these two groups, indicating that the results obtained by the two methods are driven by different sets of CGIs. In addition, excluding these 70 CGIs from the analysis, the result continued to be highly significant both for HIRs and 5LSRs (data not shown). It is intriguing to note that these 70 CGIs were less methylated when compared both to the remaining HIR CGIs and 5SLR CGIs, but the differences were not statistical significant (data not shown). To further define regions under selective pressure, we decided to use a third and last approach that looks for sequences that are conserved across species [26]. By this approach, conserved regions are defined as genomic regions with a reduced rate of evolution compared to what is expected under neutral drift. Several methods for detecting conserved regions in multiple alignments have been described. We used data downloaded from UCSC Genome Browser Conservation (cons46way) Track, which lists 725,627 Conserved Elements (CEs) that were predicted to be conserved among primates [27]. We Identified 26,936 CEs located inside 14,391 CGIs, by filtering all genomic CEs by CGIs. Excluding CGIs corresponding to sequences for alternative haplotypes, we obtained 13.288 unique CGIs containing 25.362 CEs. We named ‘‘CE CpG islands (CE CGIs)’’ those CGIs that contain at least one conserved element. For each cell line, we compared the methylation of CE CGIs with the methylation level of the remaining CGIs not containing conserved elements. In all the cell lines analyzed, CE CGIs were less methylated than CGIs that do not contain conserved elements (Figure 3). The differences were highly statistical significant (Bootstrap p-val- ue,1024) in all cell lines analyzed. Table S7 reports in detail the results of this analysis. Since the number of CE CGIs is higher than that of HIR CGIS and 5SLR CGIs, it could be possible that all HIR CGIs and 5SLR CGIs are contained in the CE CGI group. In this case the results we found with HIR and 5SLR could be due to CE only. To check this possibility, we estimated the overlaps between the CGIs lists obtained by the different methods (Figure 4). We found CpG Islands Methylation and Selective Pressure that 1,385 CGIs were in common between CE and HIR (HIR+CE CGIs) and 205 were in common between CE and 5SLR (5SLR+CE CGIs). If the phenomena underlying the three signatures (CE, HIR and 5SLR) contributed independently to lower the CGIs methylation, we expected that CGIs in regions with two signatures of selective pressure showed lower methylation when compared to CGIs in regions with one signature only. We found that, in all cell lines analyzed, HIR+CE CGIs were less methylated than the remaining CE CGIs. The differences were highly statistical significant (Bootstrap p-value,1023) in 14 cell lines analyzed, but did not reach this significance in 11. In these eleven cell lines the differences were significant only at p,0.05 (Figure S2, Table S8). Also 5SLR+CE CGIs were less methylated when compared to the remaining CE CGIs, in all cell lines analyzed. The differences were highly statistical significant (Bootstrap p-value,1023) in 17 cell lines, but did not reach this significance in 8. In these eight cell lines the differences were significant only at p,0.05 (Figure S3, Table S9). Also in these two cases the joint analysis of all cell lines yielded a combined statistical significance much less than 1023. In the genome, CGIs are located in 59,39 or in other gene regions, as well as in intergenic regions. We decided to estimate the methylation of CGIs located in these different locations to assess if the CGIs undermethylation that we found in regions under selective pressure is restricted to CGIs with a specific localization. We used the 4 classes of CpG islands described by Medvedeva et al. [28] : 59 CGIs (in 59-flank region, 59 UTR-exon , 59UTR- intron , initial coding exon and initial intron), intragenic CGIs (in internal exons and internal introns), 39 CGIs (in final exons, final introns, 39 UTR exons and 39 UTR introns) and intergenic CGIs (located at least 3 kb from any known gene upstream and downstream). In particular, 59 CGIs are located in regions that, starting 3 kb upstream Transcription Start Site, extend till the first intron. Considering all cell lines, 59 CGIs showed the lowest methylation level (weighted mean = 9.01) , intragenic and 39 CGIs showed the highest values (respectively, weighted mean = 55.21 Figure 3. Methylation of CE CGIs compared to methylation of CGIs that do not contain conserved elements. For each cell line, the mean methylation value of CE CGIs (open bars) and of the CGIs that do not contain conserved elements (closed bars) are reported. Inset shows the same data summarized by cell group (Cancer Transformed = CT, EBV transformed = EBV, Normal Untransformed = NU). Values are means +/2 SE. doi:10.1371/journal.pone.0023156.g003 PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23156