Illuminating Epigenetics and Inheritance in the Immune System with Bioluminescence.

The remarkable process of light emission by living organisms has fascinated mankind for thousands of years. A recent expansion in the repertoire of catalytic luciferase enzymes, coupled with the discovery of the genes and pathways that encode different luciferin substrates, means that bioluminescence imaging (BLI) is set to revolutionize longitudinal and dynamic studies of gene control within biomedicine, including the regulation of immune responses. In this review article, we summarize recent advances in bioluminescence-based imaging approaches that promise to enlighten our understanding of in vivo gene and epigenetic control within the immune system.

Genomic imprinting and its effects on postnatal growth and adult metabolism.

Imprinted genes display parent-of-origin-specific expression with this epigenetic system of regulation found exclusively in therian mammals. Historically, defined imprinted gene functions were almost solely focused on pregnancy and the influence on the growth parameters of the developing embryo and placenta. More recently, a number of postnatal functions have been identified which converge on resource allocation, both for animals in the nest and in adults. While many of the prenatal functions of imprinted genes that have so far been described adhere to the "parental conflict" hypothesis, no clear picture has yet emerged on the functional role of imprints on postnatal metabolism. As these roles are uncovered, interest in the potential for these genes to influence postnatal metabolism and associated adult-onset disease outcomes when dysregulated has gathered pace. Here, we review the published data on imprinted genes and their influence on postnatal metabolism, starting in the nest, and then progressing through to adulthood. When observing the functional effects of these genes on adult metabolism, we must always be careful to acknowledge the influence both of direct expression in the relevant metabolic tissue, but also indirect metabolic programming effects caused by their modulation of both in utero and postnatal growth trajectories.

The Role of CDKs and CDKIs in Murine Development.

Cyclin-dependent kinases (CDKs) and their inhibitors (CDKIs) play pivotal roles in the regulation of the cell cycle. As a result of these functions, it may be extrapolated that they are essential for appropriate embryonic development. The twenty known mouse CDKs and eight CDKIs have been studied to varying degrees in the developing mouse, but only a handful of CDKs and a single CDKI have been shown to be absolutely required for murine embryonic development. What has become apparent, as more studies have shone light on these family members, is that in addition to their primary functional role in regulating the cell cycle, many of these genes are also controlling specific cell fates by directing differentiation in various tissues. Here we review the extensive mouse models that have been generated to study the functions of CDKs and CDKIs, and discuss their varying roles in murine embryonic development, with a particular focus on the brain, pancreas and fertility.

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.

Protein restriction during pregnancy alters Cdkn1c silencing, dopamine circuitry and offspring behaviour without changing expression of key neuronal marker genes.

We tracked the consequences of in utero protein restriction in mice throughout their development and life course using a luciferase-based allelic reporter of imprinted Cdkn1c. Exposure to gestational low-protein diet (LPD) results in the inappropriate expression of paternally inherited Cdkn1c in the brains of embryonic and juvenile mice. These animals were characterised by a developmental delay in motor skills, and by behavioural alterations indicative of reduced anxiety. Exposure to LPD in utero resulted in significantly more tyrosine hydroxylase positive (dopaminergic) neurons in the midbrain of adult offspring as compared to age-matched, control-diet equivalents. Positron emission tomography (PET) imaging revealed an increase in striatal dopamine synthesis capacity in LPD-exposed offspring, where elevated levels of dopamine correlated with an enhanced sensitivity to cocaine. These data highlight a profound sensitivity of the developing epigenome to gestational protein restriction. Our data also suggest that loss of Cdkn1c imprinting and p57KIP2 upregulation alters the cellular composition of the developing midbrain, compromises dopamine circuitry, and thereby provokes behavioural abnormalities in early postnatal life. Molecular analyses showed that despite this phenotype, exposure to LPD solely during pregnancy did not significantly change the expression of key neuronal- or dopamine-associated marker genes in adult offspring.

Endogenous bioluminescent reporters reveal a sustained increase in utrophin gene expression upon EZH2 and ERK1/2 inhibition.

Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by loss of function mutations in the dystrophin gene (Dmd), resulting in progressive muscle weakening. Here we modelled the longitudinal expression of endogenous Dmd, and its paralogue Utrn, in mice and in myoblasts by generating bespoke bioluminescent gene reporters. As utrophin can partially compensate for Dmd-deficiency, these reporters were used as tools to ask whether chromatin-modifying drugs can enhance Utrn expression in developing muscle. Myoblasts treated with different PRC2 inhibitors showed significant increases in Utrn transcripts and bioluminescent signals, and these responses were independently verified by conditional Ezh2 deletion. Inhibition of ERK1/2 signalling provoked an additional increase in Utrn expression that was also seen in Dmd-mutant cells, and maintained as myoblasts differentiate. These data reveal PRC2 and ERK1/2 to be negative regulators of Utrn expression and provide specialised molecular imaging tools to monitor utrophin expression as a therapeutic strategy for DMD.

Drug-induced loss of imprinting revealed using bioluminescent reporters of Cdkn1c.

Genomic imprinting is an epigenetically mediated mechanism that regulates allelic expression of genes based upon parent-of-origin and provides a paradigm for studying epigenetic silencing and release. Here, bioluminescent reporters for the maternally-expressed imprinted gene Cdkn1c are used to examine the capacity of chromatin-modifying drugs to reverse paternal Cdkn1c silencing. Exposure of reporter mouse embryonic stem cells (mESCs) to 5-Azacytidine, HDAC inhibitors, BET inhibitors or GSK-J4 (KDM6A/B inhibitor) relieved repression of paternal Cdkn1c, either selectively or by inducing biallelic effects. Treatment of reporter fibroblasts with HDAC inhibitors or GSK-J4 resulted in similar paternal Cdkn1c activation, whereas BET inhibitor-induced loss of imprinting was specific to mESCs. Changes in allelic expression were generally not sustained in dividing cultures upon drug removal, indicating that the underlying epigenetic memory of silencing was maintained. In contrast, Cdkn1c de-repression by GSK-J4 was retained in both mESCs and fibroblasts following inhibitor removal, although this impact may be linked to cellular stress and DNA damage. Taken together, these data introduce bioluminescent reporter cells as tools for studying epigenetic silencing and disruption, and demonstrate that Cdkn1c imprinting requires distinct and cell-type specific chromatin features and modifying enzymes to enact and propagate a memory of silencing.

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.

Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice.

Transmission of epigenetic information between generations occurs in nematodes, flies and plants, mediated by specialised small RNA pathways, modified histones and DNA methylation. Similar processes in mammals can also affect phenotype through intergenerational or trans-generational mechanisms. Here we generate a luciferase knock-in reporter mouse for the imprinted Dlk1 locus to visualise and track epigenetic fidelity across generations. Exposure to high-fat diet in pregnancy provokes sustained re-expression of the normally silent maternal Dlk1 in offspring (loss of imprinting) and increased DNA methylation at the somatic differentially methylated region (sDMR). In the next generation heterogeneous Dlk1 mis-expression is seen exclusively among animals born to F1-exposed females. Oocytes from these females show altered gene and microRNA expression without changes in DNA methylation, and correct imprinting is restored in subsequent generations. Our results illustrate how diet impacts the foetal epigenome, disturbing canonical and non-canonical imprinting mechanisms to modulate the properties of successive generations of offspring.

Fetal growth restriction in a genetic model of sporadic Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a complex imprinting disorder involving fetal overgrowth and placentomegaly, and is associated with a variety of genetic and epigenetic mutations affecting the expression of imprinted genes on human chromosome 11p15.5. Most BWS cases are linked to loss of methylation at the imprint control region 2 (ICR2) within this domain, which in mice regulates the silencing of several maternally expressed imprinted genes. Modelling this disorder in mice is confounded by the unique embryonic requirement for Ascl2, which is imprinted in mice but not in humans. To overcome this issue, we generated a novel model combining a truncation of distal chromosome 7 allele (DelTel7) with transgenic rescue of Ascl2 expression. This novel model recapitulated placentomegaly associated with BWS, but did not lead to fetal overgrowth.

Neuronatin deletion causes postnatal growth restriction and adult obesity in 129S2/Sv mice.

OBJECTIVE: Imprinted genes are crucial for the growth and development of fetal and juvenile mammals. Altered imprinted gene dosage causes a variety of human disorders, with growth and development during these crucial early stages strongly linked with future metabolic health in adulthood. Neuronatin (Nnat) is a paternally expressed imprinted gene found in neuroendocrine systems and white adipose tissue and is regulated by the diet and leptin. Neuronatin expression is downregulated in obese children and has been associated with stochastic obesity in C57BL/6 mice. However, our recent studies of Nnat null mice on this genetic background failed to display any body weight or feeding phenotypes but revealed a defect in glucose-stimulated insulin secretion due to the ability of neuronatin to potentiate signal peptidase cleavage of preproinsulin. Nnat deficiency in beta cells therefore caused a lack of appropriate storage and secretion of mature insulin. METHODS: To further explore the potential role of Nnat in the regulation of body weight and adiposity, we studied classical imprinting-related phenotypes such as placental, fetal, and postnatal growth trajectory patterns that may impact upon subsequent adult metabolic phenotypes. RESULTS: Here we find that, in contrast to the lack of any body weight or feeding phenotypes on the C57BL/6J background, deletion of Nnat in mice on 129S2/Sv background causes a postnatal growth restriction with reduced adipose tissue accumulation, followed by catch up growth after weaning. This was in the absence of any effect on fetal growth or placental development. In adult 129S2/Sv mice, Nnat deletion was associated with hyperphagia, reduced energy expenditure, and partial leptin resistance. Lack of neuronatin also potentiated obesity caused by either aging or high fat diet feeding. CONCLUSIONS: The imprinted gene Nnat plays a key role in postnatal growth, adult energy homeostasis, and the pathogenesis of obesity via catch up growth effects, but this role is dependent upon genetic background.

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults.

Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact.

Isolating the role of elevated Phlda2 in asymmetric late fetal growth restriction in mice.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is a maternally expressed imprinted gene whose elevated expression has been linked to fetal growth restriction in a number of human studies. In mice, Phlda2 negatively regulates placental growth and limits the accumulation of placental glycogen. We previously reported that a three-copy transgene spanning the Phlda2 locus drove a fetal growth restriction phenotype late in gestation, suggesting a causative role for PHLDA2 in human growth restriction. However, in this mouse model, Phlda2 was overexpressed by fourfold, alongside overexpression of a second imprinted gene, Slc22a18. Here, we genetically isolate the role of Phlda2 in driving late fetal growth restriction in mice. We furthermore show that this Phlda2-driven growth restriction is asymmetrical, with a relative sparing of the brain, followed by rapid catch-up growth after birth, classic features of placental insufficiency. Strikingly, fetal growth restriction showed strain-specific differences, being apparent on the 129S2/SvHsd (129) genetic background and absent on the C57BL6 (BL6) background. A key difference between these two strains is the placenta. Specifically, BL6 placentae possess a more extensive endocrine compartment and substantially greater stores of placental glycogen. Taken together, these data support a direct role for elevated Phlda2 in limiting fetal growth but also suggest that growth restriction only manifests when there is limited placental reserve. These findings should be taken into account in interpreting the results from human studies.

Fetal overgrowth in the Cdkn1c mouse model of Beckwith-Wiedemann syndrome.

Mutations in the imprinted CDKN1C gene are associated with the childhood developmental disorder Beckwith-Wiedemann syndrome (BWS). Multiple mouse models with deficiency of Cdkn1c recapitulate some aspects of BWS but do not exhibit overgrowth of the newborn, a cardinal feature of patients with BWS. In this study, we found that Cdkn1c mutants attained a 20% increase in weight during gestation but experienced a rapid reversal of this positive growth trajectory very late in gestation. We observed a marked effect on placental development concurrently with this loss of growth potential, with the appearance of large thrombotic lesions in the labyrinth zone. The trilaminar trophoblast layer that separates the maternal blood sinusoids from fetal capillaries was disordered with a loss of sinusoidal giant cells, suggesting a role for Cdkn1c in maintaining the integrity of the maternal-fetal interface. Furthermore, the overgrowth of mutant pups decreased in the face of increasing intrauterine competition, identifying a role for Cdkn1c in the allocation of the maternal resources via the placenta. This work explains one difficulty in precisely replicating BWS in this animal model: the differences in reproductive strategies between the multiparous mouse, in which intrauterine competition is high, and humans, in which singleton pregnancies are more common.