Timothy A. Hore

Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation

Methylation at the 5′ position of cytosine in DNA has important roles in genome function and is dynamically reprogrammed during early embryonic and germ cell development. The mammalian genome also contains 5-hydroxymethylcytosine (5hmC), which seems to be generated by oxidation of 5-methylcytosine (5mC) by the TET family of enzymes that are highly expressed in embryonic stem (ES) cells. Here we use antibodies against 5hmC and 5mC together with high throughput sequencing to determine genome-wide patterns of methylation and hydroxymethylation in mouse wild-type and mutant ES cells and differentiating embryoid bodies. We find that 5hmC is mostly associated with euchromatin and that whereas 5mC is under-represented at gene promoters and CpG islands, 5hmC is enriched and is associated with increased transcriptional levels. Most, if not all, 5hmC in the genome depends on pre-existing 5mC and the balance between these two modifications is different between genomic regions. Knockdown of Tet1 and Tet2 causes downregulation of a group of genes that includes pluripotency-related genes (including Esrrb, Prdm14, Dppa3, Klf2, Tcl1 and Zfp42) and a concomitant increase in methylation of their promoters, together with an increased propensity of ES cells for extraembryonic lineage differentiation. Declining levels of TETs during differentiation are associated with decreased hydroxymethylation levels at the promoters of ES cell-specific genes together with increased methylation and gene silencing. We propose that the balance between hydroxymethylation and methylation in the genome is inextricably linked with the balance between pluripotency and lineage commitment.

NANOG-dependent function of TET1 and TET2 in establishment of pluripotency.

Molecular control of the pluripotent state is thought to reside in a core circuitry of master transcription factors including the homeodomain-containing protein NANOG, which has an essential role in establishing ground state pluripotency during somatic cell reprogramming. Whereas the genomic occupancy of NANOG has been extensively investigated, comparatively little is known about NANOG-associated proteins and their contribution to the NANOG-mediated reprogramming process. Using enhanced purification techniques and a stringent computational algorithm, we identify 27 high-confidence protein interaction partners of NANOG in mouse embryonic stem cells. These consist of 19 previously unknown partners of NANOG that have not been reported before, including the ten-eleven translocation (TET) family methylcytosine hydroxylase TET1. We confirm physical association of NANOG with TET1, and demonstrate that TET1, in synergy with NANOG, enhances the efficiency of reprogramming. We also find physical association and reprogramming synergy of TET2 with NANOG, and demonstrate that knockdown of TET2 abolishes the reprogramming synergy of NANOG with a catalytically deficient mutant of TET1. These results indicate that the physical interaction between NANOG and TET1/TET2 proteins facilitates reprogramming in a manner that is dependent on the catalytic activity of TET1/TET2. TET1 and NANOG co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells, and TET1 binding is reduced upon NANOG depletion. Co-expression of NANOG and TET1 increases 5-hydroxymethylcytosine levels at the top-ranked common target loci Esrrb and Oct4 (also called Pou5f1), resulting in priming of their expression before reprogramming to naive pluripotency. We propose that TET1 is recruited by NANOG to enhance the expression of a subset of key reprogramming target genes. These results provide an insight into the reprogramming mechanism of NANOG and uncover a new role for 5-methylcytosine hydroxylases in the establishment of naive pluripotency.

In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism

Introduction The rapid global rise in metabolic disease suggests that nongenetic environmental factors contribute to disease risk. Early life represents a window of phenotypic plasticity important for adult metabolic health and that of future generations. Epigenetic inheritance has been implicated in the paternal transmission of environmentally induced phenotypes, but the mechanisms responsible remain unknown. In utero undernourishment alters the adult germ cell methylome. Undernourishment during PGC reprogramming results in hypomethylation of discrete loci in adult sperm. These regions are enriched in nucleosomes and are low-methylated regions. Although partially resistant to blastocyst reprogramming, differential methylation does not persist in the next generation. However, dysregulated expression of genes neighboring DMRs is observed in F2 offspring. Rationale We investigated the role of DNA methylation in epigenetic inheritance in an established murine model of intergenerational developmental programming. F1 offspring of undernourished dams (UN) have low birth weight and multiple metabolic defects. Metabolic phenotypic inheritance to the F2 generation is observed through the paternal line, even though the F1 mice did not experience postnatal environmental perturbation. The timing of nutritional restriction coincides with methylation reacquisition in F1 male primordial germ cells (PGCs). Therefore, we assessed F1 sperm whole-genome methylation using immunoprecipitation of methylated DNA, combined with high-throughput sequencing, followed by independent validation. We characterized the regions susceptible to methylation change and investigated the legacy of such methylation change in the phenotypic development of the next generation. Results In UN mice, 111 regions are hypomethylated relative to control sperm, and these changes are validated by bisulfite pyrosequencing. Methylation differences span multiple CpGs, with robust absolute changes of 10 to 30% (relative reduction ~50%). The absolute methylation level is consistent with differentially methylated regions (DMRs) being “low-methylated regions,” known to be enriched in regulatory elements. Indeed, luciferase assays suggest a role for these DMRs in transcriptional regulation. Hypomethylated DMRs are significantly depleted from coding and repetitive regions and enriched in intergenic regions and CpG islands. They are also enriched in nucleosome-retaining regions, which suggests that, at some loci, paternal germline hypomethylation induced by in utero undernutrition is transmitted in a chromatin context. DMRs are late to regain methylation in normal male PGCs. This may render them particularly susceptible to environmental perturbations that delay or impair remethylation in late gestation. Except for imprinted loci, gene-associated male germline methylation has generally been thought to be largely erased in the zygote,although recent studies suggest that resistance to reprogramming is more widespread. Indeed, 43% of hypomethylated DMRs persist and thus have the potential to affect development of the next generation. We show that differential methylation is lost in late-gestation F2 tissues, but considerable tissue-specific differences in expression of metabolic genes neighboring DMRs are present. Thus, it is unlikely that these expression changes are directly mediated by altered methylation; rather, the cumulative effects of dysregulated epigenetic patterns earlier in development may yield sustained alterations in chromatin architecture, transcriptional regulatory networks, differentiation, or tissue structure. Conclusion Prenatal undernutrition can compromise male germline epigenetic reprogramming and thus permanently alter DNA methylation in the sperm of adult offspring at regions resistant to zygotic reprogramming. However, persistence of altered DNA methylation into late-gestation somatic tissues of the subsequent generation is not observed. Nonetheless, alterations in gamete methylation may serve as a legacy of earlier developmental exposures and may contribute to the intergenerational transmission of environmentally induced disease. The nutritional sins of the mother… Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science, this issue p. 10.1126/science.1255903 Prenatal assaults change DNA methylation and chromatin structure in sperm and affect offspring. [Also see Perspective by Susiarjo and Bartolomei] Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers

In mammalian development, epigenetic modifications, including DNA methylation patterns, play a crucial role in defining cell fate but also represent epigenetic barriers that restrict developmental potential. At two points in the life cycle, DNA methylation marks are reprogrammed on a global scale, concomitant with restoration of developmental potency. DNA methylation patterns are subsequently re-established with the commitment towards a distinct cell fate. This reprogramming of DNA methylation takes place firstly on fertilization in the zygote, and secondly in primordial germ cells (PGCs), which are the direct progenitors of sperm or oocyte. In each reprogramming window, a unique set of mechanisms regulates DNA methylation erasure and re-establishment. Recent advances have uncovered roles for the TET3 hydroxylase and passive demethylation, together with base excision repair (BER) and the elongator complex, in methylation erasure from the zygote. Deamination by AID, BER and passive demethylation have been implicated in reprogramming in PGCs, but the process in its entirety is still poorly understood. In this review, we discuss the dynamics of DNA methylation reprogramming in PGCs and the zygote, the mechanisms involved and the biological significance of these events. Advances in our understanding of such natural epigenetic reprogramming are beginning to aid enhancement of experimental reprogramming in which the role of potential mechanisms can be investigated in vitro. Conversely, insights into in vitro reprogramming techniques may aid our understanding of epigenetic reprogramming in the germline and supply important clues in reprogramming for therapies in regenerative medicine.

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

Summary Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism.

Introduction The rapid global rise in metabolic disease suggests that nongenetic environmental factors contribute to disease risk. Early life represents a window of phenotypic plasticity important for adult metabolic health and that of future generations. Epigenetic inheritance has been implicated in the paternal transmission of environmentally induced phenotypes, but the mechanisms responsible remain unknown. In utero undernourishment alters the adult germ cell methylome. Undernourishment during PGC reprogramming results in hypomethylation of discrete loci in adult sperm. These regions are enriched in nucleosomes and are low-methylated regions. Although partially resistant to blastocyst reprogramming, differential methylation does not persist in the next generation. However, dysregulated expression of genes neighboring DMRs is observed in F2 offspring. Rationale We investigated the role of DNA methylation in epigenetic inheritance in an established murine model of intergenerational developmental programming. F1 offspring of undernourished dams (UN) have low birth weight and multiple metabolic defects. Metabolic phenotypic inheritance to the F2 generation is observed through the paternal line, even though the F1 mice did not experience postnatal environmental perturbation. The timing of nutritional restriction coincides with methylation reacquisition in F1 male primordial germ cells (PGCs). Therefore, we assessed F1 sperm whole-genome methylation using immunoprecipitation of methylated DNA, combined with high-throughput sequencing, followed by independent validation. We characterized the regions susceptible to methylation change and investigated the legacy of such methylation change in the phenotypic development of the next generation. Results In UN mice, 111 regions are hypomethylated relative to control sperm, and these changes are validated by bisulfite pyrosequencing. Methylation differences span multiple CpGs, with robust absolute changes of 10 to 30% (relative reduction ~50%). The absolute methylation level is consistent with differentially methylated regions (DMRs) being “low-methylated regions,” known to be enriched in regulatory elements. Indeed, luciferase assays suggest a role for these DMRs in transcriptional regulation. Hypomethylated DMRs are significantly depleted from coding and repetitive regions and enriched in intergenic regions and CpG islands. They are also enriched in nucleosome-retaining regions, which suggests that, at some loci, paternal germline hypomethylation induced by in utero undernutrition is transmitted in a chromatin context. DMRs are late to regain methylation in normal male PGCs. This may render them particularly susceptible to environmental perturbations that delay or impair remethylation in late gestation. Except for imprinted loci, gene-associated male germline methylation has generally been thought to be largely erased in the zygote,although recent studies suggest that resistance to reprogramming is more widespread. Indeed, 43% of hypomethylated DMRs persist and thus have the potential to affect development of the next generation. We show that differential methylation is lost in late-gestation F2 tissues, but considerable tissue-specific differences in expression of metabolic genes neighboring DMRs are present. Thus, it is unlikely that these expression changes are directly mediated by altered methylation; rather, the cumulative effects of dysregulated epigenetic patterns earlier in development may yield sustained alterations in chromatin architecture, transcriptional regulatory networks, differentiation, or tissue structure. Conclusion Prenatal undernutrition can compromise male germline epigenetic reprogramming and thus permanently alter DNA methylation in the sperm of adult offspring at regions resistant to zygotic reprogramming. However, persistence of altered DNA methylation into late-gestation somatic tissues of the subsequent generation is not observed. Nonetheless, alterations in gamete methylation may serve as a legacy of earlier developmental exposures and may contribute to the intergenerational transmission of environmentally induced disease. The nutritional sins of the mother… Prenatal exposures of a mother can affect the health of her offspring, but how? Radford et al. found that the male progeny of undernourished pregnant mice had altered DNA chemistry in their sperm. In addition, the offspring displayed compromised metabolic health. The specific affected genes not only lost DNA methylation but also lacked the normal sperm DNA packaging factors (protamines) and instead were enriched in nucleosomes. Thus, when subjected to a suboptimal prenatal environment, offspring feel the effects of the maternal assault. Science, this issue p. 10.1126/science.1255903 Prenatal assaults change DNA methylation and chromatin structure in sperm and affect offspring. [Also see Perspective by Susiarjo and Bartolomei] Adverse prenatal environments can promote metabolic disease in offspring and subsequent generations. Animal models and epidemiological data implicate epigenetic inheritance, but the mechanisms remain unknown. In an intergenerational developmental programming model affecting F2 mouse metabolism, we demonstrate that the in utero nutritional environment of F1 embryos alters the germline DNA methylome of F1 adult males in a locus-specific manner. Differentially methylated regions are hypomethylated and enriched in nucleosome-retaining regions. A substantial fraction is resistant to early embryo methylation reprogramming, which may have an impact on F2 development. Differential methylation is not maintained in F2 tissues, yet locus-specific expression is perturbed. Thus, in utero nutritional exposures during critical windows of germ cell development can impact the male germline methylome, associated with metabolic disease in offspring.

Reprogramming the Methylome: Erasing Memory and Creating Diversity

The inheritance of epigenetic marks, in particular DNA methylation, provides a molecular memory that ensures faithful commitment to transcriptional programs during mammalian development. Epigenetic reprogramming results in global hypomethylation of the genome together with a profound loss of memory, which underlies naive pluripotency. Such global reprogramming occurs in primordial germ cells, early embryos, and embryonic stem cells where reciprocal molecular links connect the methylation machinery to pluripotency. Priming for differentiation is initiated upon exit from pluripotency, and we propose that epigenetic mechanisms create diversity of transcriptional states, which help with symmetry breaking during cell fate decisions and lineage commitment.

Evolution of Genomic Imprinting: Insights from Marsupials and Monotremes

Parent-of-origin gene expression (genomic imprinting) is widespread among eutherian mammals and also occurs in marsupials. Most imprinted genes are expressed in the placenta, but the brain is also a favored site. Although imprinting evolved in therian mammals before the marsupial-eutherian split, the mechanisms have continued to evolve in each lineage to produce differences between the two groups in terms of the number and regulation of imprinted genes. As yet there is no evidence for genomic imprinting in the egg-laying monotreme mammals, although these mammals also form a placenta (albeit short-lived) and transfer nutrients from mother to embryo. Therefore, imprinting was not essential for the evolution of the placenta and its importance in nutrient transfer but the elaboration of imprinted genes in marsupials and eutherians is associated with viviparity. Here we review the recent analyses of imprinted gene clusters in marsupials and monotremes, which have served to shed light on the origin and evolution of imprinting mechanisms in mammals.

Genome-wide Bisulfite Sequencing in Zygotes Identifies Demethylation Targets and Maps the Contribution of TET3 Oxidation

Summary Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup.

Shifting behaviour: epigenetic reprogramming in eusocial insects.

Epigenetic modifications are ancient and widely utilised mechanisms that have been recruited across fungi, plants and animals for diverse but fundamental biological functions, such as cell differentiation. Recently, a functional DNA methylation system was identified in the honeybee, where it appears to underlie queen and worker caste differentiation. This discovery, along with other insights into the epigenetics of social insects, allows provocative analogies to be drawn between insect caste differentiation and cellular differentiation, particularly in mammals. Developing larvae in social insect colonies are totipotent: they retain the ability to specialise as queens or workers, in a similar way to the totipotent cells of early embryos before they differentiate into specific cell lineages. Further, both differentiating cells and insect castes lose phenotypic plasticity by committing to their lineage, losing the ability to be readily reprogrammed. Hence, a comparison of the epigenetic mechanisms underlying lineage differentiation (and reprogramming) between cells and social insects is worthwhile. Here we develop a conceptual model of how loss and regain of phenotypic plasticity might be conserved for individual specialisation in both cells and societies. This framework forges a novel link between two fields of biological research, providing predictions for a unified approach to understanding the molecular mechanisms underlying biological complexity.