TEMT, which were subsequently confirmed by quantitative RT- PCR. Found at: doi:10.1371/journal.pone.0003390.s002 (0.08 MB DOC) Table S3 Genes regulated by diet as revealed by DNA microarray analysis. Wild type mice were fed with either diet 1 or diet 2 for two months before their livers were collected for RNA extraction. Data represent means of 2 animals per subgroup. Only genes showing more than 2 fold changes are listed. Found at: doi:10.1371/journal.pone.0003390.s003 (0.85 MB XLS) Table S4 Genes regulated by RIZ1 knockout as revealed by DNA microarray analysis. The RNAs used for analysis were from wild type and knockout mice of four months of age on balanced diet. Data represent means of 2 animals per subgroup. Genes in bold were also regulated by diet. Only genes showing more than 2 fold changes are listed. Found at: doi:10.1371/journal.pone.0003390.s004 (0.06 MB XLS) Table S5 Methyltransferases with high Km value for SAM were downregulated by methyl-imbalanced diet. The Km value for SAM and Ki value for SAH are shown for various methyltrans- ferases. The ratio (fold changes) Diet 1 vs. Diet 2 in expression levels of the methyltransferases was calculated from quantitative RT-PCR. Data represent means of at least 3 animals per subgroup at 2 months of diet treatment. *P,0.05 Diet 1 vs. Diet 2 (Student’s t-test, 2 tailed). Found at: doi:10.1371/journal.pone.0003390.s005 (0.03 MB DOC) Figure S1 Western blot analysis confirming recognition of RIZ1 but not RIZ2 by the Abcam RIZ1-specific antibody ab9710. Total References 1. Doll R, Peto R (1981) The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 66: 1191–1308. 2. Mikol YB, Hoover KL, Creasia D, Poirier LA (1983) Hepatocarcinogenesis in rats fed methyl-deficient, amino acid-defined diets. Carcinogenesis 4: 1619–1629. 3. Shivapurkar N, Poirier LA (1983) Tissue levels of S-adenosylmethionine and S- adenosylhomocysteine in rats fed methyl-deficient, amino acid-defined diets for one to five weeks. Carcinogenesis 4: 1051–1057. 4. Shivapurkar N, Wilson MJ, Hoover KL, Mikol YB, Creasia D, et al. (1986) Hepatic DNA methylation and liver tumor formation in male C3H mice fed methionine- and choline-deficient diets. J Natl Cancer Inst 77: 213–217. 5. Huang S (2002) Histone methyltransferases, diet nutrients, and tumor suppressors. Nat Rev Cancer 2: 469–476. 6. Huang S (2008) Histone methylation and the initiation of cancer, Cancer Epigenetics; Tollefsbol T, ed. New York: CRC Press. 7. Steele-Perkins G, Fang W, Yang XH, Van Gele M, Carling T, et al. (2001) Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear protein-methyltransferase superfamily. Genes Dev 15: 2250–2262. 8. Kim K-C, Geng L, Huang S (2003) Inactivation of a histone methyltransferase by mutations in human cancers. Cancer Res 63: 7619–7623. 9. Garcia-Bassets I, Kwon YS, Telese F, Prefontaine GG, Hutt KR, et al. (2007) Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors. Cell 128: 505–518. 10. Liu L, Shao G, Steele-Perkins G, Huang S (1997) The retinoblastoma interacting zinc finger gene RIZ produces a PR domain lacking product through an internal promoter. J Biol Chem 272: 2984–2991. 11. Lu SC, Alvarez L, Huang ZZ, Chen L, An W, et al. (2001) Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation. Proc Natl Acad Sci U S A 98: 5560–5565. RIZ1 in Cancer Prevention protein extracts of livers from either RIZ1 wild type or knockout animals were resolved by SDS gel followed by western blot using RIZ1 antibody ab9710. Equal loading was confirmed by western blot using beta-actin antibody. Found at: doi:10.1371/journal.pone.0003390.s006 (0.07 MB PDF) Figure S2 DNA hypomethylation in mice fed with diet 2. Total genomic DNAs were isolated from livers of mice on either diet 1 or diet 2 for 15 months. The methylation levels of these DNAs were determined by the Sss1 enzyme assay. Data are the means+SD of 3 animals per subgroup. *P = 0.03 (Student’s t-test, 2 tailed). Found at: doi:10.1371/journal.pone.0003390.s007 (0.02 MB PDF) Figure S3 Analysis of methylation changes in CpG rich regions of the genome by MS-AFLP. A. Genomic DNA isolated from MCF7 cells that were either treated or not treated with the demethylating agent Aza-C were used as a positive control for the MS-AFLP method. B. The genomic DNAs used for analysis were from brain (B) and liver (L) tissues of mice on either diet 1 or diet 2 for 15 months. Found at: doi:10.1371/journal.pone.0003390.s008 (1.49 MB PDF) Acknowledgments We thank Emily Larson for tissue samples. Author Contributions Conceived and designed the experiments: SH. Performed the experiments: WZ SA DT FY SH. Analyzed the data: MP SH. Contributed reagents/ materials/analysis tools: SSL. Wrote the paper: FY MP SH. 12. Eferl R, Ricci R, Kenner L, Zenz R, David JP, et al. (2003) Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 112: 181–192. 13. Yamamoto F, Yamamoto M, Soto JL, Kojima E, Wang EN, et al. (2001) Notl- Msel methylation-sensitive amplified fragment length polymorphism for DNA methylation analysis of human cancers. Electrophoresis 22: 1946–1956. 14. Wilson MJ, Shivapurkar N, Poirier LA (1984) Hypomethylation of hepatic nuclear DNA in rats fed with a carcinogenic methyl-deficient diet. Biochem J 218: 987–990. 15. Patnaik D, Chin HG, Esteve PO, Benner J, Jacobsen SE, et al. (2004) Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltrans- ferase. J Biol Chem 279: 53248–53258. 16. Huang S, Shao G, Liu L (1998) The PR domain of the Rb-binding zinc finger protein RIZ1 is a protein binding interface and is related to the SET domain functioning in chromatin-mediated gene expression. J Biol Chem 273: 15933–15940. 17. Briknarova K, Zhou X, Satterthwait A, Hoyt DW, Ely KR, et al. (2008) Structural studies of the SET domain from RIZ1 tumor suppressor. Biochem Biophys Res Commun 366: 807–813. 18. Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AH, et al. (2005) Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 436: 660–665. 19. Buyse IM, Shao G, Huang S (1995) The retinoblastoma protein binds to RIZ, a zinc finger protein that shares an epitope with the adenovirus E1A protein. Proc Natl Acad Sci USA 92: 4467–4471. 20. De Smet C, De Backer O, Faraoni I, Lurquin C, Brasseur F, et al. (1996) The activation of human gene MAGE-1 in tumor cells is correlated with genome- wide demethylation. Proc Natl Acad Sci U S A 93: 7149–7153. PLoS ONE | www.plosone.org 6 October 2008 | Volume 3 | Issue 10 | e3390