Microarray analysis revealed that JHDM2a deficiency reduced b- oxidation and glycerol release in skeletal muscle [13]. Although histone modification has been suggested to be associated with the development of hyperlipidemia, the association between histone methylation and liver steatosis is largely unknown, and most previous studies were limited to the effect of histone methylation on selected target genes. In the present study, we investigated variations in H3K9 and H3K4 methylation during the development of NAFLD due to hyperlipidemic conditions by ChIP-on-chip and oligonucleotide DNA array analyses. Hepatic transcriptome profiling with human apolipoprotein E2 (hAPOE2) transgenic mice showed that the high- fat diet-induced lipid accumulation responsible for the develop- ment of NAFLD altered global gene expression and genes encoding chromatin-remodeling enzymes for H3K9me3 and H3K4me3. Based on the transcriptional regulation, we loaded mouse primary hepatocytes with palmitate plus oleate to induce intracellular lipid-droplet formation to mimic hyperlipidemic NAFLD status, and examined H3K9me3 and H3K4me3 varia- tions in steatotic hepatocytes compared with non-lipid-loaded control cells. Compared with histone mono- and dimethylation, the role of trimethylation in metabolic gene transcription remains poorly understood, even though it has been implicated in the development of several chronic diseases [15,16]. This study provides the first evidence that the hyperlipidemic condition in hepatocytes induces both hypo- and hypermethylation of H3K9 and H3K4 during NAFLD development. Methods Animals Six-week-old male C57BL/6J (Samtako Korea, Osan, Korea) and hAPOE2 (R158C) transgenic mice (Taconic Farms, German- town, NY, USA) were used for ChIP-on-Chip and oligonucleotide microarray analyses, respectively. The hAPOE2 mice were generated by targeted replacement of the endogenous mouse Apoe with the human APOE2 gene and are defective in clearing TG-rich lipoproteins; thus, they spontaneously develop hyperlipidemia [17] and prone to develop hepatic steatosis [18]. Therefore, this mouse strain was appropriate to study gene expression profile for hepatic lipid accumulation. Control diet-fed C57BL/6J and hAPOE2 mice were maintained on regular rodent chow (12% fat calories, Purina Laboratory Rodent Diet 38057; Dyets Inc., Bethlehem, PA, USA) and high-fat diet-fed hAPOE2 mice were provided with pelleted rodent chow in which 60% of the calories were from fat (Purina Laboratory Rodent Diet D12492; Dyets Inc.). The animals were maintained with water ad libitum on a 12-h light:dark cycle. To obtain liver tissues, mice were killed under general anesthesia with 2.5% tribromoethanol (20 ml/kg, i.p.) and the livers were removed, snap-frozen in liquid nitrogen, and stored at –80uC prior to analysis. All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of Korea University (animal protocol number: KUIACUC- 20090421-2). Preparation of Mouse Primary Hepatocytes and Lipid- loading Primary hepatocytes of C57BL/6J mice were prepared accord- ing a method reported previously [19,20]. Fasted mice were anesthetized with 2.5% tribromoethanol (20 ml/kg, i.p.), and a catheter was inserted into the inferior vena cava. The superior vena cava was clamped, and the portal vein was transected. The liver was washed with Hanks buffer salt solution (HBSS) containing 100 U/ml penicillin/streptomycin (pH 7.4) for 4 min Hepatic Lipid Accumulation Alters H3K9 and H3K4 at a flow rate of 7 ml/min and perfused with HBSS supplemented with 1 mM CaCl2 and MgCl2, 100 U/ml penicillin/streptomycin, and 0.04% collagenase type IV (pH 7.4) for 10 min. The digested liver was removed and then mechanically disrupted in collagenase solution. The cell suspension was filtered through 70-mm Falcon cell strainers (Falcon BD, Lincoln Park, NJ, USA) and centrifuged at 506 g for 2 min. The isolated hepatocytes were washed with phosphate-buffered saline (PBS) by centrifugation at 506 g for 2 min. Cells were then cultured on collagen-coated culture plates (Iwaki, Chiba, Japan) in Williams’s Medium E with 10% heat- inactivated fetal bovine serum (FBS), 100 U/ml penicillin/ streptomycin, and 1610–7 M insulin for 12 h. Williams’s Medium E was then replaced with low glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. Mouse primary hepatocytes were cul- tured on collagen-coated culture plates with DMEM containing 10% FBS, 1% penicillin/streptomycin, and 40 mM oleate plus 40 mM palmitate conjugated to 0.16% fatty acid-free bovine serum albumin for 24 h. Lipid and Hematoxylin and Eosin (H&E) Staining For lipid-droplet staining, hepatocytes cultured on collagen- coated glass slides were fixed with 3% (w/v) paraformaldehyde for 30 min and incubated with C1-BODIPY 500/510-C12 (4,4- difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid; Molecular Probes, Eugene, OR, USA) for 10 min at room temperature. After washing with PBS, coverslips were mounted on slides using the ProLong antifade solution (Invitrogen, Carlsbad, CA, USA) and lipid-droplets in hepatocytes were visualized by fluorescence microscopy (Axio observer D1; Carl Zeiss, Jena, Germany). For H&E staining, the livers of hAPOE2 mice were fixed with 10% (v/v) formaldehyde, embedded in paraffin, sectioned, and stained with H&E. The tissue sections were observed under microscopy (Eclipse Ti; Nikon Inc, Tokyo, Japan). Oligonucleotide Microarray Analysis Two-color oligonucleotide microarray experiments (n = 6) were performed with the livers of control and high-fat diet-fed hAPOE2 transgenic mice. Total RNA was extracted from liver tissue using TRIzol reagent (Invitrogen) and further purified using the RNase- free DNase I set and the RNeasy MinElute Cleanup Kit (Qiagen, Chatsworth, CA, USA). cDNA was synthesized from 8 mg purified RNA using Superscript II reverse transcriptase (Invitrogen), oligo(dT)20VN primers, and dNTPs, and subsequently labeled with the Cy3-dUTP and Cy5-dUTP (GE Healthcare, Piscataway, NJ, USA). Labeled cDNA samples were purified using the QIAquick PCR Purification kit (Qiagen) and then hybridized to 38.8 K Mouse Exonic Evidence-Based Oligonucleotide (MEEBO) arrays (Stanford Functional Genomics Facility, Stanford, CA, USA), which contain 30,125 constitutive exonic probes. Hybrid- ized arrays were scanned with the GenePix 4000B scanner (Axon Instruments, Union City, CA, USA) and the resulting images visualized using GenePix 4.0 software (Axon Instruments). Probe- level gene expression values were computed using GenePix 4.0 software and the Stanford Microarray Database. The data were normalized by NormExp background correction (offset = 350) and the Loess and Aquantile methods using the Bioconductor Limma software. Transcriptional responses to high-fat diet feeding were assessed by p,0.05 using a t-distribution. To analyze the transcriptome profile of steatotic livers, we used the HeatMap- Viewer included in the GenePattern software (http://genepattern. broadinstitute.org/gp/pages/index.jsf) with gene symbols and log2 values of genes regulated significantly. Effects of high-fat diet feeding on biological pathways were assessed by determining the PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44345