Miguel Constancia

Child Health, Developmental Plasticity, and Epigenetic Programming

Plasticity in developmental programming has evolved in order to provide the best chances of survival and reproductive success to the organism under changing environments. Environmental conditions that are experienced in early life can profoundly influence human biology and long-term health. Developmental origins of health and disease and life-history transitions are purported to use placental, nutritional, and endocrine cues for setting long-term biological, mental, and behavioral strategies in response to local ecological and/or social conditions. The window of developmental plasticity extends from preconception to early childhood and involves epigenetic responses to environmental changes, which exert their effects during life-history phase transitions. These epigenetic responses influence development, cell- and tissue-specific gene expression, and sexual dimorphism, and, in exceptional cases, could be transmitted transgenerationally. Translational epigenetic research in child health is a reiterative process that ranges from research in the basic sciences, preclinical research, and pediatric clinical research. Identifying the epigenetic consequences of fetal programming creates potential applications in clinical practice: the development of epigenetic biomarkers for early diagnosis of disease, the ability to identify susceptible individuals at risk for adult diseases, and the development of novel preventive and curative measures that are based on diet and/or novel epigenetic drugs.

Maternal diet and aging alter the epigenetic control of a promoter-enhancer interaction at the Hnf4a gene in rat pancreatic islets

Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a, which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter–enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health.

Imprinted Genes, Placental Development and Fetal Growth

In mammals, imprinted genes have an important role in feto-placental development. They affect the growth, morphology and nutrient transfer capacity of the placenta and, thereby, control the nutrient supply for fetal growth. In particular, the reciprocally imprinted Igf2–H19 gene complex has a central role in these processes and matches the placental nutrient supply to the fetal nutrient demands for growth. Comparison of Igf2P0 and complete Igf2 null mice has shown that interplay between placental and fetal Igf2 regulates both placental growth and nutrient transporter abundance. In turn, epigenetic modification of imprinted genes via changes in DNA methylation may provide a mechanism linking environmental cues to placental phenotype, with consequences for development both before and after birth. Changes in expression of imprinted genes, therefore, have major implications for developmental programming and may explain the poor prognosis of the infant born small for gestational age and the wide spectrum of adult-onset diseases that originate in utero.

Programming placental nutrient transport capacity

Many animal studies and human epidemiological findings have shown that impaired growth in utero is associated with physiological abnormalities in later life and have linked this to tissue programming during suboptimal intrauterine conditions at critical periods of development. However, few of these studies have considered the contribution of the placenta to the ensuing adult phenotype. In mammals, the major determinant of intrauterine growth is the placental nutrient supply, which, in turn, depends on the size, morphology, blood supply and transporter abundance of the placenta and on synthesis and metabolism of nutrients and hormones by the uteroplacental tissues. This review examines the regulation of placental nutrient transfer capacity and the potential programming effects of nutrition and glucocorticoid over‐exposure on placental phenotype with particular emphasis on the role of the Igf2 gene in these processes.

Placental efficiency and adaptation: endocrine regulation

Size at birth is critical in determining life expectancy and is dependent primarily on the placental supply of nutrients. However, the fetus is not just a passive recipient of nutrients from the placenta. It exerts a significant acquisitive drive for nutrients, which acts through morphological and functional adaptations in the placenta, particularly when the genetically determined drive for fetal growth is compromised by adverse intrauterine conditions. These adaptations alter the efficiency with which the placenta supports fetal growth, which results in optimal growth for prevailing conditions in utero. This review examines placental efficiency as a means of altering fetal growth, the morphological and functional adaptations that influence placental efficiency and the endocrine regulation of these processes.

Adaptations in placental phenotype support fetal growth during undernutrition of pregnant mice

Undernutrition during pregnancy reduces birth weight and programmes adult phenotype with consequences for life expectancy, but its effects on the phenotype of the placenta, responsible for supplying nutrients for fetal growth, remain largely unknown. Using molecular, morphological and functional analyses, placental phenotype was examined in mice during restriction of dietary intake to 80% of control from day 3 of pregnancy. At day 16, undernutrition reduced placental, but not fetal, weight in association with decreased junctional zone volume and placental expression of glucose transporter Slc2a1. At day 19, both placental and fetal weights were reduced in undernourished mice (91% and 87% of control, respectively, P < 0.01), as were the volume and surface area of the labyrinthine zone responsible for placental nutrient transfer (85% and 86%, respectively, P < 0.03). However, unidirectional materno‐fetal clearance of tracer glucose was maintained and methyl‐aminoisobutyric acid increased 166% (P < 0.005) per gram of undernourished placenta, relative to controls. This was associated with an 18% and 27% increased placental expression of glucose and system A amino acid transporters Slc2a1 and Slc38a2, respectively, at day 19 (P < 0.04). At both ages, undernutrition decreased expression of the placental specific transcript of the Igf2 gene by 35% (P < 0.01), although methylation of its promoter was unaffected. The placenta, therefore, adapts to help maintain fetal growth when its own growth is compromised by maternal undernutrition. Consequently, placental phenotype is responsive to environmental conditions and may help predict the risk of adult disease programmed in utero.

Periconceptional maternal micronutrient supplementation is associated with widespread gender related changes in the epigenome: a study of a unique resource in the Gambia

In addition to the genetic constitution inherited by an organism, the developmental trajectory and resulting mature phenotype are also determined by mechanisms acting during critical windows in early life that influence and establish stable patterns of gene expression. This is the crux of the developmental origins of health and disease hypothesis that suggests undernutrition during gestation and infancy predisposes to ill health in later life. The hypothesis that periconceptional maternal micronutrient supplementation might affect fetal genome-wide methylation within gene promoters was explored in cord blood samples from offspring of Gambian women enrolled into a unique randomized, double blind controlled trial. Significant changes in the epigenome in cord blood DNA samples were further explored in a subset of offspring at 9 months. Gender-specific changes related to periconceptional nutritional supplementation were identified in cord blood DNA samples, some of which showed persistent changes in infant blood DNA samples. Significant effects of periconceptional micronutrient supplementation were also observed in postnatal samples which were not evident in cord blood. In this Gambian population, the increased death rate of individuals born in nutritionally poor seasons has been related to infection and it is of interest that we identified differential methylation at genes associated with defence against infection and immune response. Although the sample size was relatively small, these pilot data suggest that periconceptional nutrition in humans is an important determinant of newborn whole genome methylation patterns but may also influence postnatal developmental patterns of gene promoter methylation linking early with disease risk.

An upstream repressor element plays a role in Igf2 imprinting

The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans‐ acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo.

Igf2 imprinting in development and disease.

Igf2 is one of the first imprinted genes discovered and occupies a centre stage in the study of imprinting. This is because it has dramatic effects on the control of fetal growth, it is involved in growth disorders and in cancer, it interacts with products of other imprinted genes, and its imprinting status is under complex regulation in a cluster of tightly linked imprinted genes. Here we review briefly the key features of Igf2 imprinting in normal development and in disease, and hope to show what a fascinating subject of study this gene and its biology provides.

Imprinted genes and the epigenetic regulation of placental phenotype.

Imprinted genes are expressed in a parent-of-origin manner by epigenetic modifications that silence either the paternal or maternal allele. They are widely expressed in fetal and placental tissues and are essential for normal placental development. In general, paternally expressed genes enhance feto-placental growth while maternally expressed genes limit conceptus growth, consistent with the hypothesis that imprinting evolved in response to the conflict between parental genomes in the allocation of maternal resources to fetal growth. Using targeted deletion, uniparental duplication, loss of imprinting and transgenic approaches, imprinted genes have been shown to determine the transport capacity of the definitive mouse placenta by regulating its growth, morphology and transporter abundance. Imprinted genes in the placenta are also responsive to environmental challenges and adapt placental phenotype to the prevailing nutritional conditions, in part, by varying their epigenetic status. In addition, interplay between placental and fetal imprinted genes is important in regulating resource partitioning via the placenta both developmentally and in response to environmental factors. By balancing the opposing parental drives on resource allocation with the environmental signals of nutrient availability, imprinted genes, like the Igf2-H19 locus, may act as nutrient sensors and optimise the fetal acquisition of nutrients for growth. These genes, therefore, have a major role in the epigenetic regulation of placental phenotype with long term consequences for the developmental programming of adult health and disease.