CpG Islands Methylation and Selective Pressure Figure 4. Venn diagram showing the overlaps among CGIs localized in the regions under selective pressure detected by the three methods used. doi:10.1371/journal.pone.0023156.g004 and 42.59) and intergenic CGIs showed intermediate methylation values (weighted mean = 21.31). For each cell line, the differences among CGIs methylation of different genomic regions were high statistical significant (Kruskal-Wallis Test, p-value#2.2 10216) (Table S10). Next we divided CGIs with signature of selective pressure according the above described classes. Unfortunately, for intra- genic and 39 classes, we did not obtain a number of HIR CGIs and 5LSR CGIs sufficient to perform a consistent statistical analysis. In particular, in these classes we found about 80 HIR CGIs and less than 10 5LSR CGIs. We were able to perform statistical analysis only by using CE as signature of selective pressure. In all cell lines but 2 (which were both cancer cell lines), 59 CGIs in CE regions were under- methylated when compared to 59 CGIs located outside CE regions (Bootstrap p-value,1024). Intragenic and 39 CGIs located in CE regions showed no differences in methylation when compared to intragenic and 39 CGIs outside CE regions. In all cell lines, intergenic CGIs in CE regions were severely undermethylated when compared to intergenic CGIs located outside CE regions (Bootstrap p-value p,1024) (Table S11). This first set of experiments suggested that, in different cell lines, the GCIs localized in genomic regions under selective pressure are undermethylated. 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. Further- more, at least for CE, the CGIs undermethylation that we found in genomic regions under selective pressure is specifically due by CGIs located at the 59 and in the intergenic regions. Genetic variation inside CpG islands and signatures of selective pressure We decided to estimate the CGIs methylation by a different, indirect approach. It is well settled that 5-methylcytosine is the initial molecule in the deamination reaction that generates thymine; thus, methylation may be required for increased mutation rates at CpG sequences. We predicted that CGIs localized in regions under selective pressure, being less methylated, would be less likely to mutate. Under this hypothesis, these CGIs should show a lower degree of genetic variation among individuals. To evaluate the degree of genetic variation in CGIs, we calculated the frequency of SNPs present in each CGI. Among the 26,033,053 SNPs from dbSNP (build 131), we selected the 199,514 SNPs that were located inside CGIs. To obtain a normalized value of SNP frequency for each CGI, we divided the number of SNPs present in each CGI by its size. By this method we were able to calculate the SNP frequencies for 25,558 CGIs. We found that, on average, each CGI contained 1.04 SNP/ 100 bp (range 0.04–63.28). Then we compared the SNP frequency of CGIs inside the regions under selective pressure with the SNP frequency of CGIs localized in the other regions of the genome. Figure 5 reports the results obtained. The 2,345 CGIs localized in HIRs showed a mean of 0.89 SNP/100 bp in comparison with 1.05 of the other 23,213 CGIs (Bootstrap p-value,1024). The 309 CGIs localized in 5LSRs showed a mean of 0.67 SNP/100 bp in comparison with 1.04 of the other 25,249 CGIs (Bootstrap p- value,1024). The 13,286 CE CGIs showed a mean of 0.76 SNP/ 100 bp in comparison with 1.34 of the other 12,272 CGIs (Bootstrap p-value,1024). Also for this approach we checked if CGIs in regions with two signatures of selective pressure (HIR+CE or 5SLS+CE) showed differences compared to CGIs in regions showing only a signature (CE). We found that the 205 5SLR+CE CGIs contained less SNPs than the remaining 13,081 CE CGIs (mean = 0.61 SNP/100 bp vs. 0.76 SNP/100 bp, Bootstrap p-value,1024). On contrary, the 1,386 HIR-CE CGIs did not show any difference in SNP content PLoS ONE | www.plosone.org 5 August 2011 | Volume 6 | Issue 8 | e23156