Social exclusion changes histone modifications H3K4me3 and H3K27ac in liver tissue of wild house mice.

Wild house mice form social hierarchies with aggressive males defending territories, in which females, young mice and submissive adult males share nests. In contrast, socially excluded males are barred from breeding groups, have numerous bite wounds and patches of thinning fur. Since their feeding times are often disrupted, we investigated whether social exclusion leads to changes in epigenetic marks of metabolic genes in liver tissue. We used chromatin immunoprecipitation and quantitative PCR to measure enrichment of two activating histone marks at 15 candidate loci. The epigenetic profiles of healthy males sampled from nest boxes differed significantly from the profiles of ostracized males caught outside of nests and showing bite wounds indicative of social exclusion. Enrichment of histone-3 lysine-4 trimethylation (H3K4me3) changed significantly at genes Cyp4a14, Gapdh, Nr3c1, Pck1, Ppara, and Sqle. Changes at histone-3 lysine-27 acetylation (H3K27ac) marks were detected at genes Fasn, Nr3c1, and Plin5. A principal components analysis separated the socialized from the ostracized mice. This was independent of body weight for the H3K4me3 mark, and partially dependent for H3K27ac. There was no separation, however, between healthy males that had been sampled from two different nests. A hierarchical cluster analysis also separated the two phenotypes, which was independent of body weight for both markers. Our study shows that a period of social exclusion during adult life leads to quantitative changes in histone modification patterns in mouse liver tissue. Similar epigenetic changes might occur during the development of stress-induced metabolic disorders in humans

Enrichment of DNA in histone immunoprecipitates in relation to the input.

<p>(A) H3K4me3 enrichment was analyzed at 15 loci for 24 socialized males (housed and healthy) and 15 ostracized males (free roaming and wounded). (B) H3K27ac enrichment was analyzed for 25 socialized and 16 ostracized males. Significant differences in means were determined by a permutation test and corrected for multiple testing. Results are indicated by * for p ≤ 0.05 and by ** for p ≤ 0.01.</p

P-values of testing H3K4me and H3K27ac enrichment data for equality of means by permutation.

<p>Phenotype comparisons were carried out between 24 socialized and 15 ostracized males (H3K4me3) and between 25 socialized and 16 ostracized males (H3K27ac). Weight comparisons were carried out between 16 light and 16 heavy males. Permutation tests for equality of means were performed with 10,000,000 iterations per run; given is the mean P-value from 3 runs. P-values were adjusted using the Benjamini-Hochberg method.</p><p>^aSince the enrichment at locus <i>Cd36</i> was < 2-fold over negative control, results for this locus need to be treated with caution.</p><p>P-values of testing H3K4me and H3K27ac enrichment data for equality of means by permutation.</p

Population structure at time point of sampling of healthy/socialized and wounded/ostracized males.

<p>^aA-J: nest boxes; Z: free-roaming mice at the time of monitoring.</p><p>^bSex ratio is the fraction of males.</p><p>Population structure at time point of sampling of healthy/socialized and wounded/ostracized males.</p

PCA of H3K4me3 and H3K27ac enrichment data.

<p>The percentage of the PC axis label is the fraction of variance that is explained by the PC. (A) Separation of the H3K4me3 enrichment data from 39 mice and 14 loci into two phenotypes: black are the socialized (n = 24) and red are the ostracized (n = 15) males. (B) Separation of the H3K27ac enrichment data from 41 mice and 14 loci into two phenotypes: black are the socialized (n = 25) and purple are the ostracized (n = 16) males. The dashed lines indicate the split into two groups that is used to test significance by permutation. (C) Correlation of PC1 prediction values from H3K4me3 enrichment and body weight. (D) Correlation of PC2 prediction values from H3K27ac enrichment and body weight. The black lines are linear regression lines and r is the Pearson's product-moment correlation; neither correlation was significant.</p

Genome-Wide Quantitative Analysis of Histone H3 Lysine 4 Trimethylation in Wild House Mouse Liver: Environmental Change Causes Epigenetic Plasticity

<div><p>In mammals, exposure to toxic or disease-causing environments can change epigenetic marks that are inherited independently of the intrauterine environment. Such inheritance of molecular phenotypes may be adaptive. However, studies demonstrating molecular evidence for epigenetic inheritance have so far relied on extreme treatments, and are confined to inbred animals. We therefore investigated whether epigenomic changes could be detected after a non-drastic change in the environment of an outbred organism. We kept two populations of wild-caught house mice (<i>Mus musculus domesticus</i>) for several generations in semi-natural enclosures on either standard diet and light cycle, or on an energy-enriched diet with longer daylight to simulate summer. As epigenetic marker for active chromatin we quantified genome-wide histone-3 lysine-4 trimethylation (H3K4me3) from liver samples by chromatin immunoprecipitation and high-throughput sequencing as well as by quantitative polymerase chain reaction. The treatment caused a significant increase of H3K4me3 at metabolic genes such as lipid and cholesterol regulators, monooxygenases, and a bile acid transporter. In addition, genes involved in immune processes, cell cycle, and transcription and translation processes were also differently marked. When we transferred young mice of both populations to cages and bred them under standard conditions, most of the H3K4me3 differences were lost. The few loci with stable H3K4me3 changes did not cluster in metabolic functional categories. This is, to our knowledge, the first quantitative study of an epigenetic marker in an outbred mammalian organism. We demonstrate genome-wide epigenetic plasticity in response to a realistic environmental stimulus. In contrast to disease models, the bulk of the epigenomic changes we observed were not heritable.</p></div

Quantification of H3K4me3 enrichment by qPCR.

<p>ChIP-DNA was prepared from liver tissues of (A) 8 young males, or (B) 6 to 12 females. Enrichment of selected genes was analyzed with respect to the input control (before IP) and normalized to <i>Gapdh</i>. Shown are means and standard deviation. Statistical significance was tested by a two-sided T-test.</p

Experimental setup.

<p>Mouse populations A and B were started with 10 males (m) and 10 females (f) each (F₀). Siblings from out-bred wild house mice were evenly distributed to the two enclosures to ensure similar genetic diversity. Population A is the control (standard breeding conditions), population B the treatment group (high-energy diet, prolonged light period and, from week 17 onwards, higher mouse density). After week 13, founders (F₀) were removed from the experiment so that only animals that were born in the enclosures produced further offspring. After 36 weeks, eight young males of generations F₂ and higher were chosen for ChIP-Seq (orange shades). At the same time, young males and females (body weight 14–15 g) were transferred to cages and bred under standard conditions. Their offspring (F_n+1) are populations A′ and B′ of which eight males were selected for ChIP-Seq (blue shade).</p

Examples of quantitative H3K4me3 differences between the experimental populations.

<p>(A) H3K4me3 peaks were upmarked in population B at cholesterol biosynthesis regulator <i>Insig2</i> (position chr1∶123,229,166), <i>Plin5</i> (intracellular lipid storage droplet protein), <i>Agxt</i> (glyoxylate detoxifier), <i>Cyp4a14</i> (an arachidonic acid monooxygenase), and <i>Slco1a1</i> (bile acid/organic anion transporter). (B) H3K4me3 marks were reduced in population B at the TSS of insulin-like growth factor binding protein 2 (<i>Igfbp2</i>), membrane protein <i>Klhdc7a</i>, the immune regulator <i>Bcl3</i> (position chr7∶20,408,104), and the blood glucocorticoid transport protein (alpha globulin) <i>Serpina6</i>.</p