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.     

Spatio-Temporal Plasticity in Chromatin Organization in Mouse Cell Differentiation and during Drosophila Embryogenesis

Cellular differentiation and developmental programs require changing patterns of gene expression. Recent experiments have revealed that chromatin organization is highly dynamic within living cells, suggesting possible mechanisms to alter gene expression programs, yet the physical basis of this organization is unclear. In this article, we contrast the differences in the dynamic organization of nuclear architecture between undifferentiated mouse embryonic stem cells and terminally differentiated primary mouse embryonic fibroblasts. Live-cell confocal tracking of nuclear lamina evidences highly flexible nuclear architecture within embryonic stem cells as compared to primary mouse embryonic fibroblasts. These cells also exhibit significant changes in histone and heterochromatin binding proteins correlated with their distinct epigenetic signatures as quantified by immunofluorescence analysis. Further, we follow histone dynamics during the development of the Drosophila melanogaster embryo, which gives an insight into spatio-temporal evolution of chromatin plasticity in an organismal context. Core histone dynamics visualized by fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and fluorescence anisotropy within the developing embryo, revealed an intriguing transition from plastic to frozen chromatin assembly synchronous with cellular differentiation. In the embryo, core histone proteins are highly mobile before cellularization, actively exchanging with the pool in the yolk. This hyperdynamic mobility decreases as cellularization and differentiation programs set in. These findings reveal a direct correlation between the dynamic transitions in chromatin assembly with the onset of cellular differentiation and developmental programs.     

动物早期胚胎发育中染色质结构的继承和重编程

染色质开放性和染色质三维高级结构在基因表达和调控中发挥着非常重要的作用,广泛参与分化、发育、肿瘤发生等细胞生理过程,是表观遗传研究的热点领域之一。动物胚胎发育起始于终端分化的卵子受精形成全能性的受精卵。在精卵结合的过程中,染色质开放性和染色质三维高级结构发生了剧烈的变化,经历继承、重编程、重新建立的过程,并指导调控受精卵分化发育最终成为多细胞、多器官组织的新生命个体。本文介绍了近年来研究染色质开放性和染色质三维高级结构的实验分析技术手段,染色质结构在动物早期胚胎发育过程中的变化规律及其在早期胚胎发育中的作用,染色质结构与其他表观遗传信息(甲基化、组蛋白修饰等)关系方面的重要研究进展和存在的科学问题,以期为表观遗传调控早期胚胎发育的研究提供参考。     

Relaxed 3D genome conformation facilitates the pluripotent to totipotent-like state transition in embryonic stem cells

The 3D genome organization is crucial for gene regulation. Although recent studies have revealed a uniquely relaxed genome conformation in totipotent early blastomeres of both fertilized and cloned embryos, how weakened higher-order chromatin structure is functionally linked to totipotency acquisition remains elusive. Using low-input Hi-C, ATAC-seq and ChIP-seq, we systematically examined the dynamics of 3D genome and epigenome during pluripotent to totipotent-like state transition in mouse embryonic stem cells (ESCs). The spontaneously converted 2-cell-embryo-like cells (2CLCs) exhibited more relaxed chromatin architecture compared to ESCs, including global weakening of both enhancer-promoter interactions and TAD insulation. While the former correlated with inactivation of ESC enhancers and down-regulation of pluripotent genes, the latter might facilitate contacts between the putative new enhancers arising in 2CLCs and neighboring 2C genes. Importantly, disruption of chromatin organization by depleting CTCF or the cohesin complex promoted the ESC to 2CLC transition. Our results thus establish a critical role of 3D genome organization in totipotency acquisition.     

Techniques for and challenges in reconstructing 3D genome structures from 2D chromosome conformation capture data

Chromosome conformation capture technologies that provide frequency information for contacts between genomic regions have been crucial for increasing our understanding of genome folding and regulation. However, such data do not provide direct evidence of the spatial 3D organization of chromatin. In this opinion article, we discuss the development and application of computational methods to reconstruct chromatin 3D structures from experimental 2D contact data, highlighting how such modeling provides biological insights and can suggest mechanisms anchored to experimental data. By applying different reconstruction methods to the same contact data, we illustrate some state-of-the-art of these techniques and discuss our gene resolution approach based on Brownian dynamics and Monte Carlo sampling.     

CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.     

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.     

Spatial mechanisms of gene regulation in metazoan embryos.

The basic characteristics of embryonic process throughout Metazoa are considered with focus on those aspects that provide insight into how cell specification occurs in the initial stages of development. There appear to be three major types of embryogenesis: Type 1, a general form characteristic of most invertebrate taxa of today, in which lineage plays an important role in the spatial organization of the early embryo, and cell specification occurs in situ, by both autonomous and conditional mechanisms; Type 2, the vertebrate form of embryogenesis, which proceeds by mechanisms that are essentially independent of cell lineage, in which diffusible morphogens and extensive early cell migration are particularly important; Type 3, the form exemplified by long germ band insects in which several different regulatory mechanisms are used to generate precise patterns of nuclear gene expression prior to cellularization. Evolutionary implications of the phylogenetic distribution of these types of embryogenesis are considered. Regionally expressed homeodomain regulators are utilized in all three types of embryo, in similar ways in later and postembryonic development, but in different ways in early embryonic development. A specific downstream molecular function for this class of regulator is proposed, based on evidence obtained in vertebrate systems. This provides a route by which to approach the comparative regulatory strategies underlying the three major types of embryogenesis.     

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.     

Embryonic stem cell differentiation: A chromatin perspective

Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.     

Epigenetic regulation of genes during development： A conserved theme from flies to mammals

Eukaryotic genome is organized in form of chromatin within the nucleus. This organization is important for compaction of DNA as well as for the proper expression of the genes. During early embryonic development, genomic packaging receives variety of signals to eventually set up cell type specific expression patterns of genes. This process of regulated chromatinization leads to "cell type specific epigenomes". The expression states attained during differentiation process need to be maintained subsequently throughout the life of the organism. Epigenetie modifications are responsible for chromatin dependent regulatory mechanism and play a key role in maintenance of the expression state-a process referred to as cellular memory. Another key feature in the packaging of the genome is formation of chro- matin domains that are thought to be structural as well as functional units of the higher order chromatin organization. Boundary elements that function to define such domains set the limits of regulatory elements and that of epigenetie modifications. This connection of epige- netic modification, chromatin structure and genome organization has emerged from several studies. Hox genes are among the best studied in this context and have led to the significant understanding of the epigenetic regulation during development. Here we discuss the evolu- tionarily conserved features of epigenetic mechanisms emerged from studies on homeotic gene clusters.     

Self-renewal vs. differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells are typically derived from the inner cell mass of the preimplantation blastocyst and can both self-renew and differentiate into all the cells and tissues of the embryo. Because they are pluripotent, ES cells have been used extensively to analyze gene function in development via gene targeting. The embryonic stem cell is also an unsurpassed starting material to begin to understand a critical, largely inaccessible period of development. If their differentiation could be controlled, they would also be an important source of cells for transplantation to replace cells lost through disease or injury or to replace missing hormones or genes. Traditionally, ES cells have been differentiated in suspension culture as embryoid bodies, named because of their similarity to the early postimplantation-staged embryo. Unlike the pristine organization of the early embryo, differentiation in embryoid bodies appears to be largely unpatterned, although multiple cell types form. It has recently been possible to separate the desired cell types from differentiating ES cells in embryoid bodies by using cell-type-restricted promoters driving expression of either antibiotic resistance genes or fluorophores such as EGFP. In combination with growth factor exposure, highly differentiated cell types have successfully been derived from ES cells. Recent technological advances such as RNA interference to knock down gene expression in ES cells are also producing enriched populations of cells and elucidating gene function in early development.     

多组学大数据整合分析揭示早期胚胎发育过程中基因表达调控信息的变化规律

摘要 目的：探究哺乳动物早期胚胎发育过程中基因表达调控信息的变化规律。方法：收集小鼠早期胚胎发育各时期的RNA-seq,ATAC-seq,MethylC-Seq和H3K4me3 ChIP-seq数据进行整合分析,观察小鼠早期胚胎发育各时期转录因子表达量的变化,计算各时期基因表达量与转录因子结合位点数量及染色质可及性的相关性,筛选各时期表达量前10%的基因,统计其表达量和转录因子占比,并进行启动子可及性分析。根据前期报道的转录因子三节点调控网络,对早期胚胎各时期转录因子调控网络的富集模式进行分析。根据多组学数据分析结果,推测早期胚胎发育调控过程中转录因子和表观遗传修饰信息的共调控模型。结果：转录因子数量和调控关系变化以及染色质可及性、DNA甲基化修饰、组蛋白修饰等表观遗传修饰共同调控早期胚胎发育各时期的基因表达,这些因素在不同时期发挥不同程度的调控作用。结论：转录因子和表观遗传修饰在早期胚胎发育过程中动态调控基因表达。