Hepatic Lipid Accumulation Alters H3K9 and H3K4 Figure 3. Effect of H3K4me3 and H3K9me3 on the PPARa-network in lipid-accumulated mouse primary hepatocytes. Lipid metabolism-associated H3K9me3 and H3K4me3 targets (p,0.05) were selected based on Gene Ontology annotation, and their biological relationship was analyzed using the PubGene database. Red indicates H3K9me3 and H3K4me3 targets detected by ChIP-on-chip analysis and dark red represents those genes possessing a potential biological relationship with targets detected by ChIP-on-chip analysis based on previous reports. doi:10.1371/journal.pone.0044345.g003 Metabolic pathway analysis using transcriptome data revealed that genes encoding chromatin-remodeling enzymes, such as jumonji C-domain-containing histone demethylases that regulate H3K9me3 and H3K4me3 [24], were significantly altered in steatotic livers. Thus, we further investigated the genome-wide H3K9me3 and H3K4me3 status in primary hepatocytes from C57BL/6J mice by ChIP-on-chip analysis. For ChIP-on-chip analysis, isolated mouse primary hepatocytes were incubated with palmitate plus oleate to induce accumulation of lipid droplets and mimic hyperlipidemic conditions. This is a widely used method of inducing cellular lipid droplet formation [25]. H3K9me3 and H3K4me3 variations in lipid-loaded cells were compared to non-lipid-loaded control hepatocytes. Lipid accumulation in the ChIP-on-chip assay mimics a relatively early stage of NAFLD, which ranges from simple hepatic steatosis to a potentially progressive form, nonalcoholic steatohepatitis, and the highest end of the severity spectrum, cirrhosis [9]. Thus, our findings suggest that H3K9 and H3K4 methylation status of genes involved in steatosis and steatohepatitis may be associated with early-stage NAFLD. Additionally, Ppara, phosphatase and tensin homolog (Pten), cyclin-dependent kinase inhibitor 1A (Cdkn1a), spectrin beta, non-erythrocytic 1 (Sptbn1), and mediator complex subunit 1 (Med1), all of which are involved in biological pathways responsible for the development of NAFLD, exhibited altered H3K9me3 and H3K4me3 status in lipid-loaded hepatocytes. Histone modification plays a key role in gene transcription by inducing changes in chromatin structure. In general, euchromatin states lead to gene expression, while heterochromatin states facilitate gene silencing. Early studies showed that H3K9me3 is largely associated with heterochromatin and gene silencing [26], while H3K4me3 is linked to euchromatin [27], where actively transcribed genes are located, with exceptions such as Vakoc, the transcription of which is associated with H3K9 trimethylation [28]. The biological functions of histone methylation with regard to gene promoters and coding regions remain incompletely understood. Thus more research, especially on a genomic scale, is required to better understand histone trimethylation, such as H3K4me3 and H3K9me3. According to early studies of histone methylation and target gene expression, the H3K9me3 and H3K4me3 profile should overlap only slightly; however, over 1,000 genes were affected by both H3K9 and H3K4 hypo- or hypertrimethylation in our ChIP-on-chip results. In addition, many genes affected by hypertrimethylated H3K4 were not influenced by H3K9me3. Moreover, this pattern was evident in lipid metabolism- and hepatic steatohepatitis-related genes select- ed according to Gene Ontology (p,0.05). Note that under hyperlipidemic conditions, the influence of H3 trimethylation on Ppara was mirrored by other lipid catabolism- related genes, including Lipe, Atf4, Nr5a2, and Cidea, all of which are associated with the pathophysiology of NAFLD. Moreover, the expression of all of these genes was moderately reduced in lipid- loaded primary hepatocytes with marked H3K4 or H3K9 hypertrimethylation. First, Ppara is highly expressed in the liver and is a critical transcription factor responsible for the regulation of PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44345