Network of methylation changes directly correlated with transcriptional activity.

<p>Methylation changes directly correlated with transcriptional activity. Methylation-expression relationships were measured by beta regression of differentially methylated regions (DMRs, uncorrected p-value <0.05, n = 6,927) associated with HDM-treated C57BL/6^{<i>Mthfr</i>-/-} mice and all expression probes found within 1Mb of each DMR. Ingenuity Pathway Analysis (IPA) on the 503 significant methylation-expression correlations (adjusted p-value <0.05) identified a significant inflammatory response network (score = 52). Green indicates lower methylation or expression and red indicates higher methylation or expression in HDM-treated C57BL/6^{<i>Mthfr</i>-/-} mice. Methylation values are colored based on relative methylation change between HDM-treated C57BL/6^{<i>Mthfr</i>-/-} and HDM-treated C57BL/6 mice; colors of expression values are based on fold change between HDM-treated C57BL/6^{<i>Mthfr</i>-/-} and HDM-treated C57BL/6 mice. Molecule shapes: horizontal oval = transcriptional regulator; vertical oval = transmembrane receptor; diamond = enzyme; up triangle = phosphatase; down triangle = kinase; trapezoid = transporter; circle = other; double circle = group; rectangle = G-protein coupled receptor. This analysis was restricted to only direct relationships. The network score is based on the hypergeometric distribution, and is calculated with the right-tailed Fisher’s exact test to identify enrichment of correlated methylated/expressed genes in the network relative to IPA database. The other network with a score greater than 40 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190916#pone.0190916.s006" target="_blank">S5 Fig</a>.</p

Methylene-tetrahydrofolate reductase contributes to allergic airway disease

<div><p>Rationale</p><p>Environmental exposures strongly influence the development and progression of asthma. We have previously demonstrated that mice exposed to a diet enriched with methyl donors during vulnerable periods of fetal development can enhance the heritable risk of allergic airway disease through epigenetic changes. There is conflicting evidence on the role of folate (one of the primary methyl donors) in modifying allergic airway disease.</p><p>Objectives</p><p>We hypothesized that blocking folate metabolism through the loss of methylene-tetrahydrofolate reductase (Mthfr) activity would reduce the allergic airway disease phenotype through epigenetic mechanisms.</p><p>Methods</p><p>Allergic airway disease was induced in C57BL/6 and C57BL/6^{<i>Mthfr</i>-/-} mice through house dust mite (HDM) exposure. Airway inflammation and airway hyperresponsiveness (AHR) were measured between the two groups. Gene expression and methylation profiles were generated for whole lung tissue. Disease and molecular outcomes were evaluated in C57BL/6 and C57BL/6^{<i>Mthfr</i>-/-} mice supplemented with betaine.</p><p>Measurements and main results</p><p>Loss of Mthfr alters single carbon metabolite levels in the lung and serum including elevated homocysteine and cystathionine and reduced methionine. HDM-treated C57BL/6^{<i>Mthfr</i>-/-} mice demonstrated significantly less airway hyperreactivity (AHR) compared to HDM-treated C57BL/6 mice. Furthermore, HDM-treated C57BL/6^{<i>Mthfr</i>-/-} mice compared to HDM-treated C57BL/6 mice have reduced whole lung lavage (WLL) cellularity, eosinophilia, and Il-4/Il-5 cytokine concentrations. Betaine supplementation reversed parts of the HDM-induced allergic airway disease that are modified by Mthfr loss. 737 genes are differentially expressed and 146 regions are differentially methylated in lung tissue from HDM-treated C57BL/6^{M<i>thfr</i>-/-} mice and HDM-treated C57BL/6 mice. Additionally, analysis of methylation/expression relationships identified 503 significant correlations.</p><p>Conclusion</p><p>Collectively, these findings indicate that the loss of folate as a methyl donor is a modifier of allergic airway disease, and that epigenetic and expression changes correlate with this modification. Further investigation into the mechanisms that drive this observation is warranted.</p></div

Expression and methylation results were confirmed.

<p>(A) 8 differentially expressed genes from the array data were quantified through RT-PCR. 6 out of 8 genes validated. (B) <i>Rasgrp4</i>, <i>Tle4</i>, and <i>Fcer1g</i> DMRs were validated through pyrosequencing. (* p-value <0.05, ** p-value <0.01 *** p-value <0.001).</p

Single carbon metabolite levels differ between C57BL/6^{<i>Mthfr</i>-/-} and C57BL/6 mice.

<p>(A) Single Carbon metabolism pathway. C57BL/6^{M<i>thfr</i>-/-} mice demonstrate altered levels of single carbon metabolites (homocysteine, HCYS; cystathionine, CYSTAT; dimethylglycine, DMG; methylglycine, MG; methionine, METH; cysteine, CYS; glycine, GLY; alpha aminobutyrate, ABUT; serine, SER) in (B) serum and (C) whole lung tissue compared to C57BL/6 mice (C57BL/6 black bars; C57Bl/6^{<i>Mthfr</i>-/-} white bars, * p-value <0.05, *** p-value <0.001).</p

Hierarchal clustering of differentially expressed genes between HDM-treated C57BL/6^{<i>Mthfr</i>-/-} and HDM-treated C57BL/6.

<p>ANOVA analysis identified 2,588 transcripts that are differentially expressed between the four experimental groups. Gene clustering by similarity in expression as quantified by the Pearson correlation using average linkage hierarchical clustering show groups of genes related to <i>Mthfr</i> status as well as treatment dependent clusters.</p

Novel alternations did not differ but mNPC treatment increased Trisomic performance above chance performance.

<p>Overall, trisomic mice showed significantly fewer novel alternations compared to disomic mice (* p<0.05). Treatment with either saline or mNPC did not significantly change the number of novel alternations in either karyotype compared to their respective controls. However, trisomic mice treated with mNPC alternated significantly above chance alone (solid line), while untreated and saline treated trisomic mice did not. Mean ± SEM shown. * Significant main effect of karyotype. ∧ Novel alternations were significantly above chance.</p

Betaine supplementation exacerbated allergic airway disease.

<p>Betaine supplementation affected single carbon metabolites (A) HYCS, CYSTAT, and METH (not supplemented C57BL/6 black bars, supplemented C57BL/6 vertical striped bars, not supplemented C57Bl/6^{<i>Mthfr</i>-/-} white bars, supplemented C57BL/6^{<i>Mthfr</i>-/-} checkered bars). Betaine supplementation exacerbated the severity of HDM-induced allergic airway disease and abolished the effect of Mthfr loss. (B) Airway hyperresponsiveness (not supplemented HDM-treated C57BL/6^{<i>Mthfr</i>-/-} solid line closed circle, and supplemented C57BL/6^{<i>Mthfr</i>-/-} dashed line open circle and X), and (C) total cells, (D) eosinophils, (E) percentage of eosinophils in WLL. (F) Expression and (G) methylation of <i>Tle4</i> is altered by betaine supplementation. (* p-value <0.05, ** p-value <0.01 *** p-value <0.001).</p

Trisomic mice continued to drink significantly more CS on second exposure than disomic mice.

<p>There was no significant difference in degree of preference during first exposure between disomic and trisomic animals. During the second exposure disomic mice had a greater avoidance of the CS than the trisomic animals (* p<0.05). Treatment did not preferentially affect CTA learning and affected both karyotypes to the same degree. Mean ± SEM shown.</p

All groups recognized the novel object.

<p>All groups exhibited a preference for the novel object, as indicated by a DI above zero. While there is apparent variability, the differences in the DI between groups did not reach significance (p>0.05). Mean ± SEM shown.</p

GFP+ cells found in areas outside of the hippocampus.

<p>A. Half of trisomic mice had GFP+ cells in the entorhinal cortex (arrows). B. GFP+ cells were found near the lateral ventricles in the septum (arrows). C. GFP+ mNPC also were found in the CC and again in the underlying septal areas (arrows). rf, rhinal fissure; lv, lateral ventricles; CC, corpus callosum. Scale Bars in A = 100 µm and in B–C = 200 µm.</p