cancer prevention may be the maintenance of RIZ1 activity in most cells of a target tissue [6]. Materials and Methods Animals, diets, and tissues RIZ1 knockout mice in 129Sv/C57Bl6 background were crossed to 129Sv mice for 8 generations to produce RIZ1 knockout mice in 99.625% 129Sv background. These RIZ12/2 mice were then crossed with p53+/2 129Sv mice (from Jackson Laboratory) to generate RIZ1+/2p53+/2 mouse in 129Sv background. Animals of the following genotypes were maintained for experimental use: RIZ12/2p53+/2 and RIZ1+/+p53+/2. The introduction of p53 heterozygous mutation was intended to shorten the latency of tumor development in the RIZ1 knockout mice [7]. Male animals of each genotype were randomized into two groups of ,30 each. Starting at about 4 weeks of age, all animals were fed with a methyl-balanced and amino acid-defined diet or diet 1. After on this diet for one week, one group of animals continued to stay on diet 1 for the remaining period of the experiment. The other group were fed with methionine and choline deficient diet or diet 2. The compositions of the diets (see Supplementary Table S1) were essentially the same as those commonly used in the field [2,3,4]. Diets were stored at 4uC and given ad libitum with biweekly replacement. Animal growth was followed by monthly body weight measurement for upto 15 months. No significant difference in body weight was noted for different genotypes. Animals on diet 2 showed reduced (15% less at 15 months of age) body weight compared to animals on diet 1. This slight effect of diet 2 on body weight was similar to what others have found [2]. Kaplan-Meyer survival curves were plotted using the Prism statistics program (GraphPad software). The liver tissues were fixed for histological analyses or snap frozen in liquid nitrogen and stored at 280uC until used for biochemical analyses. Tissues fixed in 10% formalin were routinely processed for paraffin embedding, sectioned, and stained with hematoxylin and eosin. For measurements of dietary effects on gene expression and histone methylation, additional animals were fed diet 1 or 2 for 1 to 6 months before being sacrificed for tissue collection. Ethical approval for all works on animals was obtained from the Animal Research Committee of The Burnham Institute for Medical Research. Western blotting Tissues were homoginized in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4, plus proteinase inhibitors). The whole lysates were then mixed with SDS gel loading buffer followed by SDS gel fractionation and western blotting. RIZ antibody was rabbit serum KG7.1S against RIZ1 aa 245–573 that reacts with both RIZ1 and RIZ2, as described previously (available from Abcam ab3790) [19]. For western blot against RIZ1 but not RIZ2, we used the abcam RIZ1 antibody ab9710. Antibodies for methylated histones were from Abcam and Upstate. Quantitative RT-PCR Total RNAs were extracted from tissues using the MagNa Lyser Green Beads (Roche) and the RNAmini kit (Qiagen). Quantitative RT-PCR analyses (SYBR green) were performed using the Mx3000P QPCR system of Stratagene. All gene specific primers were confirmed to give a single band of expected size. Cyclophilin A (PPIA) gene served as a control for RNA amount. This gene was not regulated by diet or RIZ1 knockout as indicated by microarray analysis. RIZ1 in Cancer Prevention DNA microarray analysis cDNAs synthesized from total RNAs were hybridized with Sentrix Mouse-6 Expression BeadChip from Illumina containing 48,000 gene arrays. Data were obtained from replicate biological samples. Data normalization was performed using cubic spline normalization. ChIP analysis Rabbit antibody specific for RIZ1 but not RIZ2 from Abcam (ab9710) was used for ChIP analysis of RIZ1 binding to target genes. Antibodies for methylationed histones were from Abcam (ab9045 for H3K9me1, ab8898 for H3K9me3, ab9051 for H4K20me1) and Upstate (07-212 for H3K9me2). Liver tissues (,30 mg per antibody immunoprecipitation) were chopped to pieces using razor blades and cross linked in 1% formaldehyde at room temperature for 15 min. Chromatin was fragmented to an average size of 700 bp using a Misonix XL2020 sonicator. The ChIP assay kit from Upstate was used. For PCR, we used the following primer pairs: 59-AAATCTCTGGTTTCCAGGTA- CAGC-39 (2801 to 2777) and 59- GAGAAAGGGCTGAAT- GATCTGAGT-39 (2595 to 2571) for c-Jun proximal promoter region, and 59-GTGTGGGGGTAGAGGAGTGA-39 (24337 to 24317) and 59- AGTGTCACTGGACCCTCACC -39 (24128 to 24108) for c-Jun distal region. For PCR of Elovl3 gene promoter, we used the primer pairs 59-CCCCATTTTTCTCTCCAACA-39 (2768 to 2748) and 59-CCAAGCTGGACCATAAGGAA-39 (2578 to 2558) for the Elovl3 promoter region, and 59- CCAGGCTGTCCTGAAACTAATTCT-39 (28325 to 28301) and 59-CTGTAGACCAGGTGACTGCAAACT-39 (28132 to 28108) for the Elovl3 distal region. DNA methylation analysis Genomic DNAs were extracted from tissues using standard procedures. Analysis of methylation in CpG rich regions by Not1- Mse1 MS-AFLP method was as described previously [13]. Genomic methyl-cytosine content was assayed by the methylation enzyme Sss1 method [20]. Supporting Information Table S1 Composition of the amino acid-defined and methyl- balanced basal diet (Teklad Product No. TD 99366). This is referred as Diet 1. The methyl-imbalanced diet or diet 2 formulation ((Teklad Product No. TD 01513) is the same as diet 1 except that it contains 9.0 g/Kg DL-Homocystine and lacks methionine and choline bitartrate. Found at: doi:10.1371/journal.pone.0003390.s001 (0.05 MB DOC) Table S2 Quantitative RT-PCR and microarray analysis of some histone methyltransferases and related enzymes. Wild type male animals were treated with diet 1 or diet 2 (starting from 3–4 weeks of age) for 1, 2, 4, and 6 months before RNA extraction from livers. Levels of 25 histone methyltransferases and 8 other related enzymes were determined by either quantitative RT-PCR or microarray or both, and the ratio (fold change) Diet 1 versus Diet 2 calculated. Data from quantitative RT-PCR represent means of at least 3 animals per subgroup. *P,0.05 Diet 1 vs. Diet 2 (Student’s t-test, 2 tailed). Histone methyltransferases that are not listed here did not show significant expression in the liver as revealed by DNA microarray analysis. Data from microarray analysis represent means of 2 animals per subgroup. DNA microarray analysis revealed downregulation by diet 2 of four small molecule methyltransferases, GNMT, GAMT, NNMT, and PLoS ONE | www.plosone.org 5 October 2008 | Volume 3 | Issue 10 | e3390