Life Sciences Research for Lifelong Health

Publications

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Title / Authors / Details Open Access Download

DNA methylation homeostasis in human and mouse development.
Iurlaro M, von Meyenn F, Reik W

The molecular pathways that regulate gain and loss of DNA methylation during mammalian development need to be tightly balanced to maintain a physiological equilibrium. Here we explore the relative contributions of the different pathways and enzymatic activities involved in methylation homeostasis in the context of genome-wide and locus-specific epigenetic reprogramming in mammals. An adaptable epigenetic machinery allows global epigenetic reprogramming to concur with local maintenance of critical epigenetic memory in the genome, and appears to regulate the tempo of global reprogramming in different cell lineages and species.

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Current opinion in genetics & development, 43, 1879-0380, 101-109, 2017

PMID: 28260631

Aging yeast gain a competitive advantage on non-optimal carbon sources.
Frenk S, Pizza G, Walker RV, Houseley J

Animals, plants and fungi undergo an aging process with remarkable physiological and molecular similarities, suggesting that aging has long been a fact of life for eukaryotes and one to which our unicellular ancestors were subject. Key biochemical pathways that impact longevity evolved prior to multicellularity, and the interactions between these pathways and the aging process therefore emerged in ancient single-celled eukaryotes. Nevertheless, we do not fully understand how aging impacts the fitness of unicellular organisms, and whether such cells gain a benefit from modulating rather than simply suppressing the aging process. We hypothesized that age-related loss of fitness in single-celled eukaryotes may be counterbalanced, partly or wholly, by a transition from a specialist to a generalist life-history strategy that enhances adaptability to other environments. We tested this hypothesis in budding yeast using competition assays and found that while young cells are more successful in glucose, highly aged cells outcompete young cells on other carbon sources such as galactose. This occurs because aged yeast divide faster than young cells in galactose, reversing the normal association between age and fitness. The impact of aging on single-celled organisms is therefore complex and may be regulated in ways that anticipate changing nutrient availability. We propose that pathways connecting nutrient availability with aging arose in unicellular eukaryotes to capitalize on age-linked diversity in growth strategy and that individual cells in higher eukaryotes may similarly diversify during aging to the detriment of the organism as a whole.

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Aging cell, , 1474-9726, , 2017

PMID: 28247585

Open Access

The Ageing Brain: Effects on DNA Repair and DNA Methylation in Mice.
Langie SA, Cameron KM, Ficz G, Oxley D, Tomaszewski B, Gorniak JP, Maas LM, Godschalk RW, van Schooten FJ, Reik W, von Zglinicki T, Mathers JC

Base excision repair (BER) may become less effective with ageing resulting in accumulation of DNA lesions, genome instability and altered gene expression that contribute to age-related degenerative diseases. The brain is particularly vulnerable to the accumulation of DNA lesions; hence, proper functioning of DNA repair mechanisms is important for neuronal survival. Although the mechanism of age-related decline in DNA repair capacity is unknown, growing evidence suggests that epigenetic events (e.g., DNA methylation) contribute to the ageing process and may be functionally important through the regulation of the expression of DNA repair genes. We hypothesize that epigenetic mechanisms are involved in mediating the age-related decline in BER in the brain. Brains from male mice were isolated at 3-32 months of age. Pyrosequencing analyses revealed significantly increased Ogg1 methylation with ageing, which correlated inversely with Ogg1 expression. The reduced Ogg1 expression correlated with enhanced expression of methyl-CpG binding protein 2 and ten-eleven translocation enzyme 2. A significant inverse correlation between Neil1 methylation at CpG-site2 and expression was also observed. BER activity was significantly reduced and associated with increased 8-oxo-7,8-dihydro-2'-deoxyguanosine levels. These data indicate that Ogg1 and Neil1 expression can be epigenetically regulated, which may mediate the effects of ageing on DNA repair in the brain.

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Genes, 8, , , 2017

PMID: 28218666

Open Access

Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).
Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G

DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

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Nature protocols, 12, 1750-2799, 534-547, 2017

PMID: 28182018

Tracking the embryonic stem cell transition from ground state pluripotency.
Kalkan T, Olova N, Roode M, Mulas C, Lee HJ, Nett I, Marks H, Walker R, Stunnenberg HG, Lilley KS, Nichols J, Reik W, Bertone P, Smith A

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.

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Development (Cambridge, England), , 1477-9129, , 2017

PMID: 28174249

Open Access

Gender Differences in Global but Not Targeted Demethylation in iPSC Reprogramming.
Milagre I, Stubbs TM, King MR, Spindel J, Santos F, Krueger F, Bachman M, Segonds-Pichon A, Balasubramanian S, Andrews SR, Dean W, Reik W

Global DNA demethylation is an integral part of reprogramming processes in vivo and in vitro, but whether it occurs in the derivation of induced pluripotent stem cells (iPSCs) is not known. Here, we show that iPSC reprogramming involves both global and targeted demethylation, which are separable mechanistically and by their biological outcomes. Cells at intermediate-late stages of reprogramming undergo transient genome-wide demethylation, which is more pronounced in female cells. Global demethylation requires activation-induced cytidine deaminase (AID)-mediated downregulation of UHRF1 protein, and abolishing demethylation leaves thousands of hypermethylated regions in the iPSC genome. Independently of AID and global demethylation, regulatory regions, particularly ESC enhancers and super-enhancers, are specifically targeted for hypomethylation in association with transcription of the pluripotency network. Our results show that global and targeted DNA demethylation are conserved and distinct reprogramming processes, presumably because of their respective roles in epigenetic memory erasure and in the establishment of cell identity.

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Cell reports, 18, 2211-1247, 1079-1089, 2017

PMID: 28147265

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids.
Canovas S, Ivanova E, Romar R, García-Martínez S, Soriano-Úbeda C, García-Vázquez FA, Saadeh H, Andrews S, Kelsey G, Coy P

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability to. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

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eLife, 6, 2050-084X, , 2017

PMID: 28134613

Open Access

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.
Hassan-Zadeh V, Rugg-Gunn P, Bazett-Jones DP

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

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Chromosoma, , 1432-0886, , 2017

PMID: 28084535

Derivation and Culture of Epiblast Stem Cell (EpiSC) Lines.
Rugg-Gunn P

This protocol describes the derivation and culture of epiblast stem cells (EpiSCs) from early postimplantation epiblasts. EpiSCs can be maintained in an undifferentiated state and retain the ability to generate tissues from all three germ layers in vitro and to form teratomas in vivo. However, they seem unable to form chimeras. Whether this is due to differences in developmental status or a cellular incompatibility (e.g., cell adhesion) between EpiSCs and the host inner cell mass (ICM) is currently unclear. Other differences between mouse embryonic stem (ES) cells and EpiSCs also exist, including gene expression profiles and different growth factor requirements for self-renewal. Thus, EpiSCs provide an important in vitro model for studying the establishment and maintenance of pluripotency in postimplantation epiblast tissues.

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Cold Spring Harbor protocols, 2017, 1559-6095, pdb.prot093971, 2017

PMID: 28049783

Derivation and Culture of Extra-Embryonic Endoderm Stem Cell Lines.
Rugg-Gunn P

Whereas embryonic stem (ES) cells are isolated from the embryonic lineage of the blastocyst, other stable stem cell lines can be derived from the extraembryonic tissues of the early mouse embryo. Trophoblast stem (TS) cells are derived from trophectoderm and early postimplantation trophoblast, and extraembryonic endoderm stem (XEN) cells are derived from primitive endoderm. The derivation of XEN cell lines from 3.5-dpc mouse blastocysts, described here, is similar to the derivation of TS cell lines. TS and XEN cells can self-renew in vitro and differentiate in vitro and in chimeras (in vivo) in a lineage-appropriate manner, showing the developmental potential of their origin, thus providing important models to study the mouse extraembryonic lineages.

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Cold Spring Harbor protocols, 2017, 1559-6095, pdb.prot093963, 2017

PMID: 28049782

XACT Noncoding RNA Competes with XIST in the Control of X Chromosome Activity during Human Early Development.
Vallot C, Patrat C, Collier AJ, Huret C, Casanova M, Liyakat Ali TM, Tosolini M, Frydman N, Heard E, Rugg-Gunn PJ, Rougeulle C

Sex chromosome dosage compensation is essential in most metazoans, but the developmental timing and underlying mechanisms vary significantly, even among placental mammals. Here we identify human-specific mechanisms regulating X chromosome activity in early embryonic development. Single-cell RNA sequencing and imaging revealed co-activation and accumulation of the long noncoding RNAs (lncRNAs) XACT and XIST on active X chromosomes in both early human pre-implantation embryos and naive human embryonic stem cells. In these contexts, the XIST RNA adopts an unusual, highly dispersed organization, which may explain why it does not trigger X chromosome inactivation at this stage. Functional studies in transgenic mouse cells show that XACT influences XIST accumulation in cis. Our findings therefore suggest a mechanism involving antagonistic activity of XIST and XACT in controlling X chromosome activity in early human embryos, and they highlight the contribution of rapidly evolving lncRNAs to species-specific developmental mechanisms.

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Cell stem cell, , 1875-9777, , 2016

PMID: 27989768

Open Access

A Hox-Embedded Long Noncoding RNA: Is It All Hot Air?
Selleri L, Bartolomei MS, Bickmore WA, He L, Stubbs L, Reik W, Barsh GS

PLoS genetics, 12, 1553-7404, e1006485, 2016

PMID: 27977680

Open Access

Deletion of the Polycomb-Group Protein EZH2 Leads to Compromised Self-Renewal and Differentiation Defects in Human Embryonic Stem Cells.
Collinson A, Collier AJ, Morgan NP, Sienerth AR, Chandra T, Andrews S, Rugg-Gunn PJ

Through the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of EZH2-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon EZH2 deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in EZH2-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. EZH2-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. EZH2-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, EZH2 is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.

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Cell reports, 17, 2211-1247, 2700-2714, 2016

PMID: 27926872

Open Access

Efficient targeted DNA methylation with chimeric dCas9-Dnmt3a-Dnmt3L methyltransferase.
Stepper P, Kungulovski G, Jurkowska RZ, Chandra T, Krueger F, Reinhardt R, Reik W, Jeltsch A, Jurkowski TP

DNA methylation plays a critical role in the regulation and maintenance of cell-type specific transcriptional programs. Targeted epigenome editing is an emerging technology to specifically regulate cellular gene expression in order to modulate cell phenotypes or dissect the epigenetic mechanisms involved in their control. In this work, we employed a DNA methyltransferase Dnmt3a-Dnmt3L construct fused to the nuclease-inactivated dCas9 programmable targeting domain to introduce DNA methylation into the human genome specifically at the EpCAM, CXCR4 and TFRC gene promoters. We show that targeting of these loci with single gRNAs leads to efficient and widespread methylation of the promoters. Multiplexing of several guide RNAs does not increase the efficiency of methylation. Peaks of targeted methylation were observed around 25 bp upstream and 40 bp downstream of the PAM site, while 20-30 bp of the binding site itself are protected against methylation. Potent methylation is dependent on the multimerization of Dnmt3a/Dnmt3L complexes on the DNA. Furthermore, the introduced methylation causes transcriptional repression of the targeted genes. These new programmable epigenetic editors allow unprecedented control of the DNA methylation status in cells and will lead to further advances in the understanding of epigenetic signaling.

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Nucleic acids research, , 1362-4962, , 2016

PMID: 27899645

Open Access

The H3K9 dimethyltransferases EHMT1/2 protect against pathological cardiac hypertrophy.
Thienpont B, Aronsen JM, Robinson EL, Okkenhaug H, Loche E, Ferrini A, Brien P, Alkass K, Tomasso A, Agrawal A, Bergmann O, Sjaastad I, Reik W, Roderick HL

Cardiac hypertrophic growth in response to pathological cues is associated with reexpression of fetal genes and decreased cardiac function and is often a precursor to heart failure. In contrast, physiologically induced hypertrophy is adaptive, resulting in improved cardiac function. The processes that selectively induce these hypertrophic states are poorly understood. Here, we have profiled 2 repressive epigenetic marks, H3K9me2 and H3K27me3, which are involved in stable cellular differentiation, specifically in cardiomyocytes from physiologically and pathologically hypertrophied rat hearts, and correlated these marks with their associated transcriptomes. This analysis revealed the pervasive loss of euchromatic H3K9me2 as a conserved feature of pathological hypertrophy that was associated with reexpression of fetal genes. In hypertrophy, H3K9me2 was reduced following a miR-217-mediated decrease in expression of the H3K9 dimethyltransferases EHMT1 and EHMT2 (EHMT1/2). miR-217-mediated, genetic, or pharmacological inactivation of EHMT1/2 was sufficient to promote pathological hypertrophy and fetal gene reexpression, while suppression of this pathway protected against pathological hypertrophy both in vitro and in mice. Thus, we have established a conserved mechanism involving a departure of the cardiomyocyte epigenome from its adult cellular identity to a reprogrammed state that is accompanied by reexpression of fetal genes and pathological hypertrophy. These results suggest that targeting miR-217 and EHMT1/2 to prevent H3K9 methylation loss is a viable therapeutic approach for the treatment of heart disease.

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The Journal of clinical investigation, , 1558-8238, , 2016

PMID: 27893464

TET-dependent regulation of retrotransposable elements in mouse embryonic stem cells.
de la Rica L, Deniz Ö, Cheng KC, Todd CD, Cruz C, Houseley J, Branco MR

Ten-eleven translocation (TET) enzymes oxidise DNA methylation as part of an active demethylation pathway. Despite extensive research into the role of TETs in genome regulation, little is known about their effect on transposable elements (TEs), which make up nearly half of the mouse and human genomes. Epigenetic mechanisms controlling TEs have the potential to affect their mobility and to drive the co-adoption of TEs for the benefit of the host.

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Genome biology, 17, 1474-760X, 234, 2016

PMID: 27863519

From the stem of the placental tree: trophoblast stem cells and their progeny.
Latos PA, Hemberger M

Trophoblast stem cells (TSCs) retain the capacity to self-renew indefinitely and harbour the potential to differentiate into all trophoblast subtypes of the placenta. Recent studies have shown how signalling cascades integrate with transcription factor circuits to govern the fine balance between TSC self-renewal and differentiation. In addition, breakthroughs in reprogramming strategies have enabled the generation of TSCs from fibroblasts, opening up exciting new avenues that may allow the isolation of this stem cell type from other species, notably humans. Here, we review these recent advances in light of their importance for understanding placental pathologies and developing personalised medicine approaches for pregnancy complications.

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Development (Cambridge, England), 143, 1477-9129, 3650-3660, 2016

PMID: 27802134

Crosstalk between pluripotency factors and higher-order chromatin organization.
Lopes Novo C, Rugg-Gunn PJ

Pluripotent cells are characterized by a globally open and accessible chromatin organization that is thought to contribute to cellular plasticity and developmental decision-making. We recently identified the pluripotency factor Nanog as a key regulator of this form of chromatin architecture in mouse embryonic stem cells. In particular, we demonstrated that the transcription factors Nanog and Sall1 co-dependently mediate the epigenetic state of pericentromeric heterochromatin to reinforce a more open and accessible organization in pluripotent cells. Here, we summarize our main findings and place the work into a broader context. We explore how heterochromatin domains could be targets of transcriptional networks in pluripotent cells and are coordinated with cell state. We propose this integration may be to balance the requirement for a dynamic and plastic chromatin organization in pluripotent cells, together with priming for a more restrictive nuclear compartmentalization that is triggered rapidly upon lineage commitment.

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Nucleus (Austin, Tex.), , 1949-1042, 0, 2016

PMID: 27759487

Open Access

Epigenetic inheritance of proteostasis and ageing.
Li C, Casanueva O

Abundant evidence shows that the genome is not as static as once thought and that gene expression can be reversibly modulated by the environment. In some cases, these changes can be transmitted to the next generation even if the environment has reverted. Such transgenerational epigenetic inheritance requires that information be stored in the germline in response to exogenous stressors. One of the most elusive questions in the field of epigenetic inheritance is the identity of such inherited factor(s). Answering this question would allow us to understand how the environment can shape human populations for multiple generations and may help to explain the rapid rise in obesity and neurodegenerative diseases in modern society. It will also provide clues on how we might be able to reprogramme the epigenome to prevent transmission of detrimental phenotypes and identify individuals who might be at increased risk of disease. In this article, we aim to review recent developments in this field, focusing on research conducted mostly in the nematode Caenorhabditis elegans and mice, that link environmental modulators with the transgenerational inheritance of phenotypes that affect protein-folding homoeostasis and ageing.

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Essays in biochemistry, 60, 1744-1358, 191-202, 2016

PMID: 27744335

Open Access

Retinol and ascorbate drive erasure of epigenetic memory and enhance reprogramming to naïve pluripotency by complementary mechanisms.
Hore TA, von Meyenn F, Ravichandran M, Bachman M, Ficz G, Oxley D, Santos F, Balasubramanian S, Jurkowski TP, Reik W

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe(2+) recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome.

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Proceedings of the National Academy of Sciences of the United States of America, , 1091-6490, , 2016

PMID: 27729528

Open Access

Comparative Principles of DNA Methylation Reprogramming during Human and Mouse In Vitro Primordial Germ Cell Specification.
von Meyenn F, Berrens RV, Andrews S, Santos F, Collier AJ, Krueger F, Osorno R, Dean W, Rugg-Gunn PJ, Reik W

Primordial germ cell (PGC) development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state. Here we recapitulate PGC specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells. Repressive chromatin marks (H3K9me2/3) and transposable elements are enriched at demethylation-resistant regions, while active chromatin marks (H3K4me3 or H3K27ac) are more prominent at regions that demethylate faster. The dynamics of specification and epigenetic reprogramming show species-specific differences, in particular markedly slower reprogramming kinetics in the human germline. Differences in developmental kinetics may be explained by differential regulation of epigenetic modifiers. Our work establishes a robust and faithful experimental system of the early events of epigenetic reprogramming and regulation in the germline.

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Developmental cell, 39, 1878-1551, 104-115, 2016

PMID: 27728778

Open Access

MERVL/Zscan4 Network Activation Results in Transient Genome-wide DNA Demethylation of mESCs.
Eckersley-Maslin MA, Svensson V, Krueger C, Stubbs TM, Giehr P, Krueger F, Miragaia RJ, Kyriakopoulos C, Berrens RV, Milagre I, Walter J, Teichmann SA, Reik W

Mouse embryonic stem cells are dynamic and heterogeneous. For example, rare cells cycle through a state characterized by decondensed chromatin and expression of transcripts, including the Zscan4 cluster and MERVL endogenous retrovirus, which are usually restricted to preimplantation embryos. Here, we further characterize the dynamics and consequences of this transient cell state. Single-cell transcriptomics identified the earliest upregulated transcripts as cells enter the MERVL/Zscan4 state. The MERVL/Zscan4 transcriptional network was also upregulated during induced pluripotent stem cell reprogramming. Genome-wide DNA methylation and chromatin analyses revealed global DNA hypomethylation accompanying increased chromatin accessibility. This transient DNA demethylation was driven by a loss of DNA methyltransferase proteins in the cells and occurred genome-wide. While methylation levels were restored once cells exit this state, genomic imprints remained hypomethylated, demonstrating a potential global and enduring influence of endogenous retroviral activation on the epigenome.

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Cell reports, 17, 2211-1247, 179-92, 2016

PMID: 27681430

Establishment and functions of DNA methylation in the germline.
Stewart KR, Veselovska L, Kelsey G

Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm genomes, governs genomic imprinting. In this review, we describe how imprinting has informed our understanding of de novo DNA methylation mechanisms, highlight how recent genome-wide profiling studies have provided unprecedented insights into establishment of the sperm and oocyte methylomes and consider the fate and function of gametic methylation and other epigenetic modifications after fertilization.

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Epigenomics, , 1750-192X, , 2016

PMID: 27659720

In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine.
Iurlaro M, McInroy GR, Burgess HE, Dean W, Raiber EA, Bachman M, Beraldi D, Balasubramanian S, Reik W

Genome-wide methylation of cytosine can be modulated in the presence of TET and thymine DNA glycosylase (TDG) enzymes. TET is able to oxidise 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). TDG can excise the oxidative products 5fC and 5caC, initiating base excision repair. These modified bases are stable and detectable in the genome, suggesting that they could have epigenetic functions in their own right. However, functional investigation of the genome-wide distribution of 5fC has been restricted to cell culture-based systems, while its in vivo profile remains unknown.

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Genome biology, 17, 1474-760X, 141, 2016

PMID: 27356509

Open Access

Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cells.
von Meyenn F, Iurlaro M, Habibi E, Liu NQ, Salehzadeh-Yazdi A, Santos F, Petrini E, Milagre I, Yu M, Xie Z, Kroeze LI, Nesterova TB, Jansen JH, Xie H, He C, Reik W, Stunnenberg HG

Global demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally, we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition to 2i, and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently, there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.

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Molecular cell, , 1097-4164, , 2016

PMID: 27237052

Open Access