CpG Islands Undermethylation in Human Genomic Regions under Selective Pressure Sergio Cocozza1,2*, Most. Mauluda Akhtar1,2, Gennaro Miele1,3,4, Antonella Monticelli5 1 Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale, Universita` di Napoli ‘‘Federico II’’ - Universita` di Salerno, Naples, Italy, 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘‘L. Califano’’, Universita` degli Studi di Napoli ‘‘Federico II’’, Naples, Italy, 3 Dipartimento di Scienze Fisiche, Universita` degli Studi di Napoli ‘‘Federico II’’, Naples, Italy, 4 Istituto Nazionale di Fisica Nucleare – Sezione di Napoli, Naples, Italy, 5 Istituto di Endocrinologia ed Oncologia Sperimentale, CNR Napoli, Naples, Italy Abstract DNA methylation at CpG islands (CGIs) is one of the most intensively studied epigenetic mechanisms. It is fundamental for cellular differentiation and control of transcriptional potential. DNA methylation is involved also in several processes that are central to evolutionary biology, including phenotypic plasticity and evolvability. In this study, we explored the relationship between CpG islands methylation and signatures of selective pressure in Homo Sapiens, using a computational biology approach. By analyzing methylation data of 25 cell lines from the Encyclopedia of DNA Elements (ENCODE) Consortium, we compared the DNA methylation of CpG islands in genomic regions under selective pressure with the methylation of CpG islands in the remaining part of the genome. To define genomic regions under selective pressure, we used three different methods, each oriented to provide distinct information about selective events. Independently of the method and of the cell type used, we found evidences of undermethylation of CGIs in human genomic regions under selective pressure. Additionally, by analyzing SNP frequency in CpG islands, we demonstrated that CpG islands in regions under selective pressure show lower genetic variation. Our findings suggest that the CpG islands in regions under selective pressure seem to be somehow more ‘‘protected’’ from methylation when compared with other regions of the genome. Citation: Cocozza S, Akhtar MM, Miele G, Monticelli A (2011) CpG Islands Undermethylation in Human Genomic Regions under Selective Pressure. PLoS ONE 6(8): e23156. doi:10.1371/journal.pone.0023156 Editor: Amanda Ewart Toland, Ohio State University Medical Center, United States of America Received May 3, 2011; Accepted July 7, 2011; Published August 2, 2011 Copyright: 2011 Cocozza 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 work was supported by Institutional funds by University ‘‘Federico II’’, Naples. MMA is a recipient of a fellowship by Doctorate of Computational Biology and Bioinformatics, University ‘‘Federico II’’, Naples. 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: cocozza@unina.it Introduction DNA methylation at CpG sites is one of the most intensively studied epigenetic mechanisms [1] . CpG sites are DNA regions where a cytosine nucleotide occurs next to a guanine nucleotide. Cytosines in CpG dinucleotides can be methylated to form 5- methylcytosine. Human genome contains about 30 million CpGs that exist in a methylated or unmethylated state. A part of all CpG sites present in the genome are clustered into CpG islands that are defined as genomic regions with increased CpG density. These CGIs are enriched at genes, about 60% of all genes in the human genome containing a CpG island upstream [2]. The methylation status of CGIs can influence gene expression [3] [1]. The hypermethylation at promoter CGIs typically results in a decreased transcription of downstream genes [4]. Further, aberrant DNA methylation has been often reported to cause various human diseases [5] [6] [7]. Three DNA methyltransferases, namely DNMT1, DNMT3a, and DNMT3b [8] are involved in the maintenance of DNA methylation during the cell cycle. When the two parental DNA strands are separated in the S-phase of the mitosis, two hemimethylated strands are produced. DNMT1 is a component of a protein complex with high affinity with hemimethylated DNA, subsequently restoring methylation on the daughter strands [9]. Also demethylation is an important biological mechanism, as illustrated, for example, by the demethylation of the paternal and maternal genomes in the zygote after fertilization [10] or by the reprogramming of pluripotency cells to differentiated cells [11]. Nevertheless, the molecular mechanism of DNA demethylation in mammals is disputed, one possibility being that cells demethylate their genome by passive demethylation. Several evidences suggest a dependence of DNA methylation on local sequence content [12]. DNA methyltransferases within eukaryotic cells are not free, but they are compartmentalized by interaction with nuclear components [13]. Thus it is likely that chromatin structure of a genomic region will have an important impact on the maintenance of methylation of that region. It could be hypothesized that there are genomic regions somehow ‘‘protected’’ in vivo from methylation but yet readily accessible to exogenously added soluble DNA methylases [14]. Nonetheless, a complete understanding of the role of DNA methylation and the mechanisms responsible for its establishment and maintenance remain elusive [1]. Many studies focused on the interplay between epigenomic regulation and evolution, because DNA methylation is involved in several processes that are central to evolutionary biology, including phenotypic plasticity and evolvability [15]. Changes in the regulation of gene expression levels have long been hypothesized to play an important role in evolution [16]. Nevertheless, studies specifically addressed to the relation PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23156