Imprinted genes and the manipulation of parenting in mammals.

Genomic imprinting refers to the parent-of-origin expression of genes, which originates from epigenetic events in the mammalian germ line. The evolution of imprinting may reflect a conflict over resource allocation early in life, with silencing of paternal genes in offspring soliciting increased maternal provision and silencing of maternal genes limiting demands on the mother. Parental caregiving has been identified as an area of potential conflict, with several imprinted genes serendipitously found to directly influence the quality of maternal care. Recent systems biology approaches, based on single-cell RNA sequencing data, support a more deliberate relationship, which is reinforced by the finding that imprinted genes expressed in the offspring influence the quality of maternal caregiving. These bidirectional, reiterative relationships between parents and their offspring are critical both for short-term survival and for lifelong wellbeing, with clear implications for human health.

The parenting hub of the hypothalamus is a focus of imprinted gene action.

Imprinted genes are subject to germline epigenetic modification resulting in parental-specific allelic silencing. Although genomic imprinting is thought to be important for maternal behaviour, this idea is based on serendipitous findings from a small number of imprinted genes. Here, we undertook an unbiased systems biology approach, taking advantage of the recent delineation of specific neuronal populations responsible for controlling parental care, to test whether imprinted genes significantly converge to regulate parenting behaviour. Using single-cell RNA sequencing datasets, we identified a specific enrichment of imprinted gene expression in a recognised "parenting hub", the galanin-expressing neurons of the preoptic area. We tested the validity of linking enriched expression in these neurons to function by focusing on MAGE family member L2 (Magel2), an imprinted gene not previously linked to parenting behaviour. We confirmed expression of Magel2 in the preoptic area galanin expressing neurons. We then examined the parenting behaviour of Magel2-null(+/p) mice. Magel2-null mothers, fathers and virgin females demonstrated deficits in pup retrieval, nest building and pup-directed motivation, identifying a central role for this gene in parenting. Finally, we show that Magel2-null mothers and fathers have a significant reduction in POA galanin expressing cells, which in turn contributes to a reduced c-Fos response in the POA upon exposure to pups. Our findings identify a novel imprinted gene that impacts parenting behaviour and, moreover, demonstrates the utility of using single-cell RNA sequencing data to predict gene function from expression and in doing so here, have identified a purposeful role for genomic imprinting in mediating parental behaviour.

Placental endocrine insufficiency programs anxiety, deficits in cognition and atypical social behaviour in offspring.

Abnormally elevated expression of the imprinted PHLDA2 gene has been reported in the placenta of human babies that are growth restricted in utero in several studies. We previously modelled this gene alteration in mice and found that just 2-fold increased expression of Phlda2 resulted in placental endocrine insufficiency. In addition, elevated Phlda2 was found to drive fetal growth restriction (FGR) of transgenic offspring and impaired maternal care by their wildtype mothers. Being born small and being exposed to suboptimal maternal care have both been associated with the increased risk of mental health disorders in human populations. In the current study we probed behavioural consequences of elevated Phlda2 for the offspring. We discovered increased anxiety-like behaviours, deficits in cognition and atypical social behaviours, with the greatest impact on male offspring. Subsequent analysis revealed alterations in the transcriptome of the adult offspring hippocampus, hypothalamus and amygdala, regions consistent with these behavioural observations. The inclusion of a group of fully wildtype controls raised in a normal maternal environment allowed us to attribute behavioural and molecular alterations to the adverse maternal environment induced by placental endocrine insufficiency rather than the specific gene change of elevated Phlda2. Our work demonstrates that a highly common alteration reported in human FGR is associated with negative behavioural outcomes later in life. Importantly, we also establish the experimental paradigm that placental endocrine insufficiency can program atypical behaviour in offspring highlighting the under-appreciated role of placental endocrine insufficiency in driving disorders of later life behaviour.

In support of the placental programming hypothesis: Placental endocrine insufficiency programs atypical behaviour in mothers and their offspring.

NEW FINDINGS: What is the topic of this review? More than half of all pregnancies in the UK are exposed to adversity linked to increased problems in pregnancy for mothers and adverse outcomes for their children, but we do not know the mechanism(s) underpinning these relationships. What advances does it highlight? Studies in mice prove that placental endocrine insufficiency driven by genetic manipulation of imprinted genes in the offspring can concurrently drive fetal growth restriction, alterations in maternal caregiving and aberrant behaviour in wild-type offspring exposed to an adverse environment. This suggests that placental endocrine insufficiency might contribute to the co-morbidity of low birth weight, maternal depression and neurodevelopmental disorders observed in human populations. ABSTRACT: Prenatal adversity, which is estimated to impact more than half of all pregnancies in the UK, compromises fetal growth and increases the chances of stillbirth, prematurity and infant mortality. Beyond these immediate and highly visible problems, infants that survive carry the invisible burden of increased risk of some of the most common and pervasive diseases that impact human populations. In utero exposure to depression and anxiety is one adversity that has been linked to these poorer outcomes, suggesting that maternal mood disorders drive the outcomes. However, recent studies in animal models suggest that both the maternal mood disorders and the detrimental outcomes for children could be the result of the same underlying placental pathology. In these studies, genetically wild-type rodent mothers exposed to placental endocrine insufficiency engaged in less pup-focused behaviours and less self-care. Genetically wild-type rodent offspring raised in this abnormal environment exhibited increased anxiety-like behaviours, with male offspring additionally exhibiting deficits in cognition and atypical social behaviour, with some evidence of depressive-like symptoms. This work establishes experimentally that placental endocrine insufficiency alone is sufficient to drive atypical behaviour in both mothers and their offspring. Although there are some data to suggest that this phenomenon is relevant to human pregnancy, considerably more work is required.

Molecular codes and in vitro generation of hypocretin and melanin concentrating hormone neurons.

Hypocretin/orexin (HCRT) and melanin concentrating hormone (MCH) neuropeptides are exclusively produced by the lateral hypothalamus and play important roles in sleep, metabolism, reward, and motivation. Loss of HCRT (ligands or receptors) causes the sleep disorder narcolepsy with cataplexy in humans and in animal models. How these neuropeptides are produced and involved in diverse functions remain unknown. Here, we developed methods to sort and purify HCRT and MCH neurons from the mouse late embryonic hypothalamus. RNA sequencing revealed key factors of fate determination for HCRT (Peg3, Ahr1, Six6, Nr2f2, and Prrx1) and MCH (Lmx1, Gbx2, and Peg3) neurons. Loss of Peg3 in mice significantly reduces HCRT and MCH cell numbers, while knock-down of a Peg3 ortholog in zebrafish completely abolishes their expression, resulting in a 2-fold increase in sleep amount. We also found that loss of HCRT neurons in Hcrt-ataxin-3 mice results in a specific 50% decrease in another orexigenic neuropeptide, QRFP, that might explain the metabolic syndrome in narcolepsy. The transcriptome results were used to develop protocols for the production of HCRT and MCH neurons from induced pluripotent stem cells and ascorbic acid was found necessary for HCRT and BMP7 for MCH cell differentiation. Our results provide a platform to understand the development and expression of HCRT and MCH and their multiple functions in health and disease.

Prenatal Adversity Modulates the Quality of Maternal Care Via the Exposed Offspring.

Adversities in pregnancy, including poor diet and stress, are associated with increased risk of developing both metabolic and mental health disorders later in life, a phenomenon described as fetal programming or developmental origins of disease. Predominant hypotheses proposed to explain this relationship suggest that the adversity imposes direct changes to the developing fetus which are maintained after birth resulting in an increased susceptibility to ill health. However, during pregnancy the mother, the developing fetus, and the placenta are all exposed to the adversity. The same adversities linked to altered offspring outcome can also result in suboptimal maternal care, which is considered an independent adverse exposure for the offspring. Recent key experiments in mice reveal the potential of prenatal adversity to drive alterations in maternal care through abnormal maternal-pup interactions and via alterations in placental signaling. Together, these data highlight the critical importance of viewing fetal programming holistically paying attention to the intimate, bidirectional, and reiterative relationship between mothers and their offspring.

Symptoms of Prenatal Depression Associated with Shorter Telomeres in Female Placenta.

BACKGROUND: Depression is a common mood disorder during pregnancy impacting one in every seven women. Children exposed to prenatal depression are more likely to be born at a low birth weight and develop chronic diseases later in life. A proposed hypothesis for this relationship between early exposure to adversity and poor outcomes is accelerated aging. Telomere length has been used as a biomarker of cellular aging. We used high-resolution telomere length analysis to examine the relationship between placental telomere length distributions and maternal mood symptoms in pregnancy. METHODS: This study utilised samples from the longitudinal Grown in Wales (GiW) study. Women participating in this study were recruited at their presurgical appointment prior to a term elective caesarean section (ELCS). Women completed the Edinburgh Postnatal Depression Scale (EPDS) and trait subscale of the State-Trait Anxiety Inventory (STAI). Telomere length distributions were generated using single telomere length analysis (STELA) in 109 term placenta (37-42 weeks). Multiple linear regression was performed to examine the relationship between maternally reported symptoms of depression and anxiety at term and mean placental telomere length. RESULTS: Prenatal depression symptoms were significantly negatively associated with XpYp telomere length in female placenta (B = -0.098, p = 0.026, 95% CI -0.184, -0.012). There was no association between maternal depression symptoms and telomere length in male placenta (B = 0.022, p = 0.586, 95% CI -0.059, 0.103). There was no association with anxiety symptoms and telomere length for either sex. CONCLUSION: Maternal prenatal depression is associated with sex-specific differences in term placental telomeres. Telomere shortening in female placenta may indicate accelerated placental aging.

Cdkn1c Boosts the Development of Brown Adipose Tissue in a Murine Model of Silver Russell Syndrome.

The accurate diagnosis and clinical management of the growth restriction disorder Silver Russell Syndrome (SRS) has confounded researchers and clinicians for many years due to the myriad of genetic and epigenetic alterations reported in these patients and the lack of suitable animal models to test the contribution of specific gene alterations. Some genetic alterations suggest a role for increased dosage of the imprinted CYCLIN DEPENDENT KINASE INHIBITOR 1C (CDKN1C) gene, often mutated in IMAGe Syndrome and Beckwith-Wiedemann Syndrome (BWS). Cdkn1c encodes a potent negative regulator of fetal growth that also regulates placental development, consistent with a proposed role for CDKN1C in these complex childhood growth disorders. Here, we report that a mouse modelling the rare microduplications present in some SRS patients exhibited phenotypes including low birth weight with relative head sparing, neonatal hypoglycemia, absence of catch-up growth and significantly reduced adiposity as adults, all defining features of SRS. Further investigation revealed the presence of substantially more brown adipose tissue in very young mice, of both the classical or canonical type exemplified by interscapular-type brown fat depot in mice (iBAT) and a second type of non-classic BAT that develops postnatally within white adipose tissue (WAT), genetically attributable to a double dose of Cdkn1c in vivo and ex-vivo. Conversely, loss-of-function of Cdkn1c resulted in the complete developmental failure of the brown adipocyte lineage with a loss of markers of both brown adipose fate and function. We further show that Cdkn1c is required for post-transcriptional accumulation of the brown fat determinant PR domain containing 16 (PRDM16) and that CDKN1C and PRDM16 co-localise to the nucleus of rare label-retaining cell within iBAT. This study reveals a key requirement for Cdkn1c in the early development of the brown adipose lineages. Importantly, active BAT consumes high amounts of energy to generate body heat, providing a valid explanation for the persistence of thinness in our model and supporting a major role for elevated CDKN1C in SRS.

Behavioural abnormalities in a novel mouse model for Silver Russell Syndrome.

Silver Russell Syndrome (SRS) syndrome is an imprinting disorder involving low birth weight with complex genetics and diagnostics. Some rare SRS patients carry maternally inherited microduplications spanning the imprinted genes CDKN1C, PHLDA2, SLC22A18 and KCNQ1, suggesting that overexpression of one of more of these genes contributes to the SRS phenotype. While this molecular alteration is very rare, feeding difficulties are a very common feature of this condition. Given that SRS children also have very low body mass index, understanding the underpinning biology of the eating disorder is important, as well as potential co-occurring behavioural alterations. Here, we report that a mouse model of this microduplication exhibits a number of behavioural deficits. The mice had a blunted perception of the palatability of a given foodstuff. This perception may underpin the fussiness with food. We additionally report hypoactivity, unrelated to anxiety or motoric function, and a deficit in the appropriate integration of incoming sensory information. Importantly, using a second genetic model, we were able to attribute all altered behaviours to elevated expression of a single gene, Cdkn1c. This is the first report linking elevated Cdkn1c to altered behaviour in mice. Importantly, the findings from our study may have relevance for SRS and highlight a potentially underreported aspect of this disorder.

Loss of Imprinting of Cdkn1c Protects against Age and Diet-Induced Obesity.

Cyclin dependent kinase inhibitor 1c (Cdkn1c) is a maternally expressed imprinted gene with roles in embryonic development, post-natal metabolism and behaviour. Using mouse models with altered dosages of Cdkn1c, we have previously identified a role for the gene in promoting brown adipose tissue formation. Here, we use these transgenic mouse lines to model the loss of imprinting of Cdkn1c in adulthood. We demonstrate that only a two-fold increase in the expression of Cdkn1c during development is sufficient to protect against age-related weight gain in addition to glucose and insulin intolerance. Further to this, we show that the loss of imprinting of Cdkn1c protects against diet-induced obesity. Bisulphite sequencing was performed to test the stability of the two differentially methylated regions that regulate Cdkn1c imprinting, and both were found to be unaltered in aged or diet-challenged adipose tissue, despite drastic reductions in Cdkn1c expression. These data demonstrate a critical role for Cdkn1c in regulating adult adipose tissue, with modest changes in expression capable of protecting against both age and diet-induced obesity and metabolic syndrome, with a natural decline in Cdkn1c expression observed that may contribute to less healthy metabolic aging. Finally, we have observed a post-natal insensitivity of the imprint to environmental factors, in contrast to recent observations of an in utero sensitivity.

Neurodevelopmental consequences in offspring of mothers with preeclampsia during pregnancy: underlying biological mechanism via imprinting genes.

PURPOSE: Preeclampsia is known to be a leading cause of mortality and morbidity among mothers and their infants. Approximately 3-8% of all pregnancies in the US are complicated by preeclampsia and another 5-7% by hypertensive symptoms. However, less is known about its long-term influence on infant neurobehavioral development. The current review attempts to demonstrate new evidence for imprinting gene dysregulation caused by hypertension, which may explain the link between maternal preeclampsia and neurocognitive dysregulation in offspring. METHOD: Pub Med and Web of Science databases were searched using the terms "preeclampsia," "gestational hypertension," "imprinting genes," "imprinting dysregulation," and "epigenetic modification," in order to review the evidence demonstrating associations between preeclampsia and suboptimal child neurodevelopment, and suggest dysregulation of placental genomic imprinting as a potential underlying mechanism. RESULTS: The high mortality and morbidity among mothers and fetuses due to preeclampsia is well known, but there is little research on the long-term biological consequences of preeclampsia and resulting hypoxia on the fetal/child neurodevelopment. In the past decade, accumulating evidence from studies that transcend disciplinary boundaries have begun to show that imprinted genes expressed in the placenta might hold clues for a link between preeclampsia and impaired cognitive neurodevelopment. A sudden onset of maternal hypertension detected by the placenta may result in misguided biological programming of the fetus via changes in the epigenome, resulting in suboptimal infant development. CONCLUSION: Furthering our understanding of the molecular and cellular mechanisms through which neurodevelopmental trajectories of the fetus/infant are affected by preeclampsia and hypertension will represent an important first step toward preventing adverse neurodevelopment in infants.

Placental PHLDA2 expression is increased in cases of fetal growth restriction following reduced fetal movements.

BACKGROUND: Maternal perception of reduced fetal movements (RFM) is associated with increased risk of fetal growth restriction (FGR) and stillbirth, mediated by placental insufficiency. The maternally expressed imprinted gene PHLDA2 controls fetal growth, placental development and placental lactogen production in a mouse model. A number of studies have also demonstrated abnormally elevated placental PHLDA2 expression in human growth restricted pregnancies. This study examined whether PHLDA2 was aberrantly expressed in placentas of RFM pregnancies resulting in delivery of an FGR infant and explored a possible relationship between PHLDA2 expression and placental lactogen release from the human placenta. METHODS: Villous trophoblast samples were obtained from a cohort of women reporting RFM (N = 109) and PHLDA2 gene expression analysed. hPL levels were assayed in the maternal serum (N = 74). RESULTS: Placental PHLDA2 expression was significantly 2.3 fold higher in RFM pregnancies resulting in delivery of an infant with FGR (p < 0.01), with highest levels of PHLDA2 expression in the most severe cases. Placental PHLDA2 expression was associated with maternal serum hPL levels (r = -0.30, p = 0.008, n = 74) although this failed to reach statistical significance in multiple linear regression analysis controlling for birth weight (p = 0.07). CONCLUSIONS: These results further highlight a role for placental PHLDA2 in poor perinatal outcomes, specifically FGR associated with RFM. Furthermore, this study suggests a potential relationship between placental PHLDA2 expression and hPL production by the placenta, an association that requires further investigation in a larger cohort.

Deficiency of the paternally-expressed imprinted Peg3 gene in mice has sexually dimorphic consequences for offspring communication and social behaviour.

Introduction: Imprinted genes are expressed from one parental allele as a consequence of epigenetic processes initiated in the germline. Consequently, their ability to influence phenotype depends on their parent-of-origin. Recent research suggests that the sex of the individual expressing the imprinted gene is also important. We have previously reported that genetically wildtype (WT) dams carrying and caring for pups mutant for PEG3 exhibit anxiety-like behaviours and their mutant pups show a reduction in ultrasonic vocalisation when separated from their mothers. Sex-specificity was not examined. Methods: WT female mice were mated with WT, heterozygous Peg3-/+ or homozygous Peg3-/- studs to generate all WT (control), 50:50 mixed or 100% mutant litters, respectively, followed by behavioural assessment of both dams and their pups. Results: We reproduced our original finding that WT dams carrying and caring for 100% mutant litters exhibit postpartum anxiety-like symptoms and delayed pup retrieval. Additionally, these WT dams were found to allocate less time to pup-directed care behaviours relative to controls. Male Peg3-deficient pups demonstrated significantly reduced vocalisation with a more subtle communication deficit in females. Postweaning, male mutants exhibited deficits across a number of key social behaviours as did WT males sharing their environment with mutants. Only modest variations in social behaviour were detected in experimental females. Discussion: We have experimentally demonstrated that Peg3 deficiency confined to the offspring causes anxiety in mouse mothers and atypical behaviour including deficits in communication in their male offspring. A male-specific reduction in expression PEG3 in the fetally-derived placenta has previously been associated with maternal depression in human pregnancy. Maternal mood disorders such as depression and anxiety are associated with delays in language development and neuroatypical behaviour more common in sons. Peg3 deficiency could drive the association of maternal and offspring behavioural disorders reported in humans.

Epigenetic regulation of placental endocrine lineages and complications of pregnancy.

A defining feature of mammals is the development in utero of the fetus supported by the constant flow of nutrients from the mother obtained via a specialized organ: the placenta. The placenta is also a major endocrine organ that synthesizes vast quantities of hormones and cytokines to instruct both maternal and fetal physiology. Nearly 20 years ago, David Haig and colleagues proposed that placental hormones were likely targets of the epigenetic process of genomic imprinting in response to the genetic conflicts imposed by in utero development [Haig (1993) Q. Rev. Biol. 68, 495-532]. There are two simple mechanisms through which genomic imprinting could regulate placental hormones. First, imprints could directly switch on or off alleles of specific genes. Secondly, imprinted genes could alter the expression of placental hormones by regulating the development of placental endocrine lineages. In mice, the placental hormones are synthesized in the trophoblast giant cells and spongiotrophoblast cells of the mature placenta. In the present article, I review the functional role of imprinted genes in regulating these endocrine lineages, which lends support to Haig's original hypothesis. I also discuss how imprinting defects in the placenta may adversely affect the health of the fetus and its mother during pregnancy and beyond.

Chromatin signatures of pluripotent cell lines.

Epigenetic genome modifications are thought to be important for specifying the lineage and developmental stage of cells within a multicellular organism. Here, we show that the epigenetic profile of pluripotent embryonic stem cells (ES) is distinct from that of embryonic carcinoma cells, haematopoietic stem cells (HSC) and their differentiated progeny. Silent, lineage-specific genes replicated earlier in pluripotent cells than in tissue-specific stem cells or differentiated cells and had unexpectedly high levels of acetylated H3K9 and methylated H3K4. Unusually, in ES cells these markers of open chromatin were also combined with H3K27 trimethylation at some non-expressed genes. Thus, pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing expression of these genes in ES cells as premature expression occurs in embryonic ectoderm development (Eed)-deficient ES cells. Our data suggest that lineage-specific genes are primed for expression in ES cells but are held in check by opposing chromatin modifications.

Entopic overexpression of Ascl2 does not accelerate tumourigenesis in ApcMin mice.

OBJECTIVE: Expression of the Wnt target gene ASCL2 is elevated in 78% of intestinal neoplasia datasets (Oncomine), suggesting a role for deregulated ASCL2 in the aetiology of intestinal tumourigenesis. Furthermore, ectopic expression of Ascl2 has previously been shown to lead to hyperplasia in the mouse. However, elevated levels of ASCL2 does not have an impact on the overall survival or recurrence-free survival rates in colorectal cancer patients. Here the authors use a novel mouse model to analyse the role of Ascl2 in intestinal tumourigenesis and address the controversy surrounding the relevance of this gene to the aetiology of colorectal cancer. DESIGN: The authors have generated a mouse possessing a transgene carrying the Ascl2 gene together with its endogenous promoter and regulatory regions, thereby elevating Ascl2 expression in an authentic manner. The authors have further intercrossed these Ascl2 overexpressers to the classic Apc(Min) model, to study the consequence of elevated Ascl2 expression in intestinal tumourigenesis. RESULTS: Here the authors genetically demonstrate that elevated expression of Ascl2 in a Wnt signalling dependent manner specifically in the stem cell compartment of the intestine neither increases tumour formation nor diminishes survival in a well established intestinal tumour model, the Apc(min) mouse. CONCLUSION: The authors conclude that ectopic expression of Ascl2 is more important in the aetiology of neoplasia than overexpression of Ascl2.

A Quantitative Evaluation of Thin Slice Sampling for Parent-Infant Interactions.

Behavioural coding is time-intensive and laborious. Thin slice sampling provides an alternative approach, aiming to alleviate the coding burden. However, little is understood about whether different behaviours coded over thin slices are comparable to those same behaviours over entire interactions. To provide quantitative evidence for the value of thin slice sampling for a variety of behaviours. We used data from three populations of parent-infant interactions: mother-infant dyads from the Grown in Wales (GiW) cohort (n = 31), mother-infant dyads from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort (n = 14), and father-infant dyads from the ALSPAC cohort (n = 11). Mean infant ages were 13.8, 6.8, and 7.1 months, respectively. Interactions were coded using a comprehensive coding scheme comprised of 11-14 behavioural groups, with each group comprised of 3-13 mutually exclusive behaviours. We calculated frequencies of verbal and non-verbal behaviours, transition matrices (probability of transitioning between behaviours, e.g., from looking at the infant to looking at a distraction) and stationary distributions (long-term proportion of time spent within behavioural states) for 15 thin slices of full, 5-min interactions. Measures drawn from the full sessions were compared to those from 1-, 2-, 3- and 4-min slices. We identified many instances where thin slice sampling (i.e., < 5 min) was an appropriate coding method, although we observed significant variation across different behaviours. We thereby used this information to provide detailed guidance to researchers regarding how long to code for each behaviour depending on their objectives.

Characterization of DNA methylation errors in patients with imprinting disorders conceived by assisted reproduction technologies.

BACKGROUND: There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that the techniques themselves may predispose embryos to acquire imprinting errors and diseases. However, it is still unknown at what point(s) these imprinting errors arise, or the risk factors. METHODS: In 2009 we conducted a Japanese nationwide epidemiological study of four well-known imprinting diseases to determine any association with ART. Using bisulfite sequencing, we examine the DNA methylation status of 22 gametic differentially methylated regions (gDMRs) located within the known imprinted loci in patients with Beckwith-Wiedemann syndrome (BWS, n=1) and also Silver-Russell syndrome (SRS, n= 5) born after ART, and compared these with patients conceived naturally. RESULTS: We found a 10-fold increased frequency of BWS and SRS associated with ART. The majority of ART cases showed aberrant DNA methylation patterns at multiple imprinted loci both maternal and paternal gDMRs (5/6), with both hyper- and hypomethylation events (5/6) and also mosaic methylation errors (5/6). Although our study may have been limited by a small sample number, the fact that many of the changes were mosaic suggested that they occurred after fertilization. In contrast, few of the patients who were conceived naturally exhibited a similar pattern of mosaic alterations. The differences in methylation patterns between the patients who were conceived naturally or after ART did not manifest due to the differences in the disease phenotypes in these imprinting disorders. CONCLUSION: A possible association between ART and BWS/SRS was found, and we observed a more widespread disruption of genomic imprints after ART. The increased frequency of imprinting disorders after ART is perhaps not surprising given the major epigenetic events that take place during early development at a time when the epigenome is most vulnerable.

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

Developmental regulation of somatic imprints.

Genomic imprinting is an epigenetic phenomenon guiding the allele-specific marking of parental alleles. Genes regulated by imprinting are only or preferentially expressed from a single allele during development and in the adult, and the transcriptional activity of each allele is dictated by its parental origin. Consequently, active and repressed alleles of imprinted genes are marked by activating and repressive histone marks, respectively. Whether these marks are implicated in the germline imprints distinguishing maternal and paternal alleles at fertilization or indeed in the mitotic inheritance of the two transcriptional states is currently unknown. The only epigenetic modification which is known to fulfill these roles is DNA methylation. Most but not all imprinted genes are marked by regions of allele-specific DNA methylation termed differentially methylated regions (DMRs). Whereas some DMRs, the gametic DMRs, are directly inherited from the mature gametes at fertilization, others, the somatic DMRs, are only acquired in postimplantation embryos. Although all somatic imprints are thought to emerge as a consequence of the cis-activity of a nearby gametic imprint, the molecular mechanisms guiding the de novo methylation at somatic DMRs are not fully understood. Here we review the known characteristics of gametic and somatic DMRs, with an emphasis on the factors implicated in the initiation and maintenance of these epigenetic marks. The analysis of somatic DMRs offers the opportunity to study the mechanism of de novo DNA methylation outside the context of the germline and as such might help to elucidate common mechanisms implicated in epigenetic silencing during development and differentiation. Moreover, studies on genes directly silenced by somatic DMRs may be informative in understanding the significance of controlling gene dosage in the adult.