Placental contribution to the origins of sexual dimorphism in health and diseases: sex chromosomes and epigenetics

Sex differences occur in most non-communicable diseases, including metabolic diseases, hypertension, cardiovascular disease, psychiatric and neurological disorders and cancer. In many cases, the susceptibility to these diseases begins early in development. The observed differences between the sexes may result from genetic and hormonal differences and from differences in responses to and interactions with environmental factors, including infection, diet, drugs and stress. The placenta plays a key role in fetal growth and development and, as such, affects the fetal programming underlying subsequent adult health and accounts, in part for the developmental origin of health and disease (DOHaD). There is accumulating evidence to demonstrate the sex-specific relationships between diverse environmental influences on placental functions and the risk of disease later in life. As one of the few tissues easily collectable in humans, this organ may therefore be seen as an ideal system for studying how male and female placenta sense nutritional and other stresses, such as endocrine disruptors. Sex-specific regulatory pathways controlling sexually dimorphic characteristics in the various organs and the consequences of lifelong differences in sex hormone expression largely account for such responses. However, sex-specific changes in epigenetic marks are generated early after fertilization, thus before adrenal and gonad differentiation in the absence of sex hormones and in response to environmental conditions. Given the abundance of X-linked genes involved in placentogenesis, and the early unequal gene expression by the sex chromosomes between males and females, the role of X- and Y-chromosome-linked genes, and especially those involved in the peculiar placenta-specific epigenetics processes, giving rise to the unusual placenta epigenetic landscapes deserve particular attention. However, even with recent developments in this field, we still know little about the mechanisms underlying the early sex-specific epigenetic marks resulting in sex-biased gene expression of pathways and networks. As a critical messenger between the maternal environment and the fetus, the placenta may play a key role not only in buffering environmental effects transmitted by the mother but also in expressing and modulating effects due to preconceptional exposure of both the mother and the father to stressful conditions

Morphometric analysis of the placenta in the New World mouse Necromys lasiurus (Rodentia, Cricetidae): a comparison of placental development in cricetids and murids

Background: Stereology is an established method to extrapolate three-dimensional quantities from two-dimensional images. It was applied to placentation in the mouse, but not yet for other rodents. Herein, we provide the first study on quantitative placental development in a sigmodontine rodent species with relatively similar gestational time. Placental structure was also compared to the mouse, in order to evaluate similarities and differences in developmental patterns at the end of gestation. Methods: Fetal and placental tissues of Necromys lasiurus were collected and weighed at 3 different stages of gestation (early, mid and late gestation) for placental stereology. The total and relative volumes of placenta and of its main layers were investigated. Volume fractions of labyrinth components were quantified by the One Stop method in 31 placentae collected from different individuals, using the Mercator® software. Data generated at the end of gestation from N. lasiurus placentae were compared to those of Mus musculus domesticus obtained at the same stage. Results: A significant increase in the total absolute volumes of the placenta and its main layers occurred from early to mid-gestation, followed by a reduction near term, with the labyrinth layer becoming the most prominent area. Moreover, at the end of gestation, the total volume of the mouse placenta was significantly increased compared to that of N. lasiurus although the proportions of the labyrinth layer and junctional zones were similar. Analysis of the volume fractions of the components in the labyrinth indicated a significant increase in fetal vessels and sinusoidal giant cells, a decrease in labyrinthine trophoblast whereas the proportion of maternal blood space remained stable in the course of gestation. On the other hand, in the mouse, volume fractions of fetal vessels and sinusoidal giant cells decreased whereas the volume fraction of labyrinthine trophoblast increased compared to N. lasiurus placenta. Conclusions: Placental development differed between N. lasiurus and M. musculus domesticus. In particular, the low placental efficiency in N. lasiurus seemed to induce morphological optimization of fetomaternal exchanges. In conclusion, despite similar structural aspects of placentation in these species, the quantitative dynamics showed important differences.For technical support we thank Marie-Christine Aubrière, Michèle Dahirel of the Institut National de la Recherche Agronomique, Jouy-en-Josas, as well as members of the Universidade Federal Rural do Semi-Árido, Mossoró. We thank also Yves Maurin for Nanozoomer facilities. This research was supported by INRA and by grants from FAPESP (Process number: 09/53392-8).INRAFAPESP [09/53392-8

Meta-analysis of genome-wide DNA methylation and integrative omics of age in human skeletal muscle

International audienceBackground: Knowledge of age-related DNA methylation changes in skeletal muscle is limited, yet this tissue is severely affected by ageing in humans.Methods: We conducted a large-scale epigenome-wide association study meta-analysis of age in human skeletal muscle from 10 studies (total n = 908 muscle methylomes from men and women aged 18-89 years old). We explored the genomic context of age-related DNA methylation changes in chromatin states, CpG islands, and transcription factor binding sites and performed gene set enrichment analysis. We then integrated the DNA methylation data with known transcriptomic and proteomic age-related changes in skeletal muscle. Finally, we updated our recently developed muscle epigenetic clock (https://bioconductor.org/packages/release/bioc/html/MEAT.html).Results: We identified 6710 differentially methylated regions at a stringent false discovery rate <0.005, spanning 6367 unique genes, many of which related to skeletal muscle structure and development. We found a strong increase in DNA methylation at Polycomb target genes and bivalent chromatin domains and a concomitant decrease in DNA methylation at enhancers. Most differentially methylated genes were not altered at the mRNA or protein level, but they were nonetheless strongly enriched for genes showing age-related differential mRNA and protein expression. After adding a substantial number of samples from five datasets (+371), the updated version of the muscle clock (MEAT 2.0, total n = 1053 samples) performed similarly to the original version of the muscle clock (median of 4.4 vs. 4.6 years in age prediction error), suggesting that the original version of the muscle clock was very accurate.Conclusions: We provide here the most comprehensive picture of DNA methylation ageing in human skeletal muscle and reveal widespread alterations of genes involved in skeletal muscle structure, development, and differentiation. We have made our results available as an open-access, user-friendly, web-based tool called MetaMeth (https://sarah-voisin.shinyapps.io/MetaMeth/)

Sex- and Diet-Specific Changes of Imprinted Gene Expression and DNA Methylation in Mouse Placenta under a High-Fat Diet

Changes in imprinted gene dosage in the placenta may compromise the prenatal control of nutritional resources. Indeed monoallelic behaviour and sensitivity to changes in regional epigenetic state render imprinted genes both vulnerable and adaptable

Postnatal Survival of Mice with Maternal Duplication of Distal Chromosome 7 Induced by a Igf2/H19 Imprinting Control Region Lacking Insulator Function

The misexpressed imprinted genes causing developmental failure of mouse parthenogenones are poorly defined. To obtain further insight, we investigated misexpressions that could cause the pronounced growth deficiency and death of fetuses with maternal duplication of distal chromosome (Chr) 7 (MatDup.dist7). Their small size could involve inactivity of Igf2, encoding a growth factor, with some contribution by over-expression of Cdkn1c, encoding a negative growth regulator. Mice lacking Igf2 expression are usually viable, and MatDup.dist7 death has been attributed to the misexpression of Cdkn1c or other imprinted genes. To examine the role of misexpressions determined by two maternal copies of the Igf2/H19 imprinting control region (ICR)—a chromatin insulator, we introduced a mutant ICR (ICRΔ) into MatDup.dist7 fetuses. This activated Igf2, with correction of H19 expression and other imprinted transcripts expected. Substantial growth enhancement and full postnatal viability was obtained, demonstrating that the aberrant MatDup.dist7 phenotype is highly dependent on the presence of two unmethylated maternal Igf2/H19 ICRs. Activation of Igf2 is likely the predominant correction that rescued growth and viability. Further experiments involved the introduction of a null allele of Cdkn1c to alleviate its over-expression. Results were not consistent with the possibility that this misexpression alone, or in combination with Igf2 inactivity, mediates MatDup.dist7 death. Rather, a network of misexpressions derived from dist7 is probably involved. Our results are consistent with the idea that reduced expression of IGF2 plays a role in the aetiology of the human imprinting-related growth-deficit disorder, Silver-Russell syndrome

H19 Antisense RNA Can Up-Regulate Igf2 Transcription by Activation of a Novel Promoter in Mouse Myoblasts

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions

Des mécanismes épigénétiques aux Origines Développementales de la Santé: Métabolisme maternel et réponse spécifique du sexe de la descendance

Early exposures to biological and/or social stressors can have a long-term impact on the health of adults. However, the biological mechanisms underlying these developmental origins of health and disease (DOHaD) are not fully understood. Epigenetic mechanisms are reliable candidates to explain how early events could permanently modify physiology and influence the phenotype in adulthood.During my career, I approached epigenetics under the aspect of the genomic imprinting and then of the memorization of the impact of the environment and its effect on the gene expression within the framework of the DOHaD.Since my engagement as an INRAE researcher, my projects have focused on the effects of obesity before and during gestation with two aspects: the possibility of preconception correction and the exposure of individuals to several stresses of different nature. But the associated epigenetic mechanisms and persistent epigenetic markings, potential long-term memory of early exposure, are often intuitively exposed in reviews of DOHaD but not fully formally demonstrated. This question is inextricably linked with questions of sex, females and males responding differently to the environment. The epigenetic aspect certainly partly underlies these differences.Les expositions précoces à des facteurs de stress biologiques et/ou sociaux peuvent avoir un impact à long terme sur la santé des adultes. Cependant, les mécanismes biologiques qui sous-tendent ces origines développementales de la santé et de la maladie (DOHaD) ne sont pas entièrement compris. Les mécanismes épigénétiques sont de bons candidats pour expliquer comment des évènements précoces pourraient modifier durablement la physiologie et influencer le phénotype à l’âge adulte.Au cours de ma carrière, j’ai abordé l’épigénétique sous l’aspect de l’empreinte parentale puis de l'archivage de l'impact de l'environnement et son effet sur l'expression des gènes dans le cadre de la DOHaD.Depuis mon recrutement comme chargée de recherche, mes projets portent sur les effets de l’obésité avant et au cours de la gestation avec deux aspects : la possibilité de correction préconceptionnelle et l’exposition des individus à plusieurs stress de nature différente. Mais les mécanismes épigénétiques associés et les marques épigénétiques rémanentes, potentielle mémoire à long-terme de l’exposition précoce sont souvent exposé de façon intuitive dans les revues de synthèse sur la DOHaD mais pas complètement formellement démontré. Cette question est indissociable des questions liées au sexe, les femelles et les mâles répondant différemment à l’environnement. L’aspect épigénétique sous- tend certainement en partie ces différences

Etude de la fonction de l'ARN non codant H19 et de la régulation de l'Empreinte Parentale au locus H19/lgf2 dans des modèles murins transgéniques

The H19 gene was first described twenty years ago and its function remains unknown. This gene is imprinted; it is expressed only from the maternal allele. The product of the gene is a 2. 3 kb non-coding RNA abundantly expressed in mesoderm and endoderm derived tissues during embryogenesis. After birth, the expression is repressed in ail tissues except skeletal muscle where it remains strongly expressed at adult stage. The H19 gene is linked genetically to the paternally expressed /g/2 gene, which encodes an embryonic growth factor, and they share the mechanism of transcriptional regulation. The deletion of the H19 gene in mice leads to an overgrowth phenotype, which is attributed to a loss of imprinting of the Igf2 gene. We addressed the questions of the role of the H19 RNA during embryogenesis and of the regulation of this imprinted locus in adult muscle tissue. The particularity of our approach is the use of in vivo transgenic mouse models. We showed in three independent models of induced tumorigenesis that the H19 gene acts as a tumour suppressor in vivo. We then analysed transgenic animals which ectopically expressed the H19 gene and found that this RNA is a trans regulator of a recently described imprinted gene network, implicated in growth regulation. Finally we observed a biallelic expression of the H19 and /g/2 genes in satellites cells, which are muscle stem cells, as well as in haematopoietic stem cells, suggesting that a relaxation of imprinting could be a characteristic of adult stem cells.Le gène H19 a été découvert il y a un vingtaine d'années et sa fonction reste encore inconnue. Ce gène est soumis à l'Empreinte Parentale, son expression est monoallélique maternelle. Par ailleurs le transcrit produit à partir de ce gène est un ARN non codant de 2,3 kb. Il est fortement exprimé au cours de l'embryogenèse dans tous les tissus issus du mésoderme et de l'endoderme et est réprimé après la naissance. Le seul organe où il reste fortement exprimé chez l'adulte est le muscle squelettique. Le gène H19 est lié au niveau de sa localisation chromosomique et de la régulation transcriptionnelle au gène /g/2, qui code un facteur de croissance embryonnaire exprimé de façon monoallélique paternelle. L'invalidation du gène H19 chez la souris entraîne un phénotype de surcroissance attribué à la perte d'Empreinte du gène /g/2. Nous avons voulu adresser deux questions portant d'une part sur la fonction de l'ARN H19 au cours de l'embryogenèse et d'autre part sur la régulation de ce locus dans le muscle adulte. L'originalité de notre approche réside dans l'étude in vivo de modèles murins transgéniques. Nos résultats montrent, à partir de trois modèles murins de tumorigenèse induite, que le gène H19 a une fonction de gène suppresseur de tumeur in vivo. Par ailleurs nous avons montré à partir de souris exprimant le gène H19 de façon ectopique que cet ARN est un trans régulateur d'un réseau de gènes soumis à l'Empreinte, impliqué dans la régulation de la croissance. Enfin, nous avons observé une expression biallélique des gènes H19 et /g/2 dans les cellules satellites, qui sont les cellules souches musculaires, ainsi que dans les cellules souches hématopoïétiques, suggérant qu'un relâchement de l'Empreinte puisse être une caractéristique des cellules souches adultes

Effects of maternal obesity on offspring health: implication of the histone aceylation pathway

National audienceAccording to the “developmental origins of health and disease” (DOHaD) concept,maternal obesity predisposes the offspring to non-communicable diseases in adulthood. Epigenetic mechanisms could be affected by maternal weight changes, perturbing expression of key developmental genes in the placenta or fetus. Our aim was to investigate the effects of chronic maternal obesity on feto-placental growth along with the underlying epigenetic mechanisms. Moreover, while a preconceptional weight loss (WL) is recommended for obese women, its benefits on the offspring have been poorly addressed. We therefore tested whether preconceptional weight loss could alleviate these effects.At embryonic day 18.5 (E18.5), fetuses from obese females (OB group) presented fetal growth restriction. After weaning, the offspring were either put on a control diet (CD) or a high-fat (HFD) and we tracked their metabolic and olfactory behavioral trajectories. After only few weeks of HFD, the offspring developed obesity, metabolic alterations and olfactory impairments, independently of maternal context. However, male offspring born to obese mother gained even more weight under HFD than their counterparts born to lean mothers. Preconceptional WL normalized the offspring growth and metabolic phenotypes but had a reduction in olfactory sensitivity, along with a lack of fasting-induced, olfactory-based motivation. We measured the expression of 60 epigenetic machinery genes and 32 metabolic genes in the fetal liver, placental labyrinth, and junctional zone. One third of the epigenetic machinery genes were differentially expressed between at least two maternal groups. Interestingly, genes involved in the histone acetylation pathway were particularly altered. In OB group, lysine acetyltransferases and Bromodomain-containing protein 2 were upregulated, while most histone deacetylases were downregulated. In WL group, the expression of only a subset of these genes was normalized.This study highlights the high sensitivity of the epigenetic machinery gene expression, and particularly the histone acetylation pathway, to maternal obesity. Preconceptional weight loss appears beneficial to fetal growth, but induces some possible adverse outcomes on olfactory-based behaviors. These obesity-induced transcriptional changes could alter the placental and the hepatic epigenome, leading to FGR. We are currently developing methodologies to study the whole genome histone acetylation profile on snap-frozen tissue from this project biobank