Shi Huang1,3* Abstract Requirement of RIZ1 for Cancer Prevention by Methyl- Balanced Diet Wenyun Zhou1, Sergio Alonso1, Daisaku Takai1, Shelly C. Lu2, Fumiichiro Yamamoto1, Manuel Perucho 1 , 1 Cancer Research Center, The Burnham Institute for Medical Research, La Jolla, California, United States of America, 2 Gastrointestinal and Liver Diseases, USC Research Center for Liver Diseases, USC-UCLA Research Center for Alcoholic Liver and Pancreatic Diseases, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America, 3 Institute of Biomedical Sciences, Center for Evolutionary Biology, Fudan University, Shanghai, China Background: The typical Western diet is not balanced in methyl nutrients that regulate the level of the methyl donor S- adenosylmethionine (SAM) and its derivative metabolite S-adenosylhomocysteine (SAH), which in turn may control the activity of certain methyltransferases. Feeding rodents with amino acid defined and methyl-imbalanced diet decreases hepatic SAM and causes liver cancers. RIZ1 (PRDM2 or KMT8) is a tumor suppressor and functions in transcriptional repression by methylating histone H3 lysine 9. Methodology/Principal Findings: Here we show that a methyl-balanced diet conferred additional survival benefits compared to a tumor-inducing methyl-imbalanced diet only in mice with wild type RIZ1 but not in mice deficient in RIZ1. While absence of RIZ1 was tumorigenic in mice fed the balanced diet, its presence did not prevent tumor formation in mice fed the imbalanced diet. Microarray and gene expression analysis showed that, unlike most of its related enzymes, RIZ1 was upregulated by methyl-balanced diet. Methyl-balanced diet did not fully repress oncogenes such as c-Jun in the absence of RIZ1. Higher RIZ1 activity was associated with greater H3 lysine 9 methylation in RIZ1 target genes as shown by chromatin immunoprecipiation analysis. Conclusions/Significance: The data identify RIZ1 as a critical target of methyl-balanced diet in cancer prevention. The molecular understanding of dietary carcinogenesis may help people make informed choices on diet, which may greatly reduce the incidence of cancer. Citation: Zhou W, Alonso S, Takai D, Lu SC, Yamamoto F, et al. (2008) Requirement of RIZ1 for Cancer Prevention by Methyl-Balanced Diet. PLoS ONE 3(10): e3390. doi:10.1371/journal.pone.0003390 Editor: Yihai Cao, Karolinska Institutet, Sweden Received June 2, 2008; Accepted September 16, 2008; Published October 13, 2008 Copyright: 2008 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grant CA105347 to S.H., CA63585 to M.P., CA087069 to F.Y., and DK51719, AA13847 and AT001576 to S.C.L. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shuangtheman@yahoo.com Introduction The typical Western diet is linked to a third of all cancer deaths in the United States [1]. The diet is rich in meat and low in vegetables and fruits. It is not balanced in methyl nutrients or low in folic acid. Dietary nutrients, and their metabolic intermediates and products, directly influence the activity of many cellular enzymes. One class of such enzymes is SAM-dependent methyltransferases, a broad group of enzymes that have one property in common, the use of S-adenosylmethionine (SAM) as methyl group donor. The cellular level of SAM depends on dietary intake of methyl group donors, such as methionine, folic acid, vitamin B6, B12, and choline. Some methylation reactions are inhibited by low level SAM or high level of the product inhibitor S-adenosylhomocysteine (SAH). Methyl imbalanced diet that is low in folic acid, methionine, or choline is known to lower SAM level and SAM/SAH ratio. Feeding rodents with amino acid defined and methyl-imbal- anced diet decreases hepatic SAM and causes liver cancers [2,3,4]. The molecular mechanisms underlying the relationship between diet and cancer remain poorly understood. We have previously proposed that methyl-balanced diet prevents cancer by activating the histone lysine methyltransferase (KMT) class tumor suppres- sors such as RIZ1 (PRDM2 or KMT8) [5,6]. The RIZ1 tumor suppressor functions in transcriptional repression by methylating histone H3 lysine 9 [7,8,9]. Here, we determined whether RIZ1 may be a critical target of methyl balanced diet in cancer prevention. We also performed microarray and gene expression analysis to study the effect of diet on RIZ1 and other genes. The effect of diet on RIZ1 methylation enzyme activity was analyzed by chromatin immunoprecipiation assay. The results suggest that RIZ1 is regulated by diet and may be a critical target of methyl- balanced diet in cancer prevention. Results We compared RIZ1 mutant and wild type mice on a methyl- balanced diet (diet 1), versus an imbalanced diet lacking methionine and choline (diet 2). The methyl-imbalanced diet 2 (see Supplementary Table S1) is well known to lower hepatic SAM and cause liver cancers in rodents [2,3,4]. Thus, this methyl- imbalanced diet caused liver tumors and decreased survival PLoS ONE | www.plosone.org 1 October 2008 | Volume 3 | Issue 10 | e3390