Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 independently derived biological replicates to contain the same or overlapping methylation peaks. Other inves- tigators have indicated that MeDIP may miss differen- tially methylated regions in regions of low CpG density [45]. Thus we may underestimate the total number of T-DMRs and DS-DMRs to some extent. Nevertheless, our study identified almost 5,000 adult T-DMRs and 10,000 DS-DMRs that represent a total of over 15,000 T-DMRs in four adult tissues (brain, heart, liver, and testis; Tables 1 and 2). Since tissues are made up of many cell types and it is likely that there are cell type specific epigenetic differences, the methylation peaks we identified probably correspond to the major cell types in the tissue, while many methylation peaks corresponding to minor cell types would be missed. Considering that we only sampled 4 tissues and 3 developmental stages (E15, NB, AD), and that the analysis does not include methylation peaks in regions outside promoter and CpGi regions (except in tiling regions), it is quite likely that there are many additional T-DMRs and DS-DMRs that were not identified in this study. These results demonstrate that there are numerous DNA methylation differences between adult tissues (T-DMRs) and within the same tissue at different stages of development (DS- DMRs), indicating that alterations in DNA methylation are a major feature of development. Genomic locations of T-DMRs and DS-DMRs Overall most T-DMRs and DS-DMRs (about 70%) are located within non-CpGi promoter regions (Table 1 and 2), which is consistent with results of other studies [12,28,30,33,39]. However, we also found that about 30% of T-DMRs (1,440/4,686) and DS-DMRs (3,014/9,336) are located in CpGi regions (i.e. the sum of all CpGi- associated DMRs vs. the total number of DMRs; Table 1 and 2). This corresponds to more than 15% of the 15,979 CpGi regions on the NimbleGen array and is a relatively large number considering the limited number of tissues and developmental stages surveyed and other factors cited above. In fact, among the 15,979 CpGi regions annotated in UCSC mm8 freeze, a total of 5,523 (34.6%) were partially or entirely methylated in one or more of the 24 samples analyzed in this study (4 tissues each with duplicated samples for 3 developmental stages; data not shown). Thus, tissue- and developmen- tal stage-specific CpGi methylation may be a very com- mon event during development. Analysis of methylation in the few tiling regions avail- able on the NimbleGen array made it possible to deter- mine whether there was significant tissue specific methylation outside of promoter and CpG island regions. Our results indicate that as much as 30% of the T-DMRs are missed by restricting analysis to promoter and CpGi regions (Additional file 3b). Page 9 of 16 The location of methylation peaks differs according to tissue distribution. About 50% of the adult common methylation sites are within CpGi regions whereas only 30% of the T-DMRs are located within CpGi regions (Table 1). Similarly, about 48% of the methylation peaks that are common among all developmental stages within a tissue are located in CpGi regions (Table 2). The sig- nificance of this difference between common methyla- tion and differential methylation is not presently clear. Possibly this reflects a difference between transient changes in methylation that are tissue specific and more stable changes in methylation that are common to tissues. The distribution of the location of T-DMRs and DS- DMRs in testis is dramatically different from somatic tissues. Almost 94% of the adult testis T-DMRs are associated with non-CpGi promoter regions (Table 1 and 2). About 68% of the testis DS-DMRs are associated with non-CpGi promoter regions, which represents an average of E15, NB, and AD developmental stages. Unlike somatic tissues there appears to be a clear pat- tern shift in the distribution of testis DS-DMRs during development. In E15 testis only about 37% of the DS- DMRs are located in non-CpGi promoters whereas the percentage increases in NB (49%) and AD (86%). This may reflect post-natal onset of meiosis and other devel- opmental changes. This general bias of DMRs towards non-CpGi promoter vs. CpGi promoter is significant since the majority (10,915 or 68.5%) of 19,528 promo- ters (all RefSeqs promoters included in the array) are CpGi promoters (based on our criteria region of 1kb flanking each side of transcription start site). T-DMRs correspond to sites of methylation that vary between tissues, whereas DS-DMRs correspond to sites of methylation within a single tissue that differ accord- ing to developmental stage. We determined the extent of overlap between DS-DMRs from different tissues by combining all DS-DMRs for the same tissue and exclud- ing common methylation sites within a tissue (Table 3 and Additional file 5a and 5b). We reasoned that the degree of overlap of DS-DMRs among tissues would shed light on the level of tissue specificity of DNA methylation during development. We found a very low level of overlap among different tissues (1,927/9,784 or 13.2%), indicating that most DS-DMRs are tissue-unique and tissue-specific (Table 3). Even for DMRs that are shared among all three developmental stages in a tissue (Additional file 5b), almost 50% are tissue unique and almost all are tissue specific (95.2%). In addition, a large fraction of the tissue specific DS-DMRs were unique to a single tissue (7,857/9,784 or 80%). However, we would expect this percentage to decrease as more tissues are added to the analysis. These results suggest that almost all DNA methylation in non-repetitive regions is tissue