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Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.

Genomic imprinting, a phenomenon where the two parental alleles are regulated differently, is observed in mammals, marsupials and a few other species, including seed-bearing plants. Dysregulation of genomic imprinting can cause developmental disorders such as Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). In this review, we discuss 1) how various (epi)genetic lesions lead to the dysregulation of clinically relevant imprinted loci and 2) how such perturbations may contribute to the developmental defects in BWS and SRS. Given that the regulatory mechanisms of most imprinted clusters are well conserved between mice and humans, numerous mouse models of BWS and SRS have been generated. These mouse models are key to understanding how mutations at imprinted loci result in pathological phenotypes in humans, although there are some limitations. This review focuses on how the biological findings obtained from innovative mouse models explain clinical features of BWS and SRS.

DNA methylation is an essential epigenetic mark crucial for normal mammalian development. This modification controls the expression of a unique class of genes, designated as imprinted, which are expressed monoallelically and in a parent-of-origin-specific manner. Proper parental allele-specific DNA methylation at imprinting control regions (ICRs) is necessary for appropriate imprinting. Processes that deregulate DNA methylation of imprinted loci cause disease in humans. DNAmethylation patterns dramatically change duringmammalian development: first, the majority of the genome, with the exception of ICRs, is demethylated after fertilization, and subsequently undergoes genome-wide de novo DNA methylation. Secondly, after primordial germ cells are specified in the embryo, another wave of demethylation occurs, with ICR demethylation occurring late in the process. Lastly, ICRs reacquire DNA methylation imprints in developing germ cells. We describe the past discoveries and current literature defining these crucial dynamics in relation to imprinted genes and the rest of the genome. Summary Sentence This review summarizes the changes in DNA methylation at imprinting control regions, which regulate monoallelic imprinted gene expression, from fertilization, embryogenesis, and PGC development, which are essential for proper development.

Precise, monoallelic expression of imprinted genes is governed by cis regulatory elements called imprinting control regions (ICRs) and enhancer-promoter (E-P) interactions shaped by local chromatin architecture. The Igf2/H19 locus employs allele-specific CTCF binding at the ICR to instruct enhancer accessibility to maternal H19 and paternal Igf2 promoters. Here, we investigate the CTCF-bound centrally conserved domain (CCD), intergenic to H19 and Igf2 , and an adjacent widely expressed lncRNA. Using transgenic mice, deletion alleles reinforced CCD as a neonatal muscle-specific repressor of maternal Igf2 . However, deletion of the abutting lncRNA did not affect Igf2 levels. Unexpectedly, in adult skeletal muscle where Igf2 is normally repressed, absence of CCD resulted in remarkable, high-level activation of Igf2 from both parental alleles. Through multimodal chromatin analyses, we identified a conserved putative adult skeletal muscle enhancer (PaSME) insulated between chromatin domains at ICR and CCD. We propose that removal of CCD allows PaSME to drive robust abnormal Igf2 activation on both alleles in adult skeletal muscle. Thus, we uncover CCD as a developmental biallelic muscle-specific repressor, adding a new layer of architectural regulation to the extensively studied Igf2/H19 locus.

About the Authors: Marisa S. Bartolomei * E-mail: bartolom@pennmedicine.upenn.edu Affiliation: Epigenetics Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America ORCID logo http://orcid.org/0000-0001-9410-5222 Rebecca J. Oakey Affiliation: Department of Medical & Molecular Genetics, King’s College London, London, United Kingdom ORCID logo http://orcid.org/0000-0003-2706-8139 Anton Wutz Affiliation: D-BIOL, Institute of Molecular Health Sciences, Swiss Federal Institute of Technology, ETH Hönggerberg, Zurich, Switzerland ORCID logo http://orcid.org/0000-0002-4377-6330 Citation: Bartolomei MS, Oakey RJ, Wutz A (2020) Genomic imprinting: [...]their regulation may differ from genes that are imprinted in the soma. In this collection, Courtney Hannah discusses the function and regulation of imprinted genes in the placenta, with special consideration given to the role of endogenous retroviruses (ERVs) in mediating placental-specific imprinting [8]. Because of the unusual nature of imprinting, the identification and study of imprinted genes have driven the adaption and modification of methods and, in some cases, necessitated the development of new technology. Additionally, nuclear transplantation, haploid embryonic stem cells combined with site-directed deletions have more recently shown that the main block to uniparental embryo development is caused by imprinted gene expression.

Growth-factor receptor bound protein 10 (Grb10) is a signal adapter protein encoded by an imprinted gene that has roles in growth control, cellular proliferation, and insulin signaling. Additionally, Grb10 is critical for the normal behavior of the adult mouse. These functions are paralleled by Grb10 ’s unique tissue-specific imprinted expression; the paternal copy of Grb10 is expressed in a subset of neurons whereas the maternal copy is expressed in most other adult tissues in the mouse. The mechanism that underlies this switch between maternal and paternal expression is still unclear, as is the role for paternally expressed Grb10 in neurons. Here, we review recent work and present complementary data that contribute to the understanding of Grb10 gene regulation and function, with specific emphasis on growth and neuronal development. Additionally, we show that in vitro differentiation of mouse embryonic stem cells into alpha motor neurons recapitulates the switch from maternal to paternal expression observed during neuronal development in vivo. We postulate that this switch in allele-specific expression is related to the functional role of Grb10 in motor neurons and other neuronal tissues.

Preeclampsia (PE), a hypertensive disorder of pregnancy, is hypothesized to be associated with, if not mechanistically related to abnormal placental function. However, the exact mechanisms regulating the pathogenesis of PE remain unclear. While many studies have investigated changes in gene expression in the PE placenta, the role of epigenetics in PE associated placental dysfunction remains unclear. Using the genome-wide Illumina Infinium Methylation 450 BeadChip array, we analyzed gene-specific alterations in DNA methylation in placental biopsies collected from normal pregnant women delivering at term (n = 14), with term PE (≥37 weeks; n = 19) or with preterm PE (<37 weeks, n = 12). Of the 485,582 gene loci on the array, compared to controls, 229 loci were differentially methylated in PE placentas and 3411 loci were differentially methylated in preterm PE (step up p-value <0.05 and >5% methylation difference). Functional annotation of the differentially methylated genes in preterm PE placentas revealed a 32 gene cluster in the cadherin and cell adhesion functional groups (Benjamini p<0.00001). Hypermethylation of CDH11 (p = 0.0143), COL5A1 (p = 0.0127) and TNF (p = 0.0098) and hypomethylation of NCAM1 (p = 0.0158) was associated with altered mRNA expression in preterm PE placentas. Demethylation of first trimester extravillous trophoblast cells resulted in altered CDH11 (p = 0.0087), COL5A1 (p = 0.0043), NCAM1 (p = 0.0260) and TNF (p = 0.0022) mRNA expression. These studies demonstrate aberrant methylation, correlating with disease severity, in PE placentas. Furthermore, we provide evidence that disruption of gene-specific methylation in preterm PE placentas and first trimester trophoblasts is significantly associated with altered gene expression demonstrating that epigenetic modifications early in pregnancy can have effects on trophoblast function contributing to PE.

Maternal genomic imprinting is crucial for the maintenance of adult stem cells, which is accomplished by maintaining long-term haematopoietic stem cell quiescence. Epigenetic control of adult stem cells A new study by Linheng Li and colleagues investigates the effect of deletion of the H19 differentially methylated region (H19-DMR) in haematopoietic stem cells. The DMR is known to control expression of the imprinted H19 and Igf2 genes from the H19 – Igf2 locus, restricting H19 expression to the maternal allele and Igf2 to the paternal allele. The authors report the predominant expression of a list of maternally expressed growth-restricting imprinted genes in long-term haematopoietic stem cells (LT-HSCs), but not in proliferating short-term HSCs, suggesting a crucial role for genomic imprinting in maintaining quiescent LT-HSCs. The epigenetic regulation of imprinted genes by monoallelic DNA methylation of either maternal or paternal alleles is critical for embryonic growth and development 1 . Imprinted genes were recently shown to be expressed in mammalian adult stem cells to support self-renewal of neural and lung stem cells 2 , 3 , 4 ; however, a role for imprinting per se in adult stem cells remains elusive. Here we show upregulation of growth-restricting imprinted genes, including in the H19–Igf2 locus 5 , in long-term haematopoietic stem cells and their downregulation upon haematopoietic stem cell activation and proliferation. A differentially methylated region upstream of H19 (H19-DMR), serving as the imprinting control region, determines the reciprocal expression of H19 from the maternal allele and Igf2 from the paternal allele 1 . In addition, H19 serves as a source of miR-675, which restricts Igf1r expression 6 . We demonstrate that conditional deletion of the maternal but not the paternal H19-DMR reduces adult haematopoietic stem cell quiescence, a state required for long-term maintenance of haematopoietic stem cells, and compromises haematopoietic stem cell function. Maternal-specific H19-DMR deletion results in activation of the Igf2–Igfr1 pathway, as shown by the translocation of phosphorylated FoxO3 (an inactive form) from nucleus to cytoplasm and the release of FoxO3-mediated cell cycle arrest, thus leading to increased activation, proliferation and eventual exhaustion of haematopoietic stem cells. Mechanistically, maternal-specific H19-DMR deletion leads to Igf2 upregulation and increased translation of Igf1r, which is normally suppressed by H19 -derived miR-675. Similarly, genetic inactivation of Igf1r partly rescues the H19-DMR deletion phenotype. Our work establishes a new role for this unique form of epigenetic control at the H19–Igf2 locus in maintaining adult stem cells.

Background DNA cytosine modifications, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are key epigenetic regulators with distinct functions. Dissecting the ternary code (C, 5mC, 5hmC) across tissues and cell types remains a critical priority due to the limitations of traditional profiling methods based on bisulfite conversion. Results Here, we leverage the combined bisulfite and enzymatic (bACE) conversion with the Mouse Methylation BeadChip to generate 265 base-resolution ternary-code modification maps of 5mC and 5hmC across 29 mouse tissue types spanning 8–76 weeks of age and both sexes. Our atlas reveals a complex grammar of 5hmC distribution, jointly shaped by cell mitotic activity, chromatin states, and interplay with 5mC at the same and neighboring CpG sites. Of note, we demonstrate that 5hmC significantly complements 5mC-based biomarkers in delineating cell identity in both brain and non-brain tissues. Each modification state, including 5hmC alone, accurately discriminates tissue types, enabling high-precision machine learning classification of epigenetic identity. Furthermore, the ternary methylome variations extensively implicate gene transcriptional variation, with age-related changes correlated with gene expression in a tissue-dependent manner. Conclusions Our work reveals how tissue, sex, and age jointly govern the dynamics of the two cytosine modifications, augments the scope of DNA modification biomarker discovery, and provides a reference atlas to explore epigenetic dynamics in development and disease.

The agouti viable yellow ( A vy ) allele is an insertional mutation in the mouse genome caused by a variably methylated intracisternal A particle (VM-IAP) retrotransposon. A vy expressivity is sensitive to a range of early-life chemical exposures and nutritional interventions, suggesting that environmental perturbations can have long-lasting effects on the methylome. However, the extent to which VM-IAP elements are environmentally labile with phenotypic implications is unknown. Using a recently identified repertoire of VM-IAPs, we assessed the epigenetic effects of different environmental contexts. A longitudinal aging analysis indicated that VM-IAPs are stable across the murine lifespan, with only small increases in DNA methylation detected for a subset of loci. No significant effects were observed after maternal exposure to the endocrine disruptor bisphenol A, an obesogenic diet or methyl donor supplementation. A genetic mouse model of abnormal folate metabolism exhibited shifted VM-IAP methylation levels and altered VM-IAP-associated gene expression, yet these effects are likely largely driven by differential targeting by polymorphic KRAB zinc finger proteins. We conclude that epigenetic variability at retrotransposons is not predictive of environmental susceptibility. Variably methylated intracisternal A particle (VM-IAP) retrotransposons are stable across the murine lifespan. VM-IAP retrotransposons are unaffected by maternal exposure to the endocrine disruptor bisphenol A, an obesogenic diet or methyl donor supplementation.