Additional file 2: Figure S1. of Status of hepatic DNA methylome predetermines and modulates the severity of non-alcoholic fatty liver injury in mice

H3K4me3 ChIP-on-chip analysis in the livers of A/J and WSB/EiJ mice fed a choline- and folate-deficient diet. Heat map illustrating significant differences in hepatic H3K4me3-enrichment of CpG islands between A/J (a) and WSB/EiJ (b) mice fed the CFD diet. Unsupervised hierarchical clustering analysis was performed using one-way ANOVA with p value cut-off at 0.05. The color bar identifies high-H3K4me3-enriched (red) and low-H3K4me3-enriched (green) genes. (c) Table showing number of differentially H3K4me3-enriched CpG islands in hepatic DNA in A/J and WSB/EiJ mice fed a control or CFD diet. Ratio IP/input DNA was calculated using ArrayTrack application. (TIFF 1501 kb

Levels of 5mC and 5hmC in cerebellar genomic DNA.

<p>(<b>A</b>) Levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in genomic DNA isolated from the cerebellum of BTBR T+tf/J and C57BL/6J mice (mean ± SD, n = 5). (<b>B</b>) Levels of 5mC and 5hmC in genomic DNA isolated from the cerebellum of autism individuals and unaffected control individuals. Values were represented as open and closed circles as well as box plots. * - Significantly different from C57BL/6J mice or autism-free individuals. (<b>C</b>) Correlation plots of the 5hmC and the 8-oxo-7-hydrodeoxyguanosine (8-oxodG) levels in mouse and human cerebellar genomic DNA.</p

Cerebellar Oxidative DNA Damage and Altered DNA Methylation in the BTBR T+tf/J Mouse Model of Autism and Similarities with Human Post Mortem Cerebellum

<div><p>The molecular pathogenesis of autism is complex and involves numerous genomic, epigenomic, proteomic, metabolic, and physiological alterations. Elucidating and understanding the molecular processes underlying the pathogenesis of autism is critical for effective clinical management and prevention of this disorder. The goal of this study is to investigate key molecular alterations postulated to play a role in autism and their role in the pathophysiology of autism. In this study we demonstrate that DNA isolated from the cerebellum of BTBR T+tf/J mice, a relevant mouse model of autism, and from human post-mortem cerebellum of individuals with autism, are both characterized by an increased levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC). The increase in 8-oxodG and 5mC content was associated with a markedly reduced expression of the 8-oxoguanine DNA-glycosylase 1 (<i>Ogg1</i>) and increased expression of <i>de novo</i> DNA methyltransferases 3a and 3b (<i>Dnmt3a</i> and <i>Dnmt3b</i>). Interestingly, a rise in the level of 5hmC occurred without changes in the expression of ten-eleven translocation expression <i>1 (Tet1)</i> and <i>Tet2</i> genes, but significantly correlated with the presence of 8-oxodG in DNA. This finding and similar elevation in 8-oxodG in cerebellum of individuals with autism and in the BTBR T+tf/J mouse model warrant future large-scale studies to specifically address the role of <i>OGG1</i> alterations in pathogenesis of autism.</p></div

Western blot analysis of histone H3K4, H3K9, H3K27, and H4K20 trimethylation; H3K9, H4K20 dimethylation and H3K9, H3K27, and H4K16 acetylation in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>Densitometric analysis of the immunostaining results is shown as percent change in histone modification level in the cerebellum BTBR T+tf/J mice relative to the corresponding values in C57BL/6J mice, which was assigned a value of 100% (mean ± SD, n = 5).</p

Oxidative DNA damage in the cerebellum of BTBR mice.

<p>(<b>A</b>) Levels of 8-oxo-7-hydrodeoxyguanosine (8-oxodG) in genomic DNA isolated from the cerebellum of BTBR T+tf/J and C57BL/6J mice (mean ± SD, n = 5). (<b>B</b>) Levels of 8-oxodG in genomic DNA isolated from the cerebellum of autism individuals and unaffected control individuals (mean ± SD, n = 13). (<b>C</b>) The extent of mitochondrial DNA damage in the cerebellum of BTBR T+tf/J mice (mean ± SD, n = 5). (<b>D</b>) The expression of base excision DNA repair genes in the cerebellum of BTBR T+tf/J and C57BL/6J mice. The expression of <i>Ogg1, Ung, Apex1</i>, and <i>Polβ</i> genes was determined by qRT-PCR as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113712#s2" target="_blank">Materials and Methods</a>”. The results are presented as an average fold change in the expression of each gene in the cerebellum of BTBR T+tf/J mice relatively to that in C57BL/6J mice, which was assigned a value 1. * - Significantly different from C57BL/6J mice (mean ± SD, n = 5). (<b>E</b>) Correlation plots of the expression of <i>Ogg1</i> and the 8-oxodG level in mouse genomic DNA. Each symbol represents an individual animal.</p

Whole genome microarray analysis of gene expression in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>(<b>A</b>) Heat map illustrating significant differences in global gene expression between BTBR T+tf/J and C57BL/6J mice. The color bar identifies high-expressed (red) and low-expressed (green) genes. (<b>B</b>) Principal component analysis illustrating similarities and differences between BTBR T+tf/J and C57BL/6J mouse strains. (<b>C</b>) Venn diagram showing genes that were significantly different between BTBR T+tf/J and C57BL/6J mice. (<b>D</b>) Summary of molecular pathways that significantly differ between BTBR T+tf/J and C57BL/6J mice. The Ingenuity Pathway Analysis database (version 9.0) was used to determine and visualize molecular pathways enriched by the significant mRNA transcripts a P-value of <0.05 were considered “enriched”.</p

The expression of chromatin-modifying (A) and one carbon metabolism (B) genes in the cerebellum of BTBR T+tf/J and C57BL/6J mice.

<p>The gene expression was determined by qRT-PCR as detailed in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113712#s2" target="_blank">Materials and Methods</a>”. The results are presented as an average fold change in the expression of each gene in the cerebellum of BTBR T+tf/J mice relatively to that in C57BL/6J mice, which was assigned a value 1. * - Significantly different from C57BL/6J mice (mean ± SD, n = 5).</p