Stefanie Seisenberger

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.

The Dynamics of Genome-wide DNA Methylation Reprogramming in Mouse Primordial Germ Cells

Summary Genome-wide DNA methylation reprogramming occurs in mouse primordial germ cells (PGCs) and preimplantation embryos, but the precise dynamics and biological outcomes are largely unknown. We have carried out whole-genome bisulfite sequencing (BS-Seq) and RNA-Seq across key stages from E6.5 epiblast to E16.5 PGCs. Global loss of methylation takes place during PGC expansion and migration with evidence for passive demethylation, but sequences that carry long-term epigenetic memory (imprints, CpG islands on the X chromosome, germline-specific genes) only become demethylated upon entry of PGCs into the gonads. The transcriptional profile of PGCs is tightly controlled despite global hypomethylation, with transient expression of the pluripotency network, suggesting that reprogramming and pluripotency are inextricably linked. Our results provide a framework for the understanding of the epigenetic ground state of pluripotency in the germline.

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.

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.

Methylome analysis using MeDIP-seq with low DNA concentrations

DNA methylation is an epigenetic mark that has a crucial role in many biological processes. To understand the functional consequences of DNA methylation on phenotypic plasticity, a genome-wide analysis should be embraced. This in turn requires a technique that balances accuracy, genome coverage, resolution and cost, yet is low in DNA input in order to minimize the drain on precious samples. Methylated DNA immunoprecipitation-sequencing (MeDIP-seq) fulfils these criteria, combining MeDIP with massively parallel DNA sequencing. Here we report an improved protocol using 100-fold less genomic DNA than that commonly used. We show comparable results for specificity (>97%) and enrichment (>100-fold) over a wide range of DNA concentrations (5,000–50 ng) and demonstrate the utility of the protocol for the generation of methylomes from rare bone marrow cells using 160–300 ng of starting DNA. The protocol described here, i.e., DNA extraction to generation of MeDIP-seq library, can be completed within 3–5 d.

Conceptual links between DNA methylation reprogramming in the early embryo and primordial germ cells

DNA methylation is a carrier of important regulatory information that undergoes global reprogramming in the mammalian germ line, including pre-implantation embryos and primordial germ cells (PGCs). A flurry of recent studies have employed technical advances to generate global profiles of methylation and hydroxymethylation in these cells, unravelling the dynamics of methylation erasure at single locus resolution. Active demethylation in the zygote, involving extensive oxidation, is followed by passive loss over early cell divisions. Certain gamete-contributed methylation marks appear to have evolved non-canonical mechanisms for targeted maintenance of methylation in the face of these processes. These protected sequences include the imprinting control regions (ICRs) required for parental imprinting but also a surprising number of other regions. Such targeted maintenance mechanisms may also operate at certain sequences during early PGC migration when global passive demethylation occurs. In later gonadal PGCs, imprints must be reset and this may be achieved through the targeting of active mechanisms including oxidation. Thus, emerging evidence paints a complex picture whereby active and passive demethylation pathways operate synergistically and in parallel to ensure robust erasure in the early embryo and PGCs.

Retrotransposons and germ cells: reproduction, death, and diversity

The evolutionary success of retrotransposable elements is reflected by their abundance in mammalian genomes. To restrict their further advance, a number of defence mechanisms have been put in place by the host. These seem to be particularly effective in the germ line while somatic lineages might be more permissive to new insertions, as recent work by Kano and colleagues suggests.

In utero undernourishment perturbs the adult sperm methylome and is linked to metabolic disease transmission

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.