Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 growth of mouse neural progenitor cells in culture after extended passages results in aberrant methylation [26] and investigators found extensive differences in the genomic methylation patterns of independently isolated human ES cell lines [20]. At the present time, it is unclear whether sites of methylation in ES cells is aberrant due to extended growth in culture or whether demethylation during early differentiation in ES cells is an important process as in later stages of tissue differentiation noted above. Methylation analysis of stem cells revealed extensive Cytosine methylation in a non-CpG context [35,48,51] that was mainly located within gene bodies [35]. The non-CpG methylation appears to be mostly confined to stem cells and disappears upon differentiation. Our ana- lysis of mouse ES cells indicated a very low level of intragenic CpGi methylation. This suggests that most of the non-CpG gene body methylation was in non-CpGi intragenic regions that were not on the NimbleGen array or that our analysis did not resolve non-CpG methylation. Non-Promoter and non-CpGi methylation in the Hoxa gene cluster A 100 Kb region on chromosome 6 that includes the Hoxa gene cluster is essentially a tiling array on the Nim- bleGen promoter plus CpGi array. This region includes 16 RefSeq genes and 22 UCSC-annotated CpGi regions (Additional file 6). Somewhat surprisingly all 8 methyla- tion peaks were in non-CpGi regions and only one was in a promoter region (Additional file 6 Additional file 7). In addition, developmental analysis of Hoxa gene methyla- tion indicated stage-specific methylation differences (Figure 3). These differentially methylated regions include 3’ exons and intron regions. Since these regions are highly conserved and Hox genes are known to have important roles in development [41,42], these results sug- gest that methylation in non-promoter, non-CpGi regions may have novel, currently undefined roles in reg- ulating development. A recent report also indicates dif- ferential development-associated methylation within Hox gene clusters in human [48]. DNA methylation and alternative promoter use Interrogation of the MeDIP/NimbleGen array data sug- gests that DNA methylation may be associated with alter- native promoter use. Analysis of methylation within the Protocadherin gene clusters on chromosome 18 indicates extensive methylation within CpGi promoter and non- CpGi promoter regions, particularly within somatic tis- sues (Additional file 8 Additional file 9). Some differential tissue specific methylation in this region is also noted. Pcdha mRNAs are synthesized by the activation of one of the alternative promoters on only one of the two chro- mosomes resulting in monoallelic expression [52]. Page 11 of 16 Although the mechanistic basis for this selection is unknown, it is hypothesized that it provides a foundation for neuron adhesive diversity that is required for complex synaptic interactions [52]. Recent genome-wide methyla- tion analysis using CHARM [25] also indicates an asso- ciation between tissue specific DNA methylation and alternative transcripts. That study indicated that most tis- sue specific differentially methylated regions were located in CpGi shores and that 68% of the shores were within 500 bp of alternative promoter sites. The impact of T-DMRs and DS-DMRs on gene expression To understand the biological function of T-DMRs and DS-DMRs, one obvious approach is to examine their impact on gene expression. Efforts to address this ques- tion at a genome scale is complicated by several factors that can obscure correlations. First, one gene may be subjected to DNA methylation in multiple regions with the T-DMR or DS-DMR in question being just one of those. Therefore, the level of gene expression may depend on methylation status in other regions of the gene. Second, existence of multiple splicing isoforms, particularly those associated with alter- native promoters, as well as the use of multiple expres- sion probes for the same gene makes this one-gene vs. one DMR association analysis very challenging. Last but not least, DNA methylation is not the only factor affect- ing the gene expression. In other words, the lack of DNA methylation in one of the promoter region does not necessarily confer gene activation, as the expression can be limited by other factors, for instance the lack of the required transcriptional factor(s). These complications may be responsible for the highly diverse situations we observed between the occurrence of T-DMRs/DS-DMRs and the expression level of their associated genes on a gene-by-gene basis. Despite these complications, our pre- liminary analysis does seem to reveal a few novel insights. First, it appears that T-DMRs are associated with lower gene expression in non-CpGi promoter regions. Second, there may be some differences between T-DMRs and DS-DMRs from earlier developmental stages in the man- ner they impact gene expression. Certainly, extensive data analysis using more data sets representing more tis- sues, as well as experimental validation is needed to con- firm these observations. Conclusions Overall, our results indicate that there are extensive dif- ferences in DNA methylation between adult tissues and within tissues during development. These results indi- cate that differential DNA methylation is a dynamic pro- cess involving both methylation and demethylation events. The prominence of demethylation in adult tis- sues of genomic regions that are methylated at earlier