An unbiased assessment of the role of imprinted genes in an intergenerational model of developmental programming.

Environmental factors during early life are critical for the later metabolic health of the individual and of future progeny. In our obesogenic environment, it is of great socioeconomic importance to investigate the mechanisms that contribute to the risk of metabolic ill health. Imprinted genes, a class of functionally mono-allelic genes critical for early growth and metabolic axis development, have been proposed to be uniquely susceptible to environmental change. Furthermore, it has also been suggested that perturbation of the epigenetic reprogramming of imprinting control regions (ICRs) may play a role in phenotypic heritability following early life insults. Alternatively, the presence of multiple layers of epigenetic regulation may in fact protect imprinted genes from such perturbation. Unbiased investigation of these alternative hypotheses requires assessment of imprinted gene expression in the context of the response of the whole transcriptome to environmental assault. We therefore analyse the role of imprinted genes in multiple tissues in two affected generations of an established murine model of the developmental origins of health and disease using microarrays and quantitative RT-PCR. We demonstrate that, despite the functional mono-allelicism of imprinted genes and their unique mechanisms of epigenetic dosage control, imprinted genes as a class are neither more susceptible nor protected from expression perturbation induced by maternal undernutrition in either the F1 or the F2 generation compared to other genes. Nor do we find any evidence that the epigenetic reprogramming of ICRs in the germline is susceptible to nutritional restriction. However, we propose that those imprinted genes that are affected may play important roles in the foetal response to undernutrition and potentially its long-term sequelae. We suggest that recently described instances of dosage regulation by relaxation of imprinting are rare and likely to be highly regulated

In utero undernutrition in male mice programs liver lipid metabolism in the second-generation offspring involving altered lxra DNA methylation

SummaryObesity and type 2 diabetes have a heritable component that is not attributable to genetic factors. Instead, epigenetic mechanisms may play a role. We have developed a mouse model of intrauterine growth restriction (IUGR) by in utero malnutrition. IUGR mice developed obesity and glucose intolerance with aging. Strikingly, offspring of IUGR male mice also developed glucose intolerance. Here, we show that in utero malnutrition of F1 males influenced the expression of lipogenic genes in livers of F2 mice, partly due to altered expression of Lxra. In turn, Lxra expression is attributed to altered DNA methylation of its 5′ UTR region. We found the same epigenetic signature in the sperm of their progenitors, F1 males. Our data indicate that in utero malnutrition results in epigenetic modifications in germ cells (F1) that are subsequently transmitted and maintained in somatic cells of the F2, thereby influencing health and disease risk of the offspring

The role of nutrition on epigenetic modifications and their implications on health

Nutrition plays a key role in many aspects of health and dietary imbalances are major determinants of chronic diseases including cardiovascular disease, obesity, diabetes and cancer. Adequate nutrition is particularly essential during critical periods in early life (both pre- and postnatal). In this regard, there is extensive epidemiologic and experimental data showing that early sub-optimal nutrition can have health consequences several decades later. The hypothesis that epigenetic mechanisms may link such nutritional imbalances with altered disease risk has been gaining acceptance over recent years. Epigenetics can be defined as the study of heritable changes in gene expression that do not involve alterations in the DNA sequence. Epigenetic marks include DNA methylation, histone modifications and a variety of non-coding RNAs. Strikingly, they are plastic and respond to environmental signals, including diet. Here we will review how dietary factors modulate the establishment and maintenance of epigenetic marks, thereby influencing gene expression and, hence, disease risk and health. (C) 2012 Elsevier Masson SAS. All rights reserved

Epigenetic programming at the Mogat1 locus may link neonatal overnutrition with long-term hepatic steatosis and insulin resistance

Postnatal overfeeding increases the risk of chronic diseases later in life, including obesity, insulin resistance, hepatic steatosis, and type 2 diabetes. Epigenetic mechanisms might underlie the long-lasting effects associated with early nutrition. Here we aimed to explore the molecular pathways involved in early development of insulin resistance and hepatic steatosis, and we examined the potential contribution of DNA methylation and histone modifications to long-term programming of metabolic disease. We used a well-characterized mouse model of neonatal overfeeding and early adiposity by litter size reduction. Neonatal overfeeding led to hepatic insulin resistance very early in life that persisted throughout adulthood despite normalizing food intake. Up-regulation of monoacylglycerol O-acyltransferase (Mogat)1 conceivably mediates hepatic steatosis and insulin resistance through increasing intracellular diacylglycerol content. Early and sustained deregulation of Mogat1 was associated with a combination of histone modifications that might favor Mogat1 expression. In sum, postnatal overfeeding causes extremely rapid derangements of hepatic insulin sensitivity that remain relatively stable until adulthood. Epigenetic mechanisms, particularly histone modifications, could contribute to such long-lasting effects. Our data suggest that targeting hepatic monoacylglycerol acyltransferase activity during early life might provide a novel strategy to improve hepatic insulin sensitivity and prevent late-onset insulin resistance and fatty liver disease.Ramon-Krauel, M., Pentinat, T., Bloks, V. W., Cebria, J., Ribo, S., Perez-Wienese, R., Vila, M., Palacios-Marin, I., Fernandez-Perez, A., Vallejo, M., Tellez, N., Rodriguez, M. ., Yanes, O., Lerin, C., Diaz, R., Plosch, T., Tietge, U. J. F., Jimenez-Chillaron, J. C. Epigenetic programming at the Mogat1 locus may link neonatal overnutrition with long-term hepatic steatosis and insulin resistance

Expression of candidate imprinted genes at E16.5 in the F2 generation and assessment of F1 germline imprints.

<p>(A) Schematic of the F2 generation, expression was assessed at E16.5. (B) There are no significant differences in the F2 hepatic expression of imprinted genes at E16.5. Error bars denote SEM. (C) At E16.5 CU individuals demonstrate a significant increased in placental expression of <i>Igf2P0</i> (One-way ANOVA, Bonferroni's multiple comparison post-test. P<0.05), while UU placentas significant up-regulate <i>Snrpn</i> (One-way ANOVA, Bonferroni's multiple comparison post-test. P<0.01). Per condition n≥24, 6 litters. Error bars denote SEM. (D, E) Methylation was assessed by pyrosequencing at the paternally methylated <i>H19</i> (A) and <i>Dlk1/Dio3</i> (B) germline ICRs. Sperm from both control and <i>in utero</i> undernourished males showed the expected hypermethylation in comparison to somatic tissues (liver). Controls: n = 12, 5 litters. Undernourished n = 11, 4 litters. Error bars denote SEM. (F, G) Pyrosequencing assessment of methylation at the maternally methylated <i>Peg3</i> (C) and <i>Snrpn</i> (D) germline DMRs shows that these regions are unmethylated in the sperm of both control and <i>in utero</i> undernourished males. Controls: n = 12, 5 litters Undernourished n = 11, 4 litters. Error bars denote SEM.</p

Characterisation of the E16.5 hepatic and placental transcriptome response to undernourishment.

<p>(A) Schematic of the experimental design: F1 generation: On pregnancy day 12.5, dams were randomly assigned to either control or undernutrition groups and food intake of undernutrition mothers was restricted to 50% that of controls. After delivery litter size was equalized to eight pups and dams received 9F chow <i>ad libitum</i>. Pups nursed freely and were weaned at 3 weeks onto 9F chow <i>ad libitum</i>. F2 generation: control and undernourished females from the F1 generation were mated at age 2 months with nonsibling control or undernourished males. After confirmation of pregnancy, females were caged individually and fed <i>ad libitum</i> throughout pregnancy to produce the four experimental F2 generation groups: CC – both parents are controls. CU – control dam, <i>in utero</i> undernourished sire; UC - <i>in utero</i> undernourished dam, control sire; UU - <i>in utero</i> undernourished dam, <i>in utero</i> undernourished sire. (B, C) Gene ontology (GO) analysis of the F1 undernourished hepatic transcriptome at E16.5: Functional enrichment analysis using (B) DAVID <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002605#pgen.1002605-Dennis1" target="_blank">[27]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002605#pgen.1002605-Huangda1" target="_blank">[28]</a> and (C) Ingenuity Pathway Analysis functional analysis tools. Among genes upregulated in the liver, both tools identify significant enrichment (after Benjamini-Hochberg correction for multiple testing) of gene groups associated with metabolism, particularly of lipids. Among genes downregulated in the liver, both tools identify significant enrichment of categories related to cell-cycle and the control of proliferation. (D) Transcriptome analysis of E16.5 undernourished versus control liver. Distribution of the ranked difference in gene expression according to FDR q-value. Lists of housekeeping genes and adult hepatic fasting-response genes curated from the literature were used as negative and positive controls respectively and their rankings shown. Imprinted genes most closely resemble randomly selected genes. (E) Transcriptome analysis of E16.5 undernourished versus control placenta; distribution of genes according to FDR q-value. The placental response to maternal undernourishment is undetermined, and is different to that of the liver, as fasting response genes are largely unperturbed. Therefore a network of genes involved in RNA post-transcriptional modification, identified as enriched in undernourished placenta by IPA analysis; was used as a positive control. Imprinted genes most closely resemble randomly selected genes.</p