genes significantly regulated using the Database for Annotation, Visualization and Integrated Discovery. ChIP-on-chip Analysis Two-color ChIP-on-chip experiments (n = 2 for each histone status, H3K9me3 and H3K4me3) were performed with non- treated and lipid-accumulated mouse primary hepatocytes isolated from C57BL/6J mice. ChIP was performed using the EZ ChIP kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions, with a modification. In brief, mouse primary hepatocytes were fixed with 2% formaldehyde, scraped, collected, and resuspended in SDS lysis buffer supplemented with protease inhibitors. The cells were then sonicated to shear genomic DNA to an average fragment length of 200–1,000 bp, and pelleted by centrifugation at 12,0006 g for 10 min at 4uC. Chromatins in the supernatant were pre-cleared with protein A- agarose/Salmon Sperm DNA solution. The supernatants underwent overnight immunoprecipitation with chip-grade H3K9me3 (Millipore) and H3K4me3-specific antisera (Milli- pore). The antibody-associated DNA fragments were recovered using protein A-agarose/Salmon Sperm DNA and eluted with elution buffer (1% SDS and 0.1 M NaHCO3). Eluted samples were incubated at 65uC for 4 h to reverse formaldehyde cross- linking and digested with proteinase K (Mbiotech, Seoul, Korea) for 1 h at 45uC to remove proteins. The DNA was extracted using an AxyPrepTM PCR Cleanup Kit (Axygen, Union City, CA, USA). The quality of the ChIPed DNA was determined with a UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). To amplify genomic DNA, ligation- mediated PCR was performed and PCR product quality was verified by agarose gel electrophoresis (Figure S1). The amplified ChIPed DNA was labeled using a CGH labeling kit (Invitrogen) and hybridized to an Agilent mouse CpG array (105K; Agilent, Santa Clara, CA, USA) using an Oligo aCGH/ ChIP-on-Chip Hybridization kit (Agilent). After hybridization, the arrays were washed with an Oligo aCGH/ChIP-on-Chip Wash Buffer kit (Agilent) and scanned using an Agilent DNA microarray scanner (Agilent). Probe-level trimethylation values were computed using Feature Extraction software (Agilent). The data were normalized using the Lowess method with Gene- Spring software (Agilent) to remove systematic bias. To identify the targets of differentially trimethylated H3K9 and H3K4 under lipid-accumulated conditions, we set a value of p,0.05 as indicating statistical significance assessed using a t-distribution and selected targets whose expression was greater than 1.5-fold of the control in at least one histone trimethylation status. Chromosomal distribution of trimethylated targets was visual- ized with CGH explorer (http://www.ifi.uio.no/forskning/ grupper/bioinf/Papers/CGH/). Probe name (Agilent ID) and corresponding chromosome, start position, GenBank accession number, and mean log2 values were imported into the software. Biological pathways affected by H3K9me3 and H3K4me3 alterations were clarified by Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA, USA). Agilent ID of the selected targets and their log2 trimethylated values were uploaded as the input data set into the software. Biological pathways of H3K9me3 and H3K4me3 were algorithmically generated based on their connectivity, assigned a score, and ranked based on relevance to the input data set in the Ingenuity Pathway Knowledge database. A p value for biological pathways was calculated using a right-tailed Fisher’s test. Those biological pathways over a fixed threshold (p,0.05) were designated significantly differentially regulated. Based on Gene Ontology annotation, we selected significantly trimethylated candidates Hepatic Lipid Accumulation Alters H3K9 and H3K4 associated with lipid metabolism and analyzed their biological relationship, a gene–gene network, using the PubGene database (http://www.pubgene.org/). Quantitative Real-time PCR and RT-PCR Total RNA was extracted from mouse primary hepatocytes using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For cDNA synthesis, 2 mg total RNA was reverse- transcribed using oligo(dT) primers, M-MLV reverse transcriptase (Mbiotech), and dNTPs. Levels of gene expression were measured using the iQ5 Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and RealMasterMix SYBR ROX (5 PRIME GmbH, Hamburg, Germany). The following thermal conditions were used: 95uC for 3 min, followed by 50 cycles at 95uC for 10 s, 56uC for 15 s, and 72uC for 20 s. Melting curve analysis (71 cycles, starting at 55uC and increasing by 0.5uC every 10 s) was performed to confirm primer specificity. Primers used were designed with OligoPerfect Designer software (Invitrogen). Rela- tive gene expression levels were calculated using the iQ5 Optical System Software version 2 (Bio-Rad), with the expression of each target gene being normalized to that of b-actin. In RT-PCR analysis, synthesized cDNA was used as the template for a standard PCR reaction with PCR-EZ D-PCR Master Mix (Bionics, St. Louis, MO USA). Primer sequences are shown in Table S1. ChIP Assay ChIPed DNA was prepared as described for ChIP-on-chip analysis and used as the template for quantitative PCR reactions. The signal from each sample was normalized to that of the input control. Table S1 lists the sequences of the primers used. Statistical Analysis Data are presented as means 6 SEM. Values for two groups were compared using a t-test. Differences with p,0.05 were considered statistically significant. Results Expression of Genes Encoding Epigenetic Modifiers is Significantly Altered in the Steatotic Livers of High-fat Diet-fed Human APOE2 Transgenic Mice We confirmed that high-fat diet feeding exacerbated hepatic lipid accumulation of hAPOE2 mice compared with that of normal diet-fed hAPOE2 mice, as assessed by H&E staining (Figure 1A). In hepatic transcriptome analysis, a high-fat diet altered the levels of approximately 70% of the global transcriptome of the livers of hAPOE2 mice compared with those of normal diet-fed hAPOE2 mice (Figure 1B). Pathway analysis of genes whose expression was greater than 1.5-fold of control (p,0.05) suggested that high-fat diet feeding induced alterations in chromatin modification-related genes. In particu- lar, we found that genes encoding enzymes for regulation of H3K4me3 and H3K9me3, such as the jumonji C-domain- containing histone demethylase (JHDM) family (Kdm3b, Kdm5b, Kdm5c), were significantly induced in high-fat fed livers compared with control livers (Figure 1C). Accordingly, we further investigated whether hepatic lipid accumulation could lead to aberrant H3K4me3 and H3K9me3, and eventually contribute to development of NAFLD. To test this hypothesis, we examined global H3K4me3 and H3K9me3 alterations in lipid-accumulated mouse primary hepatocytes by ChIP-on-chip analysis. PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44345