Miguel Constância

Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19

Igf2 and H19 are closely linked, reciprocally imprinted genes on mouse distal chromosome 7. The paternally expressed Igf2 encodes a potent fetal growth factor and the maternally expressed H19 encodes a non-coding RNA (refs 1,2). Shared endoderm-specific enhancers 3′ to H19 are necessary for transcription of the maternal copy of H19 and the paternal copy of Igf2 (ref. 3), a chromatin boundary upstream of H19 preventing access of the enhancers to the maternal Igf2 promoters. Mesoderm-specific control elements have not been identified, and the role of differentially methylated regions (DMRs) in Igf2 has not been addressed. Two DMRs in Igf2 are methylated on the active paternal allele, suggesting that they contain silencers. Here we have deleted the DMR1 region in Igf2. Maternal transmission of the deletion results in biallelic expression of Igf2 in most mesodermally derived tissues without altering H19 imprinting or expression. Paternal or maternal transmission leads to continued postnatal transcription of Igf2, in contrast to the wild-type allele, which is silenced soon after birth. These results reveal a mesodermal silencer, which may be regulated by methylation and which has a major role in H19-independent expression and imprinting control of Igf2. Our results establish a new mechanistic principle for imprinted genes whereby epigenetically regulated silencers interact with enhancers to control expression, and suggest a new mechanism for loss of imprinting (LOI) of Igf2, which may be important in a number of diseases.

An intragenic methylated region in the imprinted Igf2 gene augments transcription.

DNA methylation is usually associated with transcriptional silencing, but in the imprinted mouse Igf2 gene, the paternally expressed copy is methylated in two discrete differentially methylated regions (DMRs). DMR1 is located upstream of the fetal promoters and has been shown to be a methylation sensitive silencer. Here we examine the role of the intragenic DMR2 by gene targeting. In contrast to DMR1, deletion of DMR2 on the maternal allele did not lead to activation of the silent Igf2 gene. Deletion of a 54 bp methylated core region in DMR2 on the paternal allele, however, reduced Igf2 mRNA levels and was associated with fetal growth retardation. Nuclear run‐on assays showed that the core region influenced transcription initiation, and luciferase reporter assays suggested that its methylation increases transcription. These results reveal a novel mechanism of gene expression whereby intragenic methylation can increase levels of transcription.

Adaptations in placental nutrient transfer capacity to meet fetal growth demands depend on placental size in mice

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra‐litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up‐regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.

DNA methylation profiling at imprinted loci after periconceptional micronutrient supplementation in humans: results of a pilot randomized controlled trial

Intrauterine exposures mediated by maternal diet may affect risk of cardiovascular disease, obesity, and type 2 diabetes. Recent evidence, primarily from animal studies and observational data in humans, suggests that the epigenome can be altered by maternal diet during the periconceptional period and that these programming events may underlie later disease risk. A randomized controlled trial of periconceptional micronutrient supplementation in The Gambia, where seasonal nutritional variations affect fetal growth and postnatal outcomes, provided a unique opportunity to test this hypothesis. Specifically, we targeted imprinted genes, which play important roles in allocation of maternal resources while being epigenetically regulated. DNA methylation at 12 differentially methylated regions (DMRs) was analyzed in cord blood samples from 58 offspring of women participating in a doubleblind randomized‐controlled trial of pre‐ and periconceptional micronutrient supplementation (including folate, zinc, and vitamins A, B, C, and D). We observed sex‐specific effects of micronutrient supplementation, reducing methylation levels at two of the DMRs analyzed, IGF2R in girls and GTL2‐2 in boys. This pilot study is the first to analyze DNA methylation in the context of a randomized controlled trial, and it provides suggestive evidence that periconceptional maternal nutrition alters offspring methylation at imprinted loci.—Cooper, W. N., Khulan, B., Owens, S., Elks, C. E., Seidel, V., Prentice, A. M., Belteki, G., Ong, K. K., Affara, N. A., Constância, M., Dunger, D. B. DNA methylation profiling at imprinted loci after periconceptional micronutrient supplementation in humans: results of a pilot randomized controlled trial. FASEB J. 26, 1782‐1790 (2012). www.fasebj.org

Placental adaptations to the maternal–fetal environment: implications for fetal growth and developmental programming

The placenta is a transient organ found in eutherian mammals that evolved primarily to provide nutrients for the developing fetus. The placenta exchanges a wide array of nutrients, endocrine signals, cytokines and growth factors with the mother and the fetus, thereby regulating intrauterine development. Recent studies show that the placenta is not just a passive organ mediating maternal-fetal exchange. It can adapt its capacity to supply nutrients in response to intrinsic and extrinsic variations in the maternal-fetal environment. These dynamic adaptations are thought to occur to maximize fetal growth and viability at birth in the prevailing conditions in utero. However, some of these adaptations may also affect the development of individual fetal tissues, with patho-physiological consequences long after birth. Here, this review summarizes current knowledge on the causes, possible mechanisms and consequences of placental adaptive responses, with a focus on the regulation of transporter-mediated processes for nutrients. This review also highlights the emerging roles that imprinted genes and epigenetic mechanisms of gene regulation may play in placental adaptations to the maternal-fetal environment.

Placental-specific Igf2 knockout mice exhibit hypocalcemia and adaptive changes in placental calcium transport

Evidence is emerging that the ability of the placenta to supply nutrients to the developing fetus adapts according to fetal demand. To examine this adaptation further, we tested the hypothesis that placental maternofetal transport of calcium adapts according to fetal calcium requirements. We used a mouse model of fetal growth restriction, the placental-specific Igf2 knockout (P0) mouse, shown previously to transiently adapt placental System-A amino acid transporter activity relative to fetal growth. Fetal and placental weights in P0 mice were reduced when compared with WT at both embryonic day 17 (E17) and E19. Ionized calcium concentration [Ca2+] was significantly lower in P0 fetal blood compared with both WT and maternal blood at E17 and E19, reflecting a reversal of the fetomaternal [Ca2+] gradient. Fetal calcium content was reduced in P0 mice at E17 but not at E19. Unidirectional maternofetal calcium clearance (Ca K mf) was not different between WT and P0 at E17 but increased in P0 at E19. Expression of the intracellular calcium-binding protein calbindin-D9K, previously shown to be rate-limiting for calcium transport, was increased in P0 relative to WT placentas between E17 and E19. These data show an increased placental transport of calcium from E17 to E19 in P0 compared to WT. We suggest that this is an adaptation in response to the reduced fetal calcium accumulation earlier in gestation and speculate that the ability of the placenta to adapt its supply capacity according to fetal demand may stretch across other essential nutrients.

Dietary composition programmes placental phenotype in mice

Non‐technical summary  Studies on mice using severe diets show alterations in placental function, and fetal and adult health. However, little is known about the effects of mild dietary variations on the placenta. We investigated placental growth and function in mice fed diets with similar energy, but small differences in protein and sugar content. We show that placental adaptations occur to help support fetal growth: reduced protein leads to increased glucose transport and transporter gene expression in late pregnancy; just prior to term, amino acid transport expression correlated with protein intake; the placental endocrine compartment was smaller with the least dietary protein and somewhat larger with slight reduction in protein. Placentas in mice fed the least protein were better adapted than those exposed to slight protein reduction. These results may provide a good index of conditions in the womb and have important implications for the pre‐birth programming of life expectancy.

Impact on offspring methylation patterns of maternal gestational diabetes mellitus and intrauterine growth restraint suggest common genes and pathways linked to subsequent type 2 diabetes risk

Size at birth, postnatal weight gain, and adult risk for type 2 diabetes may reflect environmental exposures during developmental plasticity and may be mediated by epigenetics. Both low birth weight (BW), as a marker of fetal growth restraint, and high birth weight (BW), especially after gestational diabetes mellitus (GDM), have been linked to increased risk of adult type 2 diabetes. We assessed DNA methylation patterns using a bead chip in cord blood samples from infants of mothers with GDM (group 1) and infants with prenatal growth restraint indicated by rapid postnatal catch‐up growth (group 2), compared with infants with normal postnatal growth (group 3). Seventy‐five CpG loci were differentially methylated in groups 1 and 2 compared with the controls (group 3), representing 72 genes, many relevant to growth and diabetes. In replication studies using similar methodology, many of these differentially methylated regions were associated with levels of maternal glucose exposure below that defined by GDM [the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study] or were identified as changes observed after randomized periconceptional nutritional supplementation in a Gambian cohort characterized by maternal deprivation. These studies provide support for the concept that similar epigenetic modifications may underpin different prenatal exposures and potentially increase long‐term risk for diseases such as type 2 diabetes.—Quilter, C. R., Cooper, W. N., Cliffe, K. M., Skinner, B. M., Prentice, P. M., Nelson, L., Bauer, J., Ong, K. K., Constância, M., Lowe, W. L., Affara, N. A., Dunger, D. B., Impact on offspring methylation patterns of maternal gestational diabetes mellitus and intrauterine growth restraint suggest common genes and pathways linked to subsequent type 2 diabetes risk. FASEB J. 28, 4868–4879 (2014). www.fasebj.org

Developmental adaptations to increased fetal nutrient demand in mouse genetic models of Igf2-mediated overgrowth

The healthy development of the fetus depends on an optimal balance between fetal genetic drive for growth and the maternal ability to provide nutrients through the placenta. Nothing is known about fetal‐placental signaling in response to increased fetal demand in the situation of overgrowth. Here, we examined this question using the H19Δ13 mouse model, shown previously to result in elevated levels of Igf2. Fetal and placental weights in H19Δ13 were increased by 23% and 45%, respectively, at E19, when compared with wild‐type mice. Unexpectedly, we found that disproportionately large H19Δ13 placentas transport 20–35% less (per gram placenta) glucose and system A amino acids and have similar reductions in passive permeability, despite a significantly greater surface area for nutrient exchange and theoretical diffusion capacity compared with wild‐type mice. Expression of key transporter genes Slc2a3 and Slc38a4 was reduced by ~20%. Decreasing the overgrowth of the H19Δ13 placenta by genetically reducing levels of IgβPO resulted in up‐regulation of system A activity and maintenance of fetal overgrowth. Our results provide direct evidence that large placentas can modify their nutrient transfer capacity to regulate fetal nutrient acquisition. Our findings are indicative of fetal‐placental signaling mechanisms that limit total demand for maternal nutrients.—Angiolini, E., Coan, P. M., Sandovici, I., Iwajomo, O. H., Peck, G., Burton, G. J., Sibley, C. P., Reik, W., Fowden, A. L., Constância, M. Developmental adaptations to increased fetal nutrient demand in mouse genetic models of Igf2‐mediated overgrowth. FASEB J. 25, 1737–1745 (2011). www.fasebj.org

Intergenerational epigenetic inheritance in models of developmental programming of adult disease.

It is now well established that the environment to which we are exposed during fetal and neonatal life can have a long-term impact on our health. This has been termed the developmental origins of health and disease. Factors known to have such programming effects include intrauterine nutrient availability (determined by maternal nutrition and placental function), endocrine disruptors, toxins and infectious agents. Epigenetic processes have emerged as a key mechanism by which the early environment can permanently influence cell function and metabolism after multiple rounds of cell division. More recently it has been suggested that programmed effects can be observed beyond the first generation and that therefore epigenetic mechanisms could form the basis of transmission of phenotype from parent to child to grandchild and beyond. Here we review the evidence for such processes.