Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 sample to sample (Additional file 4). Therefore the total number of methylation peaks would be somewhat underes- timated in samples with low relative to those with high Pearson Coefficients. We adjusted the total number of methylation peaks as if the Pearson Coefficients for all stages were the same as the best one in that tissue (see data labeled “Adjusted” in Table 2). Although the total number of peaks increases for the samples with lower Coefficients, the overall trends are comparable (Table 2 and Figure 2), indicating that our underestimation of methylation peaks does not have a significant impact on the distribution of methylation peaks into different genomic regions. There were considerably more methylation sites that were common within a tissue at the different develop- mental stages than between different adult tissues (460 common sites, Table 1). Brain, heart, and liver each had 1,903, 1,844 and 1,497 common methylation sites, respectively, whereas testis had 570 (Table 2). Similar to the common sites for the adult tissues, about 50% of developmental stage common sites are in CpGi regions (CpGi promoter + intragenic and intergenic CpGi) and about 50% are in non-CpGi promoter regions (Table 1 and 2). Somewhat surprising is the relatively high num- ber of sites in liver (and brain and testis, to some degree) that are methylated in E15 + NB but unmethy- lated in adult (1,419). Together, in somatic tissues (heart, brain, and liver), there were almost 5,000 sites of methylation that exhibit stage specific differences. Testis alone had 4,348 sites of stage specific methylated regions, most of which were methylated in adult and localized to non-CpGi promoters. Again, as for the Total number Adjusted total number 3000 2500 sk 2000 a e p fo 1500 r e b uNm 1000 500 Heart Brain Liver Testis Heart Brain Liver Unmethylated Methylated Testis Figure 2 DS-DMRs: Number of peaks that are Unmethylated or Methylated in adult Tissues. Total numbers of unmethylated peaks and methylated peaks are shown in adult heart, brain, liver and testis. The grey bar indicates the actual total number of unmethylated and methylated peaks in each tissue. The black bar indicates total number of unmethylated and methylated peaks in each tissue after adjustment for differences in the Pearson Coefficient. Page 6 of 16 adult tissues, the most frequent location for differential methylation was within non-CpGi promoter regions (54.1% to 71.4%), followed by CpGi promoter regions (13.9 to 22.4%), intragenic CpGi regions (9.3 to 15.6%), and intergenic CpGi regions (5.4 to 8.4%). In contrast to other tissues, the location of testis stage specific DS- DMRs appear to shift from a high proportion of CpGi location at early developmental stages (E15, 62.9% and NB, 51.0%) to a high proportion of non-CpGi promoter location in adult (86.2%). Many DS-DMRs are unmethylated in adult We grouped the DS-DMRs within a tissue according to whether they were unmethylated in adult (UnMe-AD) but methylated at earlier developmental stages (E15, E15 +NB, NB). These would represent sites of demethylation during development. Conversely sites that were methy- lated in adult (E15+AD, NB+AD, AD) but unmethylated at earlier developmental stages would be sites that were methylated during development. Surprisingly, there were more sites that became unmethylated (5,311) than became methylated (4,025) during development (Figure 2 and Table 2). Adjustment for differences in the Pearson Coefficient (Figure 2) does not significantly alter this gen- eral conclusion. Among the different tissues, there seem to be some significant differences with regard to the ratio of DMRs subjected to methylation and demethylation. In testis, there are an almost equal number of sites that become unmethylated (2,009) or methylated (2,339) in adult. However, in brain and liver there are a much larger number of sites undergoing demethylation in adult than that of methylation whereas in heart, there are a smaller number of sites undergoing demethylation than methyla- tion. These results indicate dynamic changes in DNA methylation during development and that epigenetic changes resulting in unmethylated DS-DMRs are a major feature of differentiation into adult tissues. Overlap between DS-DMRs in different tissues The DS-DMRs indicate differential stage-specific sites of methylation within a tissue without regard to whether it is tissue specific. To determine how many of the DS-DMRs are tissue specific, we determined the extent of overlapping DS-DMR (i.e., methylation) between tissues (Table 3). The results indicate for all four tissues together, the majority (7,857 or 80.3%) of the 9,784 DS-DMRs are unique to a single tissue whereas the remaining 1,927 DS-DMRs are present in two or more but not all tissues, and thus are also tissue-specific. Together, it indicates that all of the 9,784 DS-DMRs are also T-DMRs. If testis is excluded, there are 4,232 out of 5,519 DS-DMRs that are unique to a single tissue and 613 peaks being present from one or more but not all tissues (Additional file 5a). In this case, we did find a small number of peaks (21) that are common to