Figure 2. Regulation of RIZ1 protein expression by diet. A. Whole cell extracts of liver from animals on diet 1 and diet 2 for two months were analyzed by Western blot analysis using an antibody that reacts with both RIZ1 and RIZ2 proteins. Western blot using beta-actin antibody served as loading control. B. Quantification of protein levels by densitometry analysis. Data are the means6SD of 4 animals per subgroup. *P = 0.012 (Student’s t-test, 2 tailed). doi:10.1371/journal.pone.0003390.g002 To further determine whether RIZ1 expression is sensitive to SAM levels, we used the MATA1 knockout mice model [11]. These animals have lower (3 to 4 fold) hepatic SAM and also develop liver cancers. Quantitative RT-PCR analysis of animals at 4.5–5.5 months of age showed that RIZ1 expression in wild type livers (n = 6) was 2.0 fold higher than in the MATA1 knockouts (n = 5, P = 0.03, Student’s t test, 2 tailed), comparable to the fold reduction for the same aged wild type mice on diet 2 (Supplementary Table S2). We next examined whether RIZ1 target genes were regulated by diet. DNA microarray analysis of livers of RIZ1 wild type animals at 2 months of diet treatment revealed a list of 1636 genes that were affected by diet by more than 2 fold (Supplementary Table S3). DNA microarray analysis of livers of RIZ1 knockout animals identified 97 putative RIZ1 target genes showing more than 2 fold difference between wild type and knockout (Supplementary Table S4). Of these, 29 were also present in the list of genes regulated by diet, indicating a significant enrichment of RIZ1 target genes in the list of diet-regulated genes (29/97 versus 1636/48000, P,0.0001, Chi squared test, 2 tailed). The genes of interest that were upregulated by both RIZ1 knockout and methyl-imbalanced diet include c-Jun, c-Fos, and Ctgf. Some of these genes might play a direct role in liver cancers (i.e., c-Jun) [12]. The effect of RIZ1 knockout or diet on the expression of these genes was confirmed by quantitative RT-PCR analysis (Table 1). The results suggest that downregulation of RIZ1 by diet 2 was associated with deregulation of RIZ1 target genes. RIZ1 in Cancer Prevention Table 1. Quantitative RT-PCR analysis of genes that were upregulated by RIZ1 deficiency or by diet 2 treatment. Genes Abcc4 Alas1 Ctgf Fos Gdf15 Jun Mthfd1l KO vs. WT 2.1 4.0 2.2 2.4 2.3 2.0 1.6 Diet 2 vs. Diet 1 2 months 1 month 13.1 2.3 2.4 5.0 2.7 8.0 5.1 10.2 1.3 2.3 4.7 3.7 2.6 3.0 Fold changes in liver RNA expression of 7 putative RIZ1 target genes in RIZ1 knockout versus wild type animals were shown. Also shown are fold changes in liver RNA expression of 7 putative RIZ1-target genes in RIZ1 wild type animals on diet 2 versus diet 1 after either 1 month or 2 months of diet treatment. Data represent means of at least 3 animals per subgroup. P,0.05 (Student’s t-test, 2 tailed) for all data set except Alas1 at 1 month diet treatment. doi:10.1371/journal.pone.0003390.t001 Since RIZ1 expression was not significantly altered by diet 2 at 1 month treatment (Supplementary Table S2), any expression changes of the target genes of RIZ1 at 1 month diet treatment may reflect changes in RIZ1 activity rather than in expression level. We selected seven RIZ1 target genes and determined their expression levels at either 1 month or 2 months diet treatment. As shown in Table 1, six of the seven genes were found upregulated by diet 2 at 1 month diet treatment. The data suggest that diet 2 caused deregulation of RIZ1 target genes before significantly decreasing RIZ1 expression levels. We next used chromatin immunoprecipitation (ChIP) assay to examine changes in histone methylation on RIZ1 target genes as a result of decreased RIZ1 activity. We used a RIZ1-specific antibody ab9710 (Abcam) that does not react with RIZ2 (see Supplementary Figure S1 for a Western blot of RIZ1 by this antibody). As shown in Figure 3A, the c-Jun proximal promoter was bound by RIZ1 and showed higher levels of H3 lysine 9 monomethylation in RIZ1 wild type versus knockout livers. RIZ1 did not bind to the distal region of the c-Jun promoter or the promoter of Elovl3 gene that was not repressed by RIZ1. At 1 month diet 2 treatment, H3K9me1 of c-Jun promoter was decreased even though RIZ1 binding was not changed, suggesting again that RIZ1 activity was decreased even when RIZ1 expression was at normal levels (Figure 3). We also examined overall DNA methylation changes in response to methyl-imbalanced diet in DNA from livers of animals at 15 months of treatment. While we found that diet 2 decreased overall DNA methylation (Supplementary Figure S2), we did not find changes in DNA methylation (either hyper or hypo methylation) in individual random CpG rich regions (Supplemen- tary Figure S3) using the technique of Not1-Mse1 MS-AFLP (methylation sensitive amplified fragment length polymorphism) [13]. By Western blot analysis, we did not find significant changes (.2 fold) in methylation of H3K9me1, H3K9me2, H3K9me3, H4K20me1, and H4K20me2 in the liver of animals that were on diet 2 for six months (data not shown). Discussion Since only RIZ1 and 4 small molecule methyltransferases (GNMT, GAMT, NNMT, and TEMT) were found downregu- PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3390