Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 Table 3 Overlap among DS-DMRs from different tissues Tissue all DS-DMRs Tissue-Unique Multiple tissues# T-DMR% Brain 1973 Heart 867 Liver 3570 Testis 5469 Total 11879*/9784** 1065 533 2160 4099 7857 908 334 1410 1370 4022*/1927** 100 100 100 100 #: peak present in 2 or more but not all tissues; *: contains redundancy due to overlap; The numbers are larger than those in Table 2 due to certain peak splitting; **: a non-redundant (unique) list of all sites from the four tissues. all three tissues, suggesting that the 3 somatic tissues share more commonality among each other than with testis. We also examined the overlap for the 5,761 DMRs that are common to all stages in a tissue (Additional file 5b). The majority of these DMRs are also tissue-specific (5,487/ 5,761 or 95.2%). However, compared to DS-DMRs, the per- centage of these DMRs being tissue-unique is much lower, approximately 15% for each of the somatic tissues and only 1.9% for testis. Only a small proportion (274 or 4.8%) are common to all developmental stages and all tissues. Over- all, since all of the 9,784 DS-DMRs and 5,487 of the 5,761 DMRs common to all developmental stages in a tissue are also T-DMRs, we identified a total of 15,271 T-DMRs. Tissue-Specific differences in DNA methylation within the Hoxa gene cluster Homeobox genes have important roles in regulating devel- opment [41,42]. One of the tiled regions available on the NimbleGen array includes the Hoxa gene cluster on chro- mosome 6. Additional file 6 shows the scaled log2 ratios from the NimbleGen SignalMap view of an approximately 100 Kb region that includes the Hoxa gene cluster. There are 8 adult T-DMRs in this region that were present in both biological replicate DNA samples of the involved tis- sue(s). Even though this region is CpGi rich, none of the peaks were within CpGi regions and only one was loca- lized in a promoter region (Additional file 7; peak 7, Hoxa7). The other methylation peaks were within introns (peaks 2, 3, and 4) or associated with the 3’ end (exon) of the gene (peaks 1, 5, 6, and 8). Four of the peaks (peaks 2- 5) were testis specific, one (peak 6) was heart specific, one (peak 7) was present in heart and liver, and one (peak 8) was in heart and brain. All sequences corresponding to these peaks were highly conserved, suggesting a regulatory function. Analysis of methylation differences within the Hoxa gene cluster during development (Figure 3) indicates that two additional DS-DMRs not seen as T-DMRs for adult tissues were observed when samples from all three developmental stages were examined (Additional file 6; Additional file 7). One of these is specific to NB liver (peak N1) and the other is a large region (peak N2) methy- lated in E15 and NB of liver and testis and in AD heart, and demethylated in adult liver and testis. Overall, our Page 7 of 16 results indicate extensive tissue and developmental stage specific methylation in the Hoxa gene cluster that is not associated with promoter or CpGi regions. DNA methylation within the Protocadherin gene cluster Protocadherins are a large gene family involved in cell adhesion and signaling, particularly during neural devel- opment. There are three large clusters totaling more than 50 genes (alpha, beta and gamma) that span about 1 MB on Chromosome 18 in the mouse. Additional file 8 shows the MeDIP methylation profile from a portion of the Pcdha and Pchdhg gene clusters. Many of the alternative promoters are associated with CpG islands. Most of the alternative promoters are completely unmethylated in the adult testis, but some are differen- tially methylated in the liver (Pcdhga3 and Pcdhgb2, peaks 8 and 11; Additional file 8 and Additional file 9). Note that the promoter for Pcdhga6 (peak 13) is methy- lated in the testis but unmethylated in liver, brain, and heart. These results indicate that alternative promoters within the Protocadherin gene cluster are targets for DNA methylation. Relationship between T-DMRs/DS-DMRs and gene expression To test whether the T-DMRs and DS-DMRs have any relationship with and the expression of their associated genes, we identified (from NCBI GEO database) and analyzed two published expression data sets, which have the best match to the samples we used in this study with regard to animal strain, tissue type and develop- mental stage. For T-DMRs, we used GSE9954 [43] designed for the analysis of 22 different tissues from adult C57BL/6 mice, and we performed a small-scale manual examination, as well as computational analysis for all T-DMRs. Manual examination of individual genes revealed a variety of situa- tions, with no obvious consistent pattern, likely due to rea- sons discussed later. Nevertheless, the data from the analysis of all T-DMRs seems to reveal two general trends: 1) Genes containing T-DMRs in non-CpGi promoters tend to have lower expression in the tissue to which the T-DMRs are unique than in other three tissues (Addi- tional file 10 panel A-C); 2) Within the same tissue, genes containing T-DMRs in non-CpGi promoters have lower expression than those containing T-DMRs in either in CpGi promoters or in intragenic CpGi and those contain- ing no T-DMRs (Additional file 10 panel D). The first trend is well shown in brain and liver, but less consistent in heart and brain, while the second trend is consistently observed in all four tissues. T-DMRs in CpGi promoters do not seem to associate with a significantly lower expres- sion except for those from liver, while T-DMRs in intra- genic CpGi do not seem to have a significant impact on