence Read Archive (SRA) and processed it through the same pipeline i.e, peak identification by MACS. We found that the methylation pattern at the TSS of RefSeq genes in human and mice have patterns that are similar to rat methylome data (Figure S6A, B and C). Further, the exon intron junctions also follow a similar pattern in human and mice (Figure S7A, B and C). This is consistent with earlier reports in human embryonic stem cells and neonatal fibroblasts [20]. Thus, DNA methylation may define the splicing boundaries of a gene, thereby helping RNA polymerase II in recognizing exons in a sea of intronic DNA. We observed a marked elevation of methylation at exons which is apparent at the exon start site and similarly the decrease in methylation density at the approach of exon end site. Interestingly, while the first exon also followed the same pattern their methylation density was lower than that generally observed for all RefSeq exons (Figure 5A). The methylation at the beginning of the first and the last exon were lower than other exon (Figure 5A,B). Since, the first and last exons usually constitute the 59UTR and 39 UTR regions respectively, we checked methylation levels in first exons and last exons that either contains or lacks coding region and noted that DNA methylation marked the start sites of these exons only if they formed a part of the protein coding sequence (Figure 6A, B). This was further substantiated by the observations that methylation levels up to the second exon were substantially lower in genes where coding starts from the third exon (Figure S8). For instance, in Ccdc 75 gene where coding region starts from the 3rd exon and first two exons, that comprise the UTR, are unmethylated (Figure 7). To confirm that the pattern of cytosine methylation associated with coding part of the transcript is not restricted to rat liver only, we compared methylation levels from human brain tissue, an entirely different tissue type from phylogenetically distant species PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31621