Repeat elements are usually associated with chromosome instability, translocation and gene disruption via transposition or recombination events [6,34]. Methylation of repeat elements is known to silence the repeat region and prevent reactivation of endoparasitic sequences [35,36]. Consequently, repeat regions have been reported to account for a major proportion of genomic methylation which is substantiated by our data. Simple repeats were hypermethylated to a greater extent (,20% of total methylation peak summits) vis a vis other repeat classes and thus might hinder recombination and consequent chromosome insta- bility [37]. LINE (Long Interspersed Nuclear Elements) and LTR (Long Terminal Repeats) were also significantly methylated and accounted for 16% and 11.4% of total methylation peak summits. Methylation of LTRs is crucial in maintenance of genomic stability since transcriptional silencing at these loci via DNA methylation suppresses the initial stages of retrotransposition. A recent study has shown that one endogenous retrovirus (ERV) class i.e., ERV-K-type family contributes to genome variability in inbred rat strains [38]. Therefore, such defence mechanism is important for rodents who harbour active ERV within their genomes. Although methylation occurs at 70–80% of cytosines that are followed by guanine bases (CpG), there are regions in the genome known as CpG islands (CGI) which are generally believed to be unmethylated in spite of having high GC percentage [3]. These regions are known to play a key role in gene regulation and their aberrant methylation has been reported in various disease conditions such as cancer and several neurological/autoimmune disorders [6]. Our study for the first time provides a catalogue of methylated and unmethylated CGIs in adult rat liver which will aid in better understanding of the disease mechanism in rat models of such diseases. While earlier reports investigating CGI had established hypomethylation as a hall mark of CGIs, a recent report indicates that a small proportion of these islands might be methylated. Straussman et. al. showed that methylated CGIs are generally shorter in size. We for the first time show that these methylated CGIs are significantly enriched in exons and are shorter in size in the rat genome [27]. We also show a distinct methylation pattern upstream of CGIs irrespective of their methylation status with a dip in the methylation levels upstream (,1 Kb) of CGI start site (Figure S11). Although, this trend is recurrent in rat methylome, we could not identify any underlying specific sequence motif. It is plausible that there are sequence independent messages that bring about this pattern of methylation distribution immediately preceding CGI. Our data also shows that about 1/3rd of unmethylated CGIs are distributed within the promoter region which are important for expression of genes. Analysis of methylation status across promoter regions of RefSeq genes and expressed genes revealed a V shaped curve, due to declining methylation near TSS which is in agreement with other reports [20,39]. Expressed genes, like RefSeq genes, were less methylated at proximal promoter region while methylation at distal promoter in both categories was higher. Therefore, collectively the methylation status of proximal promoter diverges from that of the distal promoter. Such a divergence might stem from increased probability of existence of CpG Island at the proximal promoter. DNA methylation along with nucleosome positioning has been shown to be enriched at the exonic positions in the genome hinting at a role in splicing [40]. During splicing, which generally occurs co-transcriptionally, gene splicing machinery needs to accurately distinguish an exon from an intron [41]. Earlier reports have shown that splicing is influenced by chromatin structure [42]. Our observation that intron-exon-intron junctions are distinctly First High Resolution Rat Methylome marked by DNA methylation thus supports the hypothesis that chromatin modification and DNA methylation probably work in tandem to regulate splicing [42,43]. Choi et al. have shown that the coding margins constituting coding start and end boundaries are demarcated by DNA methylation [44]. However, inclusion of UTR in the final transcript has hitherto remained unexplained. Our analysis revealed that UTR recognition and retention is independent of DNA methylation. In general, UTRs were not marked by methylation unless a part of UTR was included in the coding sequence, in which case they were found to be methylated. A similar pattern was also observed on analysis of human brain tissue, thus, advocating a universal identification code conserved over different species and tissue types. In the light of this observation we suggest that protein coding region of genes harbor distinctly elevated methylation levels in comparison to the non- coding regions, which might help in splicing as postulated by earlier studies [40]. Anastasiadou et al. has recently analyzed a small data set derived from Human Epigenome Project and reported a possible link between methylation and splicing [45]. Our observation of altered methylation in alternately spliced events like cassette exons and intron retention and that of marking of UTR containing coding region with DNA methylation suggests that DNA methylation is possibly used as a mark to label these introns and exons as candidates for inclusion within the final transcript and to enable them to evade splicing. It has been reported that co- transcriptional splicing requires the recruitment of splicing factors at splice sites during transcription, even though completion of intron removal may occur post-transcriptionally [41,46]. This dynamic link between splicing and transcription has been partially explained by RNA Pol II kinetic model of alternative splicing, which states that recognition of splice sites is dependent on the rate of RNA Pol II elongation [46]. Therefore, it can be perceived that DNA methylation along with chromatin road blocks like nucleosome positioning may cause slowing down of RNA Pol II and lead to alternate splicing. This view is also supported by the fact that nucleosomes and RNA Pol II and DNA methylation (as found in our study) are enriched at the alternate splice sites [46,47]. Thus, we speculate that DNA methylation may directly or indirectly via nucleosome positioning affect splice site choice and thereby decide the sequence of the final transcript. Overall, our results show that DNA methylation is one of the marks that a cell employs to distinguish between protein coding and non-coding regions of the genome. Interestingly, methylation seems to mark all the coding exons more than the non-coding ones suggesting presence of an under-appreciated link between DNA modification and coding. While generating the first high resolution methylome map of rat liver, the present study has provided ample intriguing and critical insights into methylome organization and function of cytosine methylation in defining the coding region in a gene. Further studies to validate the functional potential can help identify key methylation signatures in diverse cellular contexts including altered, disease states, which would not only increase our knowledge base but also empower us to design better epigenetic diagnostics. Materials and Methods Genomic DNA extraction The experiment was carried out in Wistar rats in accordance with the ‘principles of laboratory animal care’ (US Department of Health, Education and Welfare: Guide for the Care and Use of Laboratory Animals. Washington, DC, U.S. Govt. Printing Office, 1985, (NIH publ. no. 85–23) and with the approval of the PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31621