High Resolution Methylome Map of Rat Indicates Role of Intragenic DNA Methylation in Identification of Coding Region Satish Sati1, Vinay Singh Tanwar1, K. Anand Kumar2, Ashok Patowary1, Vaibhav Jain1, Sourav Ghosh1, Shadab Ahmad 1 , Meghna Singh1, S. Umakar Reddy3, Giriraj Ratan Chandak3, Manchala Raghunath2, Sridhar Sivasubbu 1 , Kausik Chakraborty1, Vinod Scaria1*, Shantanu Sengupta1* 1 CSIR-Institute of Genomics and Integrative Biology, Delhi, India, 2 National Institute of Nutrition, Hyderabad, India, 3 CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India Abstract DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability. Citation: Sati S, Tanwar VS, Kumar KA, Patowary A, Jain V, et al. (2012) High Resolution Methylome Map of Rat Indicates Role of Intragenic DNA Methylation in Identification of Coding Region. PLoS ONE 7(2): e31621. doi:10.1371/journal.pone.0031621 Editor: Matteo Pellegrini, UCLA-DOE Institute for Genomics and Proteomics, United States of America Received November 2, 2011; Accepted January 10, 2012; Published February 15, 2012 Copyright: 2012 Sati et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Department of Biotechnology, India (BT/PR4525/MED/14/533/2003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vinods@igib.in (VS); shantanus@igib.res.in (S. Sengupta) Introduction The genome per se appears to be static, incorporating stable changes in sequence in spans of generations. However, higher organisms require versatile characteristics in order to maintain homeostasis with their fluctuating environmental niche [1,2]. To cater to such needs, mechanisms of chemical modifications of chromatin have evolved, that retains the genetic code but transiently alter its functional potential [2]. Such modulations are termed epigenetic modifications; key modifications include acetylation/methylation of histones and methylation of cytosine bases [1]. Aberration/dysregulation of these epigenetic signatures influences the transcription and leads to altered protein expression. The regulation and characteristics of DNA methylation remains enigmatic although it has been implicated in a range of processes like genomic integrity, X chromosome inactivation, genomic imprinting, transposon silencing and diseases like cancer, cardio- vascular diseases, etc [3–7]. Thus, for a comprehensive under- standing of these processes and manifestation of related diseases along with their prognosis, it is imperative to investigate the distribution pattern of DNA methylation at genomic level [4–6]. With the advent of newer technologies, elucidation of methylation profiles as a function of the genome is now possible. In this regard, immunoprecipitation of methylated DNA by monoclonal antibodies specific to 5-methylcytidine (5mC) (MeDIP) followed by microarray analysis (MeDIP-Chip) or direct sequencing (MeDIP-Seq) has been used as a valuable tool to map methylated DNA on a genomic scale [8,9]. The MeDIP-Seq approach provides sequence-level information that aids in distinguishing highly similar sequences as opposed to MeDIP- Chip (using microarrays) where technical drawbacks of cross hybridization, prior knowledge for probe design and low sensitivity from poorly methylated regions limits its use in the study of whole genome methylation [10–12]. However, unlike whole genome bisulfite sequencing which provides single base resolution of methylated cytosines, MeDIP-Seq gives sequences of the region that are enriched in methylation [13]. A significantly lower cost and ease of data analysis makes MeDIP-Seq an attractive method to study tissue or cell specific genome-wide methylation profiles [13]. Such studies using model systems have revealed some unique features of the methylome landscape like promoter hypomethylation and gene body hyper- PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31621