Hepatic Lipid Accumulation Alters H3K9 and H3K4 Figure 4. ChIP analysis of mRNA levels of H3K9me3 and H3K4me3 and selected Ppara network genes (n = 3). doi:10.1371/journal.pone.0044345.g004 hepatic lipid accumulation, modulating target genes related to fatty acid oxidation, including carnitine palmitoyltransferase 1 (Cpt1), acetyl-CoA synthase and acyl-CoA oxidase, fatty acid synthesis, and triglyceride hydrolysis [29]. Thus, Ppara-null mice were reportedly defective in fatty acid utilization and displayed a fatty liver phenotype [30], and activation of PPARa with an agonist compound has been suggested as a therapeutic strategy for NAFLD [31]. Second, Lipe promotes hepatic lipid oxidation in vivo. Overexpression of hepatic Lipe induces fatty acid oxidation and eventually ameliorates steatosis [32]. Third, Atf4 also influences fatty acid oxidation by regulating Cpt1 and medium chain acyl-CoA dehydrogenase in the liver [33]. Fourth, Nr5a2 is an activator of cholesterol 7 alpha-hydroxylase for cholesterol excretion [34]. Finally, the metabolic function of Cidea in hepatocytes is not clearly understood, although its expression was reduced in the livers of type 2 diabetic mice exhibiting steatosis [35]. All these suggest that aberrant modulation of PPARa and its related genes associated with lipid catabolism at an epigenetic level may contribute to hepatic lipid accumulation and eventually the development of NAFLD. The underlying cause of liver fat accumulation in NAFLD is mostly due to the inhibition of fatty acid catabolism, in which PPARa has been continuously suggested as a target molecule for pathogenesis and treatment of NAFLD [36]. Indeed, in also human study, mRNA expression levels of Ppara was suppressed in both the livers of obese and non-obese patients with NAFLD compared with normal controls [37]. Therefore, to study and establish whether the reduction of Ppara mRNA in NAFLD patients could be induced by aberrant modulation at an epigenetic level, as we showed in this study, the results found in the current study are needed to be replicate, further investigated, and expanded in the livers of NAFLD patients. We performed experiments with primary hepatocytes in conventional monolayer to investigate the sole effects on the hepatocytes, which is a major cell type responsible for hepatic lipid metabolism. Results suggested that the isolated primary hepato- cytes express high levels of transferrin and albumin, the hepatocyte-specific genes, assessed with RT-PCR (Figure S2B) indicating the cell preparation was done appropriately. Previously reported data suggested that gene expression levels in primary hepatocytes are somewhat different from those in the liver. However, when compared with cultured hepatoma cell lines, primary hepatocytes overall show much similarity in gene expression profile to those of the livers [38]. Several factors may affect the gene expressions in primary cells. First, the genes primarily expressed in nonparenchymal cells, such as genes in inflammatory reaction genes including iNOS, TNFa, IL-1b, IL- 10, are not highly expressed in primary hepatocytes [39]. Second, the three-dimensional architecture and the extracellular matrix are not well maintained in primary cells thus mRNA expression of collagen and other structural proteins is generally decreased [20]. Additionally, the expression of P450 is known to diminish in vitro over culture time [40]. However, it is widely accepted that the lipid metabolism and related gene expressions in primary hepatocytes are quite similar to the livers thus primary cells may be appropriate system for the purpose, although not perfect [20,38]. The current study provides the first evidence of genome-wide histone trimethylation changes in response to hyperlipidemic conditions during the development of NAFLD. This has potential applications in the development and assessment of drugs and nutrients for NAFLD therapeutics. Supporting Information Figure S1 ChIP verification. After H3K9me3- and H3K4me3-specific immunoprecipitation, chromatin fragmenta- tion and random-prime amplification of genomic DNA fragments PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44345