Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 DNA methyltransferase, is lethal during development [14]. Dnmt3a and Dnmt3b are required for de novo methylation during development [15]. The persistent expression of both Dnmt 3a and Dnmt 3b after gastrulation and implan- tation suggests that de novo methylation occurs during later stages of development. Restriction Landmark Genomic Scanning (RLGS) detected differences in DNA methylation between tissues more than 15 years ago [16-19]. These studies, as well as more recent reports using a variety of methods, have markedly altered our concept of differentially methylated regions (DMRs) in the mammalian genome and the pro- gression of DNA methylation changes during development [19-36]. Tissue Specific Differentially Methylated Regions (T-DMRs) include both CpGi and non-CpGi promoter regions as well as intragenic and intergenic regions [19,32,37,38]. Analysis of T-DMRs using a CpGi array indicated that T-DMRs in CpGi regions are associated with developmental gene loci and many are located in non-promoter regions [23]. CpGi amplification in con- junction with microarrays, indicated approximately 4% of dense CpGi promoters are methylated in normal periph- eral blood [31]. A number of studies have consistently observed that weak or non-CpGi promoters are major tar- gets for tissue specific DNA methylation [12,28,30,33,39]. Analysis of DNA methylation changes during develop- ment in vitro and in vivo are more limited but indicate that DNA methylation is a dynamic process involving both methylation and demethylation [20,26,32]. In vivo analysis of methylation changes within a tissue at differ- ent developmental stages is complicated by changes in cell populations [32]. In vitro analysis of DNA methyla- tion during differentiation of ES cells or progenitor cells is complicated by potential aberrant methylation due to growth of cells in tissue culture [20,26,40]. Our previous results identified a limited number of T-DMRs using RLGS, suggesting that methylation changes during development are dynamic and involve both methylation and demethylation [19,32]. The results reported here investigate this with a more comprehen- sive set of T-DMRs and Developmental Stage specific Differentially Methylated DNA Regions (DS-DMRs) identified through Methylated DNA ImmunoPrecipita- tion (MeDIP) [20,32]. Results MeDIP methylation analysis The MeDIP methylation analysis developed by Weber et al [33] was utilized to identify genomic sites of differential DNA methylation in selected 12 wk adult C57BL/6J mouse tissues (T-DMRs) and in tissues at different devel- opmental stages (DS-DMRs: 15 day embryo, E15; new born, NB; and 12 wk adult, AD). Methylated DNA was immunoprecipitated with antibody to 5-methyl-cytidine, Page 2 of 16 the input and immunoprecipitated (IP) DNA were differ- entially labeled and hybridized to a NimbleGen promoter plus CpGi array according to a protocol provided by Nim- bleGen. The NimbleGen Array covers all UCSC-annotated CpGi (15,979) as well as promoters for all RefSeq genes (19,530). Repetitive regions are excluded from the Array. Non-CpGi intragenic and intergenic regions are not cov- ered by the array except in some limited regions in which the array is essentially a tiling array (1.9 Mb on portions of chromosomes 6, 7, and 17). Assessment of the MeDIP data quality and validation of MeDIP methylation peaks A number of methods were used to validate the quality of the MeDIP methylation data. These include scatter plots of log 2 ratios between IP DNA and input DNA, analysis of Pearson Coefficients between biological replicate sam- ples, comparison of MeDIP methylation with previously identified RLGS T-DMRs, MeDIP analysis of other known methylation sites, such as imprinted genes, and analysis of both selected and randomly chosen MeDIP methylated regions by Sequenom MassARRAY quantitative methyla- tion analysis of bisulfite treated DNA. An example of a selected region analyzed by Sequenom MassARRAY is shown in Figure 1. The results indicate a close similarity between MeDIP and Sequenom MassARRAY methylation analysis. Additional details of Sequenom MassARRAY methylation analysis and other validation methods are pre- sented in the Methods section and Additional files. Identification of genomic sites of DNA methylation in adult tissues (brain, heart, liver, and testis) The IP and input DNA from 12 wk adult C57BL/6J mouse tissues (brain, heart, liver, and testis) were hybridized to a NimbleGen Promoter plus CpGi array. T-DMRs are geno- mic regions that are methylated in one or more of the four adult tissues, but unmethylated in at least one tissue. For the identification of T-DMRs, the log2 ratios of all probes for each sample were normalized and used to identify methylation peaks as described in the methods. To facili- tate the analysis of MeDIP data, an ad hoc bioinformatics tool was developed to process the methylation peak lists for individual samples and identify genomic regions that are 1) commonly methylated in all tissues; 2) methylated in a single tissue; and 3) methylated in some but not all tissues. Only those methylation peak regions that were present in both biological replicate samples and were absent from at least one other tissue were designated as T-DMRs. Thus, the frequency of T-DMRs may be under- estimated. T-DMR locations were designated as 1) CpGi Promoter; 2) Intragenic CpGi; 3) Intergenic CpGi; and 4) Non-CpGi promoter (see details in Methods). Summaries of the number and distributions of the adult T-DMRs are shown in Table 1. Overall, there are