RIZ1 in Cancer Prevention compared with the methyl-balanced diet (Figure 1A). Most of the dead or moribund animals that were suitable for autopsy analysis were found to have hepatocarcinomas. In contrast, in the absence of wild type RIZ1, there was no difference in survival regardless of diet (Figure 1B). These RIZ1 knockout animals developed mostly hepatocarcinomas regardless of diet. Therefore, while the balanced diet 1 conferred additional survival benefits compared to the imbalanced diet 2 in mice with wild type RIZ1, it failed to do so in mice deficient in RIZ1. The data also shows that, consistent with previous work [7], RIZ1+/+ mice had lower mortality and tumor incidence than RIZ12/2 mice when fed methyl-balanced diet 1 (Figure 1C). However, when fed imbalanced diet 2, RIZ1+/+ mice showed similar mortality as RIZ12/2 mice (Figure 1D). Thus the tumor suppressor function of RIZ1 is dependent on a methyl-balanced diet. The similar survival rates of RIZ1-deficient and wild type animals on diet 2 (Figure 1D) also suggests that the capacity of diet 1 to confer additional survival benefits compared to diet 2 in RIZ1 wild type (Figure 1A) but not in RIZ1-deficient animals (Figure 1B) is not because of the trivial reason that the RIZ1-deficient animals may be too sick in general to respond to diet 1. To determine the effects of diet on RIZ1, we examined RIZ1 gene expression in the liver target tissue using quantitative RT- PCR and Western blot analysis. RIZ1 mRNA level was downregulated 4.2 fold after treatment with diet 2 for 2 months (Supplementary Table S2). The downregulation was not evident at 1 month on diet 2 but became obvious at 2, 4, and 6 months (Supplementary Table S2). The downregulation of RIZ1 by the methyl-imbalanced diet was confirmed by western blot analysis (Figure 2). In contrast to RIZ1, the shorter RIZ2 protein that lacks the PR/SET domain [10] was not significantly affected by diet. We also examined other methyltransferases and related enzymes by quantitative RT-PCR and DNA microarray analysis. A total of 25 histone methyltransferases were examined, including at least one enzyme specific for each of the amino acid residues that are known to be methylated. None of these enzymes, except RIZ1, was significantly downregulated (.2 fold) at 2 months of diet 2 treatment (Supplementary Table S2). Figure 1. Survival of RIZ1 wild type and mutant animals on diet 1 versus diet 2. A. The viability of RIZ1 wild type animals on diet 1 versus diet 2. B. The viability of RIZ1 mutant animals on diet 1 versus diet 2. C. The viability of RIZ1 wild type and mutant animals on diet 1. D. The viability of RIZ1 wild type and mutant animals on diet 2. Most of the dead or moribund animals that were suitable for autopsy analysis were found to have tumors. The graph was drawn with the Prism statistics software program (GraphPad Software) based on the Kaplan-Meyer theory. P values were calculated by Fisher’s exact test (2 tailed) using the survival rate at ages 20 to 22 months. doi:10.1371/journal.pone.0003390.g001 PLoS ONE | www.plosone.org 2 October 2008 | Volume 3 | Issue 10 | e3390