Relation entre la méthylation des promoteurs du gène IGF1 et les variations de la croissance des enfants

At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...

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/)

Relationship between DNA methylation of IGF1 gene promoters and child growth variations

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...)At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role

Epigenetic contribution to obesity

Obesity is a worldwide epidemic and contributes to global morbidity and mortality mediated via the development of nonalcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), cardiovascular (CVD) and other diseases. It is a consequence of an elevated caloric intake, a sedentary lifestyle and a genetic as well as an epigenetic predisposition. This review summarizes changes in DNA methylation and microRNAs identified in blood cells and different tissues in obese human and rodent models. It includes information on epigenetic alterations which occur in response to fat-enriched diets, exercise and metabolic surgery and discusses the potential of interventions to reverse epigenetic modifications

Enriched Alternative Splicing in Islets of Diabetes-Susceptible Mice

Dysfunctional islets of Langerhans are a hallmark of type 2 diabetes (T2D). We hypothesize that differences in islet gene expression alternative splicing which can contribute to altered protein function also participate in islet dysfunction. RNA sequencing (RNAseq) data from islets of obese diabetes-resistant and diabetes-susceptible mice were analyzed for alternative splicing and its putative genetic and epigenetic modulators. We focused on the expression levels of chromatin modifiers and SNPs in regulatory sequences. We identified alternative splicing events in islets of diabetes-susceptible mice amongst others in genes linked to insulin secretion, endocytosis or ubiquitin-mediated proteolysis pathways. The expression pattern of 54 histones and chromatin modifiers, which may modulate splicing, were markedly downregulated in islets of diabetic animals. Furthermore, diabetes-susceptible mice carry SNPs in RNA-binding protein motifs and in splice sites potentially responsible for alternative splicing events. They also exhibit a larger exon skipping rate, e.g., in the diabetes gene Abcc8, which might affect protein function. Expression of the neuronal splicing factor Srrm4 which mediates inclusion of microexons in mRNA transcripts was markedly lower in islets of diabetes-prone compared to diabetes-resistant mice, correlating with a preferential skipping of SRRM4 target exons. The repression of Srrm4 expression is presumably mediated via a higher expression of miR-326-3p and miR-3547-3p in islets of diabetic mice. Thus, our study suggests that an altered splicing pattern in islets of diabetes-susceptible mice may contribute to an elevated T2D risk

Polymorphisms in miRNA binding sites involved in metabolic diseases in mice and humans

Type 2 diabetes and obesity are well-studied metabolic diseases, which are based on genetic and epigenetic alterations in combination with an obesogenic lifestyle. The aim of this study was to test whether SNPs in miRNA-mRNA binding sites that potentially disrupt binding, elevate the expression of miRNA targets, which participate in the development of metabolic diseases. A computational approach was developed that integrates transcriptomics, linkage analysis, miRNA-target prediction data, and sequence information of a mouse model of obesity and diabetes. A statistical analysis demonstrated a significant enrichment of 566 genes for a location in obesity- and diabetes-related QTL. They are expressed at higher levels in metabolically relevant tissues presumably due to altered miRNA-mRNA binding sites. Of these, 51 genes harbor conserved and impaired miRNA-mRNA-interactions in human. Among these, 38 genes have been associated to metabolic diseases according to the phenotypes of corresponding knockout mice or other results described in the literature. The remaining 13 genes (e.g. Jrk, Megf9, Slfn8 and Tmem132e) could be interesting candidates and will be investigated in the future

MiR‐205 is upregulated in islets of diabetes‐susceptible mice and targets the diabetes gene Tcf7l2

Aim: MicroRNAs play an important role in the maintenance of cellular functions by fine-tuning gene expression levels. The aim of the current study was to identify genetically caused changes in microRNA expression which associate with islet dysfunction in diabetic mice. Methods: To identify novel microRNAs involved in islet dysfunction, transcriptome and miRNome analyses were performed in islets of obese, diabetes-susceptible NZO and diabetes-resistant B6-ob/ob mice and results combined with quantitative trait loci (QTL) and functional in vitro analysis. Results: In islets of NZO and B6-ob/ob mice, 94 differentially expressed microRNAs were detected, of which 11 are located in diabetes QTL. Focusing on conserved microRNAs exhibiting the strongest expression difference and which have not been linked to islet function, miR-205-5p was selected for further analysis. According to transcriptome data and target prediction analyses, miR-205-5p affects genes involved in Wnt and calcium signalling as well as insulin secretion. Over-expression of miR-205-5p in the insulinoma cell line INS-1 increased insulin expression, left-shifted the glucose-dependence of insulin secretion and supressed the expression of the diabetes gene TCF7L2. The interaction between miR-205-5p and TCF7L2 was confirmed by luciferase reporter assay. Conclusion: MiR-205-5p was identified as relevant microRNA involved in islet dysfunction by interacting with TCF7L2

MiR-205 is up-regulated in islets of diabetes-susceptible mice and targets the diabetes gene Tcf7l2

AIM: MicroRNAs play an important role in the maintenance of cellular functions by fine-tuning gene expression levels. The aim of the current study was to identify genetically caused changes in microRNA expression which associate with islet dysfunction in diabetic mice. METHODS: To identify novel microRNAs involved in islet dysfunction, transcriptome and miRNome analyses were performed in islets of obese, diabetes-susceptible NZO and diabetes-resistant B6-ob/ob mice and results combined with quantitative trait loci (QTL) and functional in vitro analysis. RESULTS: In islets of NZO and B6-ob/ob mice, 94 differentially expressed microRNAs were detected, of which 11 are located in diabetes QTL. Focusing on conserved microRNAs exhibiting the strongest expression difference and which have not been linked to islet function, miR-205-5p was selected for further analysis. According to transcriptome data and target prediction analyses, miR-205-5p affects genes involved in Wnt and calcium signalling as well as insulin secretion. Over-expression of miR-205-5p in the insulinoma cell line INS-1 increased insulin expression, left-shifted the glucose-dependence of insulin secretion and supressed the expression of the diabetes gene TCF7L2. The interaction between miR-205-5p and TCF7L2 was confirmed by luciferase reporter assay. CONCLUSION: MiR-205-5p was identified as relevant microRNA involved in islet dysfunction by interacting with TCF7L2

Epigenetic changes in islets of langerhans preceding the onset of diabetes

The identification of individuals with a high risk of developing type 2 diabetes (T2D) is fundamental for pre-vention. Here, we used a translational approach and prediction criteria to identify changes in DNA methylation visible before the development of T2D. Islets of Langerhans were isolated from genetically identical 10-week-old female New Zealand Obese mice, which differ in their degree of hyperglycemia and in liver fat content. The application of a semiexplorative approach identified 497 differentially expressed and methylated genes (P = 6.42e-09, hypergeometric test) enriched in pathways linked to insulin secretion and extracellular matrix-receptor interaction. The comparison of mouse data with DNA methylation levels of incident T2D cases from the prospective European Prospective Investigation of Cancer (EPIC)-Potsdam cohort, revealed 105 genes with altered DNA methylation at 605 cytosine-phosphate-guanine (CpG) sites, which were associated with future T2D. AKAP13, TENM2, CTDSPL, PTPRN2, and PTPRS showed the strongest predictive potential (area under the receiver operating characteristic curve values 0.62–0.73). Among the new candidates identified in blood cells, 655 CpG sites, located in 99 genes, were differentially methylated in islets of humans with T2D. Using correction for multiple testing detected 236 genes with an altered DNA methylation in blood cells and 201 genes in diabetic islets. Thus, the introduced translational approach identified novel putative biomarkers for early pancreatic islet aberrations preceding T2D