Liang et al. BMC Genomics 2011, 12:231 http://www.biomedcentral.com/1471-2164/12/231 boxplots (panel A, B and C, respectively). Comparison was also made for T-DMRs associated genes across different location groups within liver (Panel D). The p values of pairwise t-tests between liver and each of the other tissue (panels A-C) and between each pair of location groups (panel C) are provided on the right side of the boxplots with those no great than 0.05 shown in bold font. “NDMR” in panel C refers to genes associated with all genes not associated with liver unique T-DMRs. Additional file 11: Gene ontology for T-DMRs in adult. Additional file 12: Gene ontology for DS-DMRs subjected to methylation or demethylation in adult. Additional file 13: A list of Sequenom MassARRAY primers used for Validation. Additional file 14: Scatter plots of log2 ratios of two replicates for brain tissues at E15, NB, and AD stages. Log2 ratio of all probes for the two replicate samples of brain at all stages were plotted. In each plot, data points in red rectangular area are those showing log2 ratio ≥ 1 (i.e. ratio ≥2) in both samples. Pearson Coefficient and the common methylation peak number are provided on the top of each plot. Additional file 15: MeDIP/NimbleGen Promoter + CpGi Array: Methylation analysis of RLGS T-DMR loci. The MeDIP methylation profile including scaled log2 ratio and methylation peaks of four RLGS T- DMR loci are shown (Pst4, Pst10, Pvu4 and Pvu5). The loci were identified as T-DMRs by RLGS [32]. The CpG island, genomic location, relevant gene and the direction of each transcript are indicated. The log2 ratio is the ratio of signals for the input and immunoprecipitated DNA test samples that were co-hybridized to the array. RLGS T-DMRs were also confirmed by Sequenom MassARRAY ([32], and data not shown). Additional file 16: Comparison of methylation data between MeDIP/ NimbleGen Promoter + CpGi Array and Sequenom MassARRAY: Non CpGi promoter region (randomly selected). Data similar to that presented in Figure 1 for a randomly selected locus, the non-CpGi promoter region of Gm1070 that is methylated in liver and ES, but not in testis. Additional file 17: MeDIP/NimbleGen Promoter + CpGi Array (Tiling region): Methylation analysis of Igf2r imprint control region. The MeDIP methylation profile of a region on chromosome 17 that includes Igf2r and BC009123 is shown. The numbers on the top indicate the genomic position. The CpGi in the rectangle is located close to the promoter region of BC009123 and the second intron of Igf2r. Methylation within the CpGi is associated with repression of transcription of Airn antisense RNA located somewhat downstream of TSS for BC009123. The imprinting control region (rectangle) is monoallelically methylated in somatic tissue and unmethylated in testis [57]. Acknowledgements This research was in part supported by the National Cancer Institute Grant CA102423 (WAH) and the National Cancer Institute Core Center Grant CA16056 (Roswell Park Cancer Institute), National Institute of Environmental Health Science Grant ES012249 (HN), Academic frontier project for 2006 from MEXT (HN) and grants from the Canada Research Chair program, Canadian Foundation of Innovation, Ontario Ministry of Research and Brock University (PL). We thank Arlene Morrow for the preparation of some of the DNAs used in these studies, and Michael Higgins and Dominic Smiraglia for suggestions and critical reading of the manuscript. Author details 1Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada. 2Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY, USA. 3Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY, USA. 4Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA. 5Department of Zoology, North-Eastern Hill University, Umshing Mawkynroh, Shillong, Meghalaya, India. 6Department of Bioinformatics, University of British Columbia, Vancouver, British Columbia, Canada. 7Department of Biochemistry, State University of New York at Buffalo, Buffalo, NY, USA. 8Division of Cancer Genetics, Department of Advanced Medical Science, Page 15 of 16 Nihon University School of Medicine, Tokyo, Japan. 9Division of Cancer Genetics, Chiba Cancer Center, Research Institute, Chiba, Japan. Authors’ contributions PL: performed most of the bioinformatics data analysis and interpretation and contributed in writing the manuscript; FS: performed most of the molecular analysis including preparation for MeDIP and MassARRAY analysis and contributed in writing, SG: performed validation experiments, EM: performed data analysis; MQ: developed the computational tools for identifying methylation peaks shared by or unique to different sample groups, SM: performed ES cell and DNA preparation and validation experiments, KF and IJ: performed tissue preparation, HN: performed supervision of most of molecular biological analysis and contributed in writing the manuscript, WH: performed most of writing and supervision of both bioinformatics and molecular analysis. All authors read and approved the final manuscript. Received: 25 June 2010 Accepted: 11 May 2011 Published: 11 May 2011 References 1. Bird AP: DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res 1980, 8:1499-1504. 2. Antequera F, Bird A: Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci USA 1993, 90:11995-11999. 3. Yoder JA, Walsh CP, Bestor TH: Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 1997, 13:335-340. 4. Deng J, Shoemaker R, Xie B, Gore A, LeProust EM, Antosiewicz-Bourget J, Egli D, Maherali N, Park IH, Yu J, Daley GQ, Eggan K, Hochedlinger K, Thomson J, Wang W, Gao Y, Zhang K: Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol 2009, 27:353-360. 5. Kafri T, Ariel M, Brandeis M, Shemer R, Urven L, McCarrey J, Cedar H, Razin A: Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev 1992, 6:705-714. 6. Monk M, Boubelik M, Lehnert S: Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 1987, 99:371-382. 7. Reik W, Dean W, Walter J: Epigenetic reprogramming in mammalian development. Science 2001, 293:1089-1093. 8. Ehrlich M: Expression of various genes is controlled by DNA methylation during mammalian development. J Cell Biochem 2003, 88:899-910. 9. Futscher BW, Oshiro MM, Wozniak RJ, Holtan N, Hanigan CL, Duan H, Domann FE: Role for DNA methylation in the control of cell type specific maspin expression. Nat Genet 2002, 31:175-179. 10. Riggs AD: X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet 1975, 14:9-25. 11. Walsh CP, Chaillet JR, Bestor TH: Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat Genet 1998, 20:116-117. 12. Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D: Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet 2007, 39:457-466. 13. Holliday R, Pugh JE: DNA modification mechanisms and gene activity during development. Science 1975, 187:226-232. 14. Li E, Bestor TH, Jaenisch R: Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 1992, 69:915-926. 15. Okano M, Bell DW, Haber DA, Li E: DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 1999, 99:247-257. 16. Hatada I, Hayashizaki Y, Hirotsune S, Komatsubara H, Mukai T: A genomic scanning method for higher organisms using restriction sites as landmarks. Proc Natl Acad Sci USA 1991, 88:9523-9527. 17. Kawai J, Hirotsune S, Hirose K, Fushiki S, Watanabe S, Hayashizaki Y: Methylation profiles of genomic DNA of mouse developmental brain detected by restriction landmark genomic scanning (RLGS) method. Nucleic Acids Res 1993, 21:5604-5608. 18. Shiota K, Kogo Y, Ohgane J, Imamura T, Urano A, Nishino K, Tanaka S, Hattori N: Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice. Genes Cells 2002, 7:961-969. 19. Song F, Smith JF, Kimura MT, Morrow AD, Matsuyama T, Nagase H, Held WA: Association of tissue-specific differentially methylated regions