RIZ1 in Cancer Prevention Figure 3. Regulation of histone methylation on RIZ1 target genes. A. Soluble chromatin was prepared from livers of RIZ1 wild type and knockout animals. Immunoprecipitation was performed using the indicated antibodies. DNA was amplified using primer sets that cover the proximal or the distal promoter regions of c-Jun and Elovl3 gene. B. Soluble chromatin was prepared from livers of wild type animals on either diet 1 or diet 2 for 1 month. Immunoprecipitation was performed using the indicated antibodies. DNA was amplified using primer sets that cover the proximal or the distal promoter regions of c-Jun and Elovl3 gene. One representative data set is shown out of four experiments performed. doi:10.1371/journal.pone.0003390.g003 lated by diet 2 in a non-biased screening using both DNA microarray and RT-PCR methods (see Supplementary Table S2), we examined whether these 5 enzymes shared some common properties that could explain their sensitivity to diet. We found from the literature Km values for SAM for 3 of the 4 small molecule methyltransferases and all three turned out to have high Km value (.49 mM) (see Supplementary Table S5). For the large number of methyltransferases that were not regulated by diet, we identified Km values for 7 of them and all 7 have Km values lower than 10 mM (see Supplementary Table S5). Thus, there is a significant association between high Km for SAM and downreg- ulation by diet 2 (P = 0.0083, Fisher’s exact test, 2 tailed). Hepatic SAM concentration is ,60 mM and methyl-imbal- anced diet typically causes a 1.5 to 3 fold drop in SAM levels [3,14], which would be still high enough to allow near maximum activity for most methyltransferases with low Km for SAM but would significantly inhibit those enzymes with high Km values. The preferential downregulation by diet 2 of the methyltransfer- ases with high Km for SAM may reflect a feedback inhibition on their own gene expression by unliganded or unused enzymes. The canonical SET domain shared by most KMTs has low Km for SAM [15]. But RIZ1 may be an exception since its SET domain is different from others [16,17]. Numerous effects of methyl-imbalanced diet, such as fat accumulation, protein kinase C activation, abnormal cell turnover, oxidative stress, low SAM/SAH ratio, DNA hypomethylation, and uracil incorporation into DNA or mutation, are prevented by a methyl-balanced diet. In addition, a methyl-imbalanced diet affects the expression of numerous genes as shown here. Our observation that deficiency of a single gene may neutralize the cancer preventive benefit of a methyl-balanced diet shows that at least one of the numerous effects of such diet may play a rate- limiting role in the cancer prevention process. From all these observations it can be postulated that the molecular pathway of carcinogenesis by methyl-imbalanced diet may be the following: a methyl-imbalanced diet — chronic low SAM/SAH ratio in most cells of a diet-responsive target tissue — inhibition and downregulation of RIZ1 in most cells of the target tissue — upregulation of prosurvival and progrowth oncogenes such as c-Jun in most cells of the target tissue — stochastic accumulative genetic and epigenetic changes in a single cell — clonal proliferation of the single cell — cancer. In this multi-cell origin model of cancer initiation [6], inactivation of RIZ1 and upregulation of some oncogenes such as c-Jun in most cells of a diet-responsive tissue may not be sufficient to cause clonal proliferation but may allow a large pool of less than fully normal cells to be more prone to clonal proliferation in response to stochastic strong oncogenic mutations in a single cell. In contrast, fully normal cells may undergo senescence or cell death in response to oncogenic mutations [18]. Thus, a rate-limiting step in PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3390