CpG Islands Methylation and Selective Pressure Figure 5. SNP content of CGIs in genomic regions under selective pressure compared with CGIs localized in other genomic regions. The mean SNP frequencies (SNPs/100 bp) of CGIs in genomic regions under selective pressure (open bars) and of CGIs localized in other genomic regions (closed bars) are reported. The regions are: A = HIR, B = 5SLR and C = CE). Values are means +/2 SE. doi:10.1371/journal.pone.0023156.g005 in comparison with the remaining 11,900 CE CGIs (mean = 0.74 vs. 0.76, Bootstrap p-value = 0.36). In summary, we demonstrated that the CGIs localized in regions showing signatures of selective pressures contain less SNPs than CGIs in other regions of the genome. When compared to CGIs in regions with one signature only, CGIs in regions with two signatures of selective pressure showed differences in the case of 5SLR but not for HIR. Discussion One of the most studied epigenetic modifications is the DNA methylation, which is evolutionarily ancient and associated with regulation of gene transcription [29]. DNA methylation could be central both to the ability of a population of organisms to change its phenotype in response to changes in the environment and to its ability to generate genetic diversity and evolve through natural selection [15]. The evolutionary conservation and divergence of epigenetic mechanisms in eukaryotes have started to be revealed by genetic and genomic studies of various organisms [29]. A general scenario that seems to emerge is that the epigenetic marks and the mechanisms that establish these marks are basically ancient and conserved, but the precise details of how these marks function within genomes is far to be completely clarified. An intriguing question is how evolutionary forces have adapted epigenetic mechanisms to the needs of the specific organism and, within a species, to the needs of a specific population. In this study we searched for possible differences in DNA methylation between genomic regions under selective pressure and the remaining genome. We focused on CpGs inside CpG islands and on the species Homo Sapiens. We chose a genome-wide approach using computational biology methods. One of the difficulties in this kind of study concerns the methods to be used to detect signatures left by natural selection. Despite the many methods that have been developed, up to now no method could be considered the ‘‘best one’’. Each method apparently provides distinct information about selective events [17]. To overcome this limit we decided to use three different approaches. The first one, the iHS score [23], is a population genetic approach. The general idea of this method is to search for haplotypes longer than expected, the so-called ‘‘long-range haplotype’’. An allele under selection increases in frequency so rapidly that long-range associations with neighboring polymorphisms are not disrupted by recombination. Generally this approach is thought to provide evidence for recent positive pressure [23], ‘‘recent’’ meaning after the human population separation. The second method defines as ‘‘under selective pressure’’ the regions of the human genome with a strong signal for depletion of Neanderthal-derived alleles. The presence of these signals may mark an episode of positive selection in early humans, after the separation from Neanderthal [25]. The third and last method belongs to the comparative approaches, involving data from multiple different species. Methods for detecting signatures of selection from rates and patterns of substitution have a long history in the field of molecular evolution [6]. The method that we used [26] is aimed to identify conserved elements in primates allowing to test hypotheses about selective pressures on this particular evolutionary lineage. We decided to use these three methods because they provide information about selective events happened in different evolutionary times. Independently of the method that we used, CGIs localized inside regions under selective pressure were less methylated than CGIs in other genomic regions. In addition, we found that CGIs in regions with two signatures of selective pressure (in which CE is involved) showed lower methylation when compared to CGIs in regions with one signature only. This finding suggests that each signature is providing distinct information about selective events. We observed CGIs undermethylation in all cell lines analyzed, including different types of normal cultured cells (fibroblasts, epithelial cells, myocytes etc.). It is well known that, in a multicellular organism, different cell types acquire various functional capabilities by distinct epigenetic modifications. Ac- quired during early development, the cell type-specific epigen- otype is maintained by cellular memory mechanisms. It is quite surprising that different cells showed similar methylation differ- ences. This finding may suggest that the regions under selective pressure are somehow more ‘‘protected’’ from methylation, independently of the cell type-specific epigenotype. This interpre- tation could be further supported by the analysis of EBV transformed and cancer derived cells. Epigenetics of cancer has been deeply studied, and the loss of DNA methylation at CpG dinucleotides was the first epigenetic abnormality to be identified in cancer cells [30]. The role of hypomethylation in activating oncogenes, as well as hypermethylation affects tumor-suppressor genes has been well established [30]. We found that genomic regions under selective pressure are relatively less methylated in cancer cells too. This difference persists even in a scenario of PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23156