Hepatic Lipid Accumulation Alters H3K9 and H3K4 Figure 1. Transcriptome profile of the steatotic livers of high-fat diet-fed hAPOE2 mice determined by oligonucleotide microarray analysis. (A) H&E staining of the livers of normal diet- and high-fat diet-fed hAPOE2 mice (original magnification, 6400). (B) Heat map of the transcriptome profile of the steatotic livers of high-fat diet-fed hAPOE2 mice. Columns represent individual arrays and rows indicate gene expression profiles. Red, blue, and white indicate upregulated, downregulated, and unaltered genes, respectively (p,0.05, n = 6). (C) mRNA expression of genes encoding epigenetic modifiers in the steatotic livers of high-fat diet-fed hAPOE2 mice (p,0.05). doi:10.1371/journal.pone.0044345.g001 Lipid Accumulation Induces Genome-wide H3K9me3 and H3K4me3 Variations in Mouse Primary Hepatocytes We first isolated primary hepatocytes from C57BL/6J mice livers (Figure S2A) and verified the expression of albumin and transferrin, hepatocyte-specific markers (Figure S2B) [21]. To induce lipid accumulation in primary hepatocytes for mimicking steatotic liver, palmitate and oleate were treated and then lipid droplet formation in the hepatocytes was verified (Figure 2A). The expressions of the jumonji C-domain-containing histone demethy- lase (JHDM) family (Kdm3b, Kdm5b, Kdm5c) were significantly induced in lipid accumulated primary hepatocytes compared with those in control cells (Table S2) We performed ChIP-on-chip analysis to investigate the H3K9me3 and H3K4me3 alterations caused by lipid accumulation in mouse primary hepatocytes. A total of 1,830 targets on the 405 K CpG array displayed a $1.5- fold change of at least one histone trimethylation status, showing that target hyper- and hypotrimethylated H3K9me3 and H3K4me3 were evenly chromosomally distributed in lipid-loaded hepatocytes, with diverse signal intensities (Figure S3); 332 and 810 targets were commonly up- or downregulated in both H3K9me3 and H3K4me3, respectively. However, 688 targets showed an inverse H3K9 and H3K4 trimethylation pattern: a total of 180 targets displayed increased H3K4me3 and decreased H3K9me3, whereas 508 exhibited the opposite pattern (Figure 2B). These target genes had many biological functions, including gene expression, cellular development, assembly/organization, growth/ proliferation, cell death, and lipid metabolism, as assessed by Ingenuity Pathway Analysis (Figure 2C). Furthermore, hepatic lipid accumulation induced H3K9me3 and H3K4me3-related alterations in the expression of genes associated with liver hepatomegaly, proliferation, steatohepatitis, necrosis, hyperplasia, hyperproliferation, steatosis, regeneration, dysplasia, hypertrophy, damage, and degeneration, all of which are associated with the pathophysiology of NAFLD, although the number of targeted genes was small and thus the pathway was not significant (data not shown). PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44345