Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 specific. We have previously shown, using RLGS, that methylation of some genomic regions containing repeti- tive sequences is also tissue-specific [32]. Recent studies [22,25] identified 16,379 T-DMRs in four human adult tissues (brain, liver, spleen, and colon) using a method termed “Comprehensive High-throughput Arrays for Relative Methylation” or CHARM. Although the methods, species and tissues used for this analysis were different from those presented here, the total number of T-DMRs is surprisingly similar (15,271 vs. 16,379), emphasizing extensive tissue specific DNA differences in DNA methylation. However, the CHARM analysis of human tissues found that 76% of the T-DMRs were located within 2 kb of CpGi regions that were denoted CpGi shores. The array design used in our study limits analysis to promoter, CpGi regions, and very limited tiled genomic regions and would appear to exclude most CpGi shores. However, we reason that many CpGi shores may be within the promoter regions in our studies. For both the promoter and tiling regions, we observed a slightly higher density of DS-DMRs in CpGi shores than in CpGi regions (data not shown). Since at the genome scale, the CpGi shore region is much larger (~6X) than the CpGi region, we can expect to have a larger number of DS- DMRs in the CpGi shores than in the CpGi, supporting the conclusion of Irizarry et al [25] in principle that there are more T-DMRs in CpGi shores. Gene ontology of T-DMRs and DS-DMRs Gene ontology (GO) analysis was performed to identify any common theme among the identified T-DMRs. In this analysis, we divided all DMRs associated with genes into three groups (non-CpGi promoter, CpGi promoter, and intragenic CpGi), since as noted by others [37,38], methylation in different gene locations may impact the gene expression regulation differently. We found more or less similar enrichment categories for different tissues, especially among somatic tissues. For both T-DMRs and DS-DMRs, the most consistent GO enrichment pattern is seen among those in non-CpGi promoters (Additional file 11 and Additional file 12) with enrichment for “membrane proteins, G-proteins, olfactory proteins” among UnMe-AD (DS-DMRs) and “extracellular space/region” for both T-DMRs and DS- DMRS (Me-AD). Apparently, these enrichment patterns reflect a pattern that is observed for all genes with non- CpGi promoters (data not shown). Additional GO enrichment was observed for somatic DS-DMRs in intragenic CpGi regions on “regulation of biological and cellular processes, ion-binding and transport”. This data supports the notion that intra-genic CpGi methylation tends to promote the expression of the involved gene [46,47], and may participate in gene regulation during differentiation and development. Page 10 of 16 “Demethylation” of previously methylated sites (DS- DMRs) is a common feature of tissue differentiation Quite surprisingly, our studies indicate that many DS- DMRs that are methylated at early stages of development (E15 and NB) are unmethylated in adult tissues (Table 2 and Figure 2). This is particularly evident in brain and liver where there are almost 3 times as many DS-DMRs that become “unmethylated” in adult as become “methy- lated” in adult. This is somewhat contrary to expectations that differentiation into adult tissues would reflect pro- moter methylation and silencing of genes not associated with the final gene expression pattern. In contrast, it sug- gests that the final gene expression pattern depends on extensive demethylation events during differentiation. Although methylation of gene promoter regions is asso- ciated with gene repression, gene body methylation is associated with gene expression [37,38,48]. In addition, recent reports indicate that there are extensive allelic dif- ferences in gene expression in human that result from allelic differences in methylation, due to imprinting or DNA sequence variation [49,50]. Thus, demethylation of previously methylated sites could reflect either increased or decreased gene expression, depending on site location. Our observations that demethylation is a common fea- ture of tissue differentiation are consistent with the recent report that liver development in human is charac- terized predominantly by demethylation [20]. Methylation of ES cells ES cells are pluripotent and are derived from the blasto- meres of the early embryo that are thought to be exten- sively demethylated due to active and passive demethylation that follows fertilization. Therefore, we decided to compare the methylation peaks from ES cells with those found in E15 embryonic tissues (brain, heart, liver, and testis) to determine the extent of methylation differences during early stages of tissue differentiation (Additional file 1). We found that the number of E15 T- DMRs (1,769) is less than half of that found in adult (4,686), which is consistent with E15 tissues being gener- ally less methylated than adult. ES cells also had a low level of methylation with 981 T-DMRs. However, some- what surprisingly, ES cells had more methylation peaks (981, Additional file 1) than any of the E15 tissues. This would suggest that many genomic sites that are methy- lated in ES cells become demethylated during early devel- opment. We previously found that many (60%) of the adult T-DMRs identified by RLGS were methylated in ES cells, suggesting these were demethylated during tissue dif- ferentiation [32]. It is also possible that some or many of the sites that are methylated in ES cells are due to growth in tissue culture. It has previously been shown that tumor cells grown in tissue culture accumulate excessive aberrant methylation that is unrelated to tumorigenesis [40]. Also,