Hepatic Lipid Accumulation Alters H3K9 and H3K4 Figure 2. Genome-wide H3K9me3 and H3K4me3 variations in lipid-accumulated mouse primary hepatocytes determined by ChIP- on-chip analysis. (A) BODIPY-labeled lipid droplets in non- and palmitate plus oleate-treated mouse primary hepatocytes. (B) Expression pattern of H3K9me3 and H3K4me3 targets (fold change $1.5 in at least one histone trimethylation status, p,0.05, n = 2 for each histone status, H3K9me3 and H3K4me3) in lipid-accumulated hepatocytes. (C) Biological pathways affected by H3K9me3 and H3K4me3 targets in response to lipid accumulation in hepatocytes (p,0.05). doi:10.1371/journal.pone.0044345.g002 Lipid Accumulation Specifically Alters the Effect of H3K9me3 and H3K4me3 on the Peroxisome Proliferator- activated Receptor Alpha (PPARa)-network Based on the ChIP-on-chip data, we selected 22 lipid metabolism genes with altered H3K9me3 and H3K4me3 status in the promoter, as identified by Gene Ontology annotation (Table S3). We then investigated whether these genes were involved in cellular metabolic pathways associated with NAFLD development. A PubGene analysis for biological networks revealed that 16 of the 22 gene targets were biologically closely associated. In particular, we found that Ppara and related lipid catabolism genes, including Apoa5, nuclear receptor subfamily 5, group A, member 2 (Nr5a2), lipase, hormone-sensitive (Lipe), isocitrate + dehydrogenase 3 (NAD ) alpha (Idh3a), aconitase 2 (Aco2), activating transcription factor 4 (Atf4), succinate dehydrogenase complex, subunit B, iron sulfur (Sdhb), cell death-inducing DNA fragmentation factor, and alpha subunit-like effector A (Cidea), were associated with an altered H3K9me3 or H3K4me3 status in lipid-accumulated hepatocytes (Figure 3 and Table S3). We selected five genes, Ppara, Nr5a2, Lipe, Atf4, and Cidea, which are involved in hepatic lipid metabolism, and validated the ChIP- on-chip results with a conventional ChIP assay. The histone status of the selected genes determined by a conventional ChIP assay was similar to the results of the ChIP-on-chip analysis (Figure 4). In addition, since histone methylation plays a key role in the transcriptional regulation of gene expression, we also determined mRNA expression of Ppara and its related genes selected in the ChIP-on-chip analysis. Quantitative real-time analysis showed reduced expression of all five genes in lipid-accumulated hepatocytes compared with non-treated control cells (Figure 4). This may induce defects in lipid catabolism under hyperlipidemic conditions and eventually contribute to the development of NAFLD. Discussion NAFLD refers to accumulation of hepatic steatosis not due to excess alcohol consumption [9] and is the most common liver condition globally [22]. The pathogenesis of NAFLD is related to increased lipid influx into the liver and increased de novo hepatic lipogenesis, promoting hepatic triglyceride accumulation [9]. Defects in lipid utilization via mitochondrial oxidation and lipid export may also contribute to hepatic lipid buildup [9]. Subjects with NAFLD have a higher mortality rate than the general population and are at increased risk of developing cardiovascular disease and diabetes [23]. Histologically, NAFLD occurs as a spectrum from mild hepatic steatosis only, to nonalcoholic steatohepatitis characterized by hepatocellular injury and inflam- mation, to cirrhosis [9]. The molecular epigenetic mechanisms behind each symptom have begun to be investigated. In this study, we aimed to identify molecular markers altered in lipid-loaded hepatocytes in the early stage of NAFLD. PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e44345