Epigenetic reprogramming in mouse primordial germ cells

Genome-wide epigenetic reprogramming in mammalian germ cells, zygote and early embryos, plays a crucial role in regulating genome functions at critical stages of development. We show here that mouse primordial germ cells (PGCs) exhibit dynamic changes in epigenetic modifications between days 10.5 and 12.5 post coitum (dpc). First, contrary to previous suggestions, we show that PGCs do indeed acquire genome-wide de novo methylation during early development and migration into the genital ridge. However, following their entry into the genital ridge, there is rapid erasure of DNA methylation of regions within imprinted and non-imprinted loci. For most genes, the erasure commences simultaneously in PGCs in both male and female embryos, which is completed within 1 day of development. Based on the kinetics of this process, we suggest that this is an active demethylation process initiated upon the entry of PGCs into the gonadal anlagen. The timing of reprogramming in PGCs is crucial since it ensures that germ cells of both sexes acquire an equivalent epigenetic state prior to the differentiation of the definitive male and female germ cells in which new parental imprints are established subsequently. Some repetitive elements, however, show incomplete erasure, which may be essential for chromosome stability and for preventing activation of transposons to reduce the risk of germline mutations. Aberrant epigenetic reprogramming in the germ line would cause the inheritance of epimutations that may have consequences for human diseases as suggested by studies on mouse models.     

生殖细胞及早期胚胎基因组印记的表观调控

基因组印记是一种区别父母等位基因的表观遗传过程,可导致父源和母源基因特异性表达。印记是在配子发生过程中全基因组表观重编程时获得的,且在早期胚胎发育过程中得以维持。因此,在全基因组重编程过程中,对印记的识别和维持十分重要。本文概述了原始生殖细胞的印记清除、双亲原始生殖细胞的印记获得以及早期胚胎发育过程中印记维持的相关过程,并对在印记区域内保护印记基因免受全基因组DNA去甲基化的表观遗传因子的相关作用机制进行了讨论。     

Parallel mechanisms of epigenetic reprogramming in the germline

Germ cells possess the extraordinary and unique capacity to give rise to a new organism and create an enduring link between all generations. To acquire this property, primordial germ cells (PGCs) transit through an unprecedented programme of sequential epigenetic events that culminates in an epigenomic basal state that is the foundation of totipotency. This process is underpinned by genome-wide DNA demethylation, which may occur through several overlapping pathways, including conversion to 5-hydroxymethylcytosine. We propose that the epigenetic programme in PGCs operates through multiple parallel mechanisms to ensure robustness at the level of individual cells while also being flexible through functional redundancy to guarantee high fidelity of the process. Gaining a better understanding of the molecular mechanisms that direct epigenetic reprogramming in PGCs will enhance our ability to manipulate epigenetic memory, cell-fate decisions and applications in regenerative medicine.     

Early loss of Xist RNA expression and inactive X chromosome associated chromatin modification in developing primordial germ cells

BACKGROUND: The inactive X chromosome characteristic of female somatic lineages is reactivated during development of the female germ cell lineage. In mouse, analysis of protein products of X-linked genes and/or transgenes located on the X chromosome has indicated that reactivation occurs after primordial germ cells reach the genital ridges. PRINCIPAL FINDINGS/METHODOLOGY: We present evidence that the epigenetic reprogramming of the inactive X-chromosome is initiated earlier than was previously thought, around the time that primordial germ cells (PGCs) migrate through the hindgut. Specifically, we find that Xist RNA expression, the primary signal for establishment of chromosome silencing, is extinguished in migrating PGCs. This is accompanied by displacement of Polycomb-group repressor proteins Eed and Suz(12), and loss of the inactive X associated histone modification, methylation of histone H3 lysine 27. CONCLUSIONS/SIGNIFICANCE: We conclude that X reactivation in primordial germ cells occurs progressively, initiated by extinction of Xist RNA around the time that germ cells migrate through the hindgut to the genital ridges. The events that we observe are reminiscent of X reactivation of the paternal X chromosome in inner cell mass cells of mouse pre-implantation embryos and suggest a unified model in which execution of the pluripotency program represses Xist RNA thereby triggering progressive reversal of epigenetic silencing of the X chromosome.     

Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells.

Genomic reprogramming of primordial germ cells (PGCs), which includes genome-wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG-thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non-imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re-activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG-somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG-somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development.     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

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.     

Epiblast stem cell-based system reveals reprogramming synergy of germline factors

Epigenetic reprogramming in early germ cells is critical toward the establishment of totipotency, but investigations of the germline events are intractable. An objective cell culture-based system could provide mechanistic insight on how the key determinants of primordial germ cells (PGCs), including Prdm14, induce reprogramming in germ cells to an epigenetic ground state. Here we show a Prdm14-Klf2 synergistic effect that can accelerate and enhance reversion of mouse epiblast stem cells (epiSCs) to a naive pluripotent state, including X reactivation and DNA demethylation. Notably, Prdm14 alone has little effect on epiSC reversion, but it enhances the competence for reprogramming and potentially PGC specification. Reprogramming of epiSCs by the combinatorial effect of Prdm14-Klf2 involves key epigenetic changes, which might have an analogous role in PGCs. Our study provides a paradigm toward a systematic analysis of how other key genes contribute to complex and dynamic events of reprogramming in the germline.     

Germline-derived DNA methylation and early embryo epigenetic reprogramming: The selected survival of imprints

DNA methylation is an essential epigenetic mechanism involved in many essential cellular processes. During development epigenetic reprograming takes place during gametogenesis and then again in the pre-implantation embryo. These two reprograming windows ensure genome-wide removal of methylation in the primordial germ cells so that sex-specific signatures can be acquired in the sperm and oocyte. Following fertilization the majority of this epigenetic information is erased to give the developing embryo an epigenetic profile coherent with pluripotency. It is estimated that ∼65% of the genome is differentially methylated between the gametes, however following embryonic reprogramming only parent-of-origin methylation at known imprinted loci remains. This suggests that trans-acting factors such as Zfp57 can discriminate imprinted differentially methylated regions (DMRs) from the thousands of CpG rich regions that are differentially marked in the gametes. Recently transient imprinted DMRs have been identified suggesting that these loci are also protected from pre-implantation reprograming but succumb to de novo remethylation at the implantation stage. This highlights that “ubiquitous” imprinted loci are also resilient to gaining methylation by protecting their unmethylated alleles. In this review I examine the processes involved in epigenetic reprograming and the mechanisms that ensure allelic methylation at imprinted loci is retained throughout the life of the organism, discussing the critical differences between mouse and humans.This article is part of a Directed Issue entitled: Epigenetics Dynamics in development and disease.     

Nuclear transfer: Progress and quandaries

Cloning mammals by nuclear transfer is a powerful technique that is quickly advancing the development of genetically defined animal models. However, the overall efficiency of nuclear transfer is still very low and several hurdles remain before the power of this technique will be fully harnessed. Among these hurdles include an incomplete understanding of biologic processes that control epigenetic reprogramming of the donor genome following nuclear transfer. Incomplete epigenetic reprogramming is considered the major cause of the developmental failure of cloned embryos and is frequently associated with the disregulation of specific genes. At present, little is known about the developmental mechanism of reconstructed embryos. Therefore, screening strategies to design nuclear transfer protocols that will mimic the epigenetic remodeling occurring in normal embryos and identifying molecular parameters that can assess the developmental potential of pre-implantation embryos are becoming increasingly important. A crucial need at present is to understand the molecular events required for efficient reprogramming of donor genomes after nuclear transfer. This knowledge will help to identify the molecular basis of developmental defects seen in cloned embryos and provide methods for circumventing such problems associated with cloning the future application of this technology.     

Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos

The mammalian germline is generally assumed to undergo extensive epigenetic reprogramming during embryonic development, including a nearly complete erasure of DNA methylation. This assumption does, however, to large degree rely on data from mouse, and despite a well-grounded picture the general nature of these data needs to be validated by investigations of other mammalian species. This study represents such a contribution in the examination of the germline in the domestic pig (Sus scrofa). Semiquantitative immunohistochemistry was used to investigate the level of DNA methylation in the POU5F1-positive primordial germ cells (PGCs) compared with neighboring somatic cells in porcine embryos at Embryonic Day 15 (E15), E17, E20, E21, and E28. We show that, in agreement with the mouse model, a significantly lower level of DNA methylation was observed in the early migrating PGCs. This level was decreasing until a stage coinciding with the entrance of the PGCs to the genital ridge. After this, the methylation level increased. Using whole-mount immunostaining, we determined the spatial arrangement of the porcine PGCs in the period between E15 and E28, allowing some comparison with the migration of the murine germline. The overall conclusion from the obtained data is that the DNA methylation changes in porcine PGCs, as well as the migration of these cells, parallels the picture reported for the mouse.     

Mechanisms of histone H3 lysine 27 trimethylation remodeling during early mammalian development

During fertilization, two of the most differentiated cells in the mammalian organism, a sperm and oocyte, are combined to form a pluripotent embryo. Dynamic changes in chromatin structure allow the transition of the chromatin on these specialized cells into an embryonic configuration capable of generating every cell type. Initially, this reprogramming activity is supported by oocyte-derived factors accumulated during oogenesis as proteins and mRNAs; however, the underlying molecular mechanisms that govern it remain poorly characterized. Trimethylation of histone H3 at lysine 27 (H3K27me3) is a repressive epigenetic mark that changes dynamically during pre-implantation development in mice, bovine and pig embryos. Here we present data and hypotheses related to the potential mechanisms behind H3K27me3 remodeling during early development. We postulate that the repressive H3K27me3 mark is globally erased from the parental genomes in order to remove the gametic epigenetic program and to establish a pluripotent embryonic epigenome. We discuss information gathered in mice, pigs, and bovine, with the intent of providing a comparative analysis of the reprogramming of this epigenetic mark during early mammalian development.     

Mechanisms of histone H3 lysine 27 trimethylation remodeling during early mammalian development

During fertilization, two of the most differentiated cells in the mammalian organism, a sperm and oocyte, are combined to form a pluripotent embryo. Dynamic changes in chromatin structure allow the transition of the chromatin on these specialized cells into an embryonic configuration capable of generating every cell type. Initially, this reprogramming activity is supported by oocyte-derived factors accumulated during oogenesis as proteins and mRNAs; however, the underlying molecular mechanisms that govern it remain poorly characterized. Trimethylation of histone H3 at lysine 27 (H3K27me3) is a repressive epigenetic mark that changes dynamically during pre-implantation development in mice, bovine and pig embryos. Here we present data and hypotheses related to the potential mechanisms behind H3K27me3 remodeling during early development. We postulate that the repressive H3K27me3 mark is globally erased from the parental genomes in order to remove the gametic epigenetic program and to establish a pluripotent embryonic epigenome. We discuss information gathered in mice, pigs, and bovine, with the intent of providing a comparative analysis of the reprogramming of this epigenetic mark during early mammalian development.     

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes,followed by major epigenetic remodeling leading to the formation of a totipotent embryo.During the development of the pre-implantation embryo,precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality,but the underlying molecular mechanisms remain elusive.For the past few years,unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development,taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies.The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals,including DNA methylation,histone modifications,chromatin accessibility and 3D chromatin organization.     

PR结构域蛋白质与原始生殖细胞特化

原始生殖细胞特化在精子和卵子生成过程中发挥着重要的作用,而PR结构域蛋白质(PR-domain protein,PRDM)家族部分成员参与了该过程。PRDM1可抑制体细胞程序化过程中基因的表达,而PRDM1和PRDM14共同参与了潜在的全能性细胞的重新获取和基因组范围内表观遗传学重编程。这三个过程都是原始生殖细胞特化所必需的。此外,原始生殖细胞特化还需要一些其他因素如骨形态发生蛋白4(bone morphogenetic protein4,Bmp4)和RNA结合蛋白Lin28,这些因素通过影响PRDM发挥生理作用。对原始生殖细胞特化的理解有利于生殖细胞发育和相关问题的研究。     

Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos. We previously found that some mouse PGCs develop into pluripotential cells (EG cells) when cultured on a feeder layer expressing the membrane bound form of Steel factor with culture medium containing leukemia inhibitory factor and basic fibroblast growth factor. To understand the mechanisms of the conversion of PGCs into EG cells, we attempted to identify PGC subpopulations that have the ability to develop into EG cells. Using flow cytometry, we fractionated PGCs by the expression of the cell surface antigen integrin α6, as well as by the detection of side‐population (SP) cells in which stem cells are enriched in various tissues. PGCs with negative or low integrin α6 expression and with SP cell phenotype showed higher potential to convert to EG cells. Negative or low integrin α6 expression in PGCs was also correlated with lower expression of Ddx4, which is specifically expressed in PGCs after embryonic day 10.5. The results indicate that the primitive PGC population showing the SP cell phenotype among undifferentiated PGCs has a higher ability of being converted into EG cells. Thus, conversion of PGCs into pluripotential stem cells may be regulated by being influenced by the natural status of individual PGCs as well as the reprogramming process after starting culture.