First High Resolution Rat Methylome Figure 6. Methylation distribution of first and last exons based on presence and absence of coding region. The first and last exons were further classified as coding exons and non-coding exons based on the fact that they contain coding region within them or not. (a), (b) represents the methylation of rat RefSeq first exon and last exons while (c), (d) represent the methylation pattern in Human RefSeq first exon and last exons plotted using the MeDIP-Seq data from Human brain tissue. Distribution of peak summit count in 10 bp sliding window, 200 bp upstream and downstream from the start site and end site of exons was calculated for first exon and all last exons. Smoothing of peaks was done by taking moving average of 5. doi:10.1371/journal.pone.0031621.g006 they are retained in the transcript (Figure S10A). Similarly, RT- PCR was done for constitutive introns (that were not methylated), which acted as controls. We found that the introns that are methylated are indeed retained in the transcript while the constitutive introns that were unmethylated were not a part of the final transcript (Figure S9B). This strengthens our conclusion that DNA methylation is possibly used as a mark to label the introns as candidates for inclusion within the final transcript by enabling them to evade splicing. To further prove our point, we analyzed the methylation pattern at the boundaries of cassette exons (of alternative splicing events from ASTD). Interestingly, we observed a marked elevation in methylation density at intron-exon junction in constitutive exons but only mildly higher methylation density at the intronic part of the intron-exon boundary in cassette exons compared to that of the constitutive suggestive of the role of DNA methylation in marking these exons for splicing (Figure 8 Figure S10B). Overall, our observations indicate an important role for DNA methylation in regulation of splicing events and final constitution of the protein sequence. Discussion DNA methylation has two crucial evolutionarily conserved functions. It is one of the subtle control elements that govern gene response to environmental cues and also chief defense mechanism for the genome against selfish mobile elements. Genome-wide methylation map provides a thorough quantitative and qualitative assessment of genomic methylation; a prerequisite for understand- ing its functional potential both in terms of maintenance of genomic stability as well as gene regulation. There is increasing evidence to show that in addition to its role in X-chromosome inactivation, genomic imprinting, and maintenance of cellular transcriptional memory during development, DNA methylation plays an important role in predicting the course of complex diseases [1,3,33]. To understand the importance of DNA methylation in disease causation and progression it becomes imperative to analyze the basal methylome of various tissues in different model systems. Despite rat being a well established model system for studying several complex disorders, methylome from a control rat is not PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e31621