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.     

植物胚乳发育的表观遗传学调控

被子植物的种子发育从双受精开始, 产生二倍体的胚和三倍体的胚乳。在种子发育和萌发过程中, 胚乳向胚组织提供营养物质, 因此胚乳对胚和种子的正常生长发育至关重要。开花植物发生基因组印迹的主要器官是胚乳。印迹基因的表达受表观遗传学机制的调控, 包括DNA甲基化和组蛋白H3K27甲基化修饰以及依赖于PolIV的siRNAs (p4-siRNAs)调控。基因组印迹的表观遗传学调控对胚乳的正常发育和种子育性具有不可或缺的重要作用。最新研究显示, 胚乳的整个基因组DNA甲基化水平降低, 而且去甲基化作用可能源于雌配子体的中央细胞。该文综述了种子发育的表观遗传学调控机制, 包括基因组印迹机制以及胚乳基因组DNA甲基化变化研究的最新进展。     

Reprogramming and epigenesis

The fact that the nucleus of a differentiated somatic cell can be reprogrammed in order to sustain embryonic development is now well established. Experiments of somatic cell nuclear transfer (cloning) have proved that a foreign nucleus introduced into an enucleated oocyte can give rise to physiologically normal offsprings, with a normal lifespan. Such evidence of genome expression plasticity is also observed experimentally with heterokaryons, created by the fusion or the nuclear transfer between two somatic cells, where differentiated nuclei are able to express genes characteristic of the host cell. However, the epigenetic mechanisms that permit nuclear plasticity remain poorly understood. In this paper we present the main evidences showing important modifications of the large scale organisation of chromosomal domains and of the DNA methylation pattern upon nuclear transfer and during the first cleavages. These modifications of epigenetic marks, brought by an intimate contact between the chromatin and the recipient oocyte cytoplasmic factors, appear essential for further development. They are established over the first cell cycles of development. The onset of embryonic genome activation and the first cellular differentiation events that occur over the implantation period are two additional check-points of reprogramming that appear to be also highly dependent on epigenetic alterations. Beyond those stages, defective placental functions might be directly responsible for the fetal and postnatal physiopathologies frequently observed in cloned animals. No direct link between preimplantation reprogramming defaults, placental dysfunctions and low development to term has been established yet. The epigenetics studies which are now used to characterise loci specific and probably genotype dependent alterations in cloned animals of different species will provide invaluable help to define the role of epigenesis in the achievement of a developmental program.     

Genomic imprinting and epigenetic control of development

Epigenetic mechanisms are extensively utilized during mammalian development. Specific patterns of gene expression are established during cell fate decisions, maintained as differentiation progresses, and often augmented as more specialized cell types are required. Much of what is known about these mechanisms comes from the study of two distinct epigenetic phenomena: genomic imprinting and X-chromosome inactivation. In the case of genomic imprinting, alleles are expressed in a parent-of-origin-dependent manner, whereas X-chromosome inactivation in females requires that only one X chromosome is active in each somatic nucleus. As model systems for epigenetic regulation, genomic imprinting and X-chromosome inactivation have identified and elucidated the numerous regulatory mechanisms that function throughout the genome during development.     

Establishment of male-specific epigenetic information

The setting of male-specific epigenetic information is a complex process, which involves a major global re-organisation, as well as localized changes of the nucleus structure during the pre-meiotic, meiotic and post-meiotic stages of the male germ cell differentiation. Although it has long been known that DNA methylation in targeted regions of the genome is associated with male-specific genomic imprinting, or that most core histones are hyperacetylated and then replaced by sperm-specific proteins during the post-meiotic condensation of the nucleus, many questions remain unanswered. How these changes interact, how they affect the epigenetic information and how the paternal epigenetic marks contribute to the future genome are indeed major issues remaining to be explored.     

Epigenetics across the evolutionary tree: New paradigms from non-model animals

All animals have evolved solutions to manage their genomes, enabling the efficient organization of meters of DNA strands in the nucleus and allowing for nuanced regulation of gene expression while keeping transposable elements suppressed. Epigenetic modifications are central to accomplishing all these. Recent advances in sequencing technologies and the development of techniques that profile epigenetic marks and chromatin accessibility using reagents that can be used in any species has catapulted epigenomic studies in diverse animal species, shedding light on the multitude of epigenomic mechanisms utilized across the evolutionary tree. Now, comparative epigenomics is a rapidly growing field that is uncovering mechanistic aspects of epigenetic modifications and chromatin organization in non-model invertebrates, ranging from octopus to sponges. This review puts recent discoveries in the epigenetics of non-model invertebrates in historical context, and describes new insight into the patterning and functions of DNA methylation and other highly conserved epigenetic modifications.     

PGC7/Stella protects against DNA demethylation in early embryogenesis

DNA methylation is an important means of epigenetic gene regulation and must be carefully controlled as a prerequisite for normal early embryogenesis. Although global demethylation occurs soon after fertilization, it is not evenly distributed throughout the genome. Genomic imprinting and epigenetic asymmetry between parental genomes, that is, delayed demethylation of the maternal genome after fertilization, are clear examples of the functional importance of DNA methylation. Here, we show that PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state of several imprinted loci and epigenetic asymmetry. After determining that PGC7/Stella binds to Ran binding protein 5 (RanBP5; a nuclear transport shuttle protein), mutant versions of the two proteins were used to examine exactly when and where PGC7/Stella functions within the cell. It is likely that PGC7/Stella protects the maternal genome from demethylation only after localizing to the nucleus, where it maintains the methylation of several imprinted genes. These results demonstrate that PGC7/Stella is indispensable for the maintenance of methylation involved in epigenetic reprogramming after fertilization.     

Cheating death at the dawn of life: developmental control of apoptotic repression in the preimplantation embryo

During early development, the mammalian embryo is resistant to pro-apoptotic signals because of biochemical properties of the mitochondrion and nucleus. Mitochondria of the bovine two-cell embryo are resistant to depolarization because of low amounts of the proapoptotic protein BAX and high concentrations of the anti-apoptotic protein BCL2. As development proceeds, BAX content increases, BCL2 content declines, and mitochondria becomes capable of pore formation and depolarization in response to pro-apoptotic signals. The nucleus of the two-cell embryo is resistant to degradation by the DNase DFFB because epigenetic modifications, including DNA methylation and histone deacetylation, mask internucleosomal sites for DNA cleavage. Blastomere DNA becomes progressively less methylated during development so that DNA becomes accessible to cleavage by DFFB.     

Inheritable histone H4 acetylation of somatic chromatins in cloned embryos

A viable cloned animal indicates that epigenetic status of the differentiated cell nucleus is reprogrammed to an embryonic totipotent state. However, molecular events regarding epigenetic reprogramming of the somatic chromatin are poorly understood. Here we provide new insight that somatic chromatins are refractory to reprogramming of histone acetylation during early development. A low level of acetylated histone H4-lysine 5 (AcH4K5) of the somatic chromatin was sustained at the pronuclear stage. Unlike in vitro fertilized (IVF) embryos, the AcH4K5 level remarkably reduced at the 8-cell stage in cloned bovine embryos. The AcH4K5 status of somatic chromatins transmitted to cloned and even recloned embryos. Differences of AcH4K5 signal intensity were more distinguishable in the metaphase chromosomes between IVF and cloned embryos. Two imprinted genes, Ndn and Xist, were aberrantly expressed in cloned embryos as compared with IVF embryos, which is partly associated with the AcH4K5 signal intensity. Our findings suggest that abnormal epigenetic reprogramming in cloned embryos may be because of a memory mechanism, the epigenetic status itself of somatic chromatins.     

RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse

Epigenetic regulation shapes normal and pathological mammalian development and physiology. Our previous work showed that Kit RNAs injected into fertilized mouse eggs can produce heritable epigenetic defects, or paramutations, with relevant loss-of-function pigmentation phenotypes, which affect adult phenotypes in multiple succeeding generations of mice. Here, we illustrate the relevance of paramutation to pathophysiology by injecting fertilized mouse eggs with RNAs targeting Cdk9, a key regulator of cardiac growth. Microinjecting fragments of either the coding region or the related microRNA miR-1 led to high levels of expression of homologous RNA, resulting in an epigenetic defect, cardiac hypertrophy, whose efficient hereditary transmission correlated with the presence of miR-1 in the sperm nucleus. In this case, paramutation increased rather than decreased expression of Cdk9. These results highlight the diversity of RNA-mediated epigenetic effects and may provide a paradigm for clinical cases of familial diseases whose inheritance is not fully explained in Mendelian terms.     

Towards an integrated understanding of the structure and mechanics of the cell nucleus

Changes in the shape and structural organization of the cell nucleus occur during many fundamental processes including development, differentiation and aging. In many of these processes, the cell responds to physical forces by altering gene expression within the nucleus. How the nucleus itself senses and responds to such mechanical cues is not well understood. In addition to these external forces, epigenetic modifications of chromatin structure inside the nucleus could also alter its physical properties. To achieve a better understanding, we need to elucidate the relationship between nuclear structure and material properties. Recently, new approaches have been developed to systematically investigate nuclear mechanical properties. These experiments provide important new insights into the disease mechanism of a growing class of tissue-specific disorders termed 'nuclear envelopathies'. Here we review our current understanding of what determines the shape and mechanical properties of the cell nucleus.     

Nuclear cloning,stem cells,and genomic reprogramming

The generation of adult animals by nuclear cloning from adult donor cells is extremely inefficient, with most clones dying soon after implantation. In contrast, cloning from embryonic stem cell donor nuclei is significanty more efficient than from adult donor cells. However, regardless of donor cell type, all clones that survive to birth and beyond suffer serious phenotypic and gene expression abnormalities. All available evidence is consistent with the notion that the anomalous phenotypes of cloned animals are caused by faulty epigenetic reprogramming of the donor nucleus. Faulty reprogramming appears to be caused by the cloning process itself as well as by the epigenetic state of the donor nucleus. In contrast to reproductive cloning, faulty reprogramming of the donor nucleus does not tend to interfere with the application of nuclear transfer technology for therapeutic purposes (therapeutic cloning).     

Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.     

DNA methyltransferase is actively retained in the cytoplasm during early development.

The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.     

A Crowdsourced nucleus: Understanding nuclear organization in terms of dynamically networked protein function

The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear function. This compartmentalization involves genome organization as well as the formation of nuclear bodies and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a common theme for nuclear proteins is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information technology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear organization and function. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.     

Genetic and epigenetic aspects of cloning and potential effects on offspring of cloned mammals

Although the biological mechanisms by which host cytoplasm and donor nuclei interact to produce a developmentally competent reconstructed embryo remain largely unknown, some advances have been made to our understanding of the genetic and epigenetic factors involved in the of reprogramming of the donor nucleus. Genetic alterations, which comprise changes to the genetic information in both the nuclear and cytoplasm compartments, are passed on to subsequent generations at fertilization and are a potential source of variation among cloned animals and their offspring. Apart from the major chromosomal anomalies found in developmentally arrested embryos and fetuses, less detrimental rearrangements and/or mutations are likely to go unnoticed in most donor cell karyotypes, suggesting that such problems could lead to inheritable anomalies among clones and their offspring. Mitochondrial DNA is also relevant to cloning because most animals inherit most or all of their mitochondria from the host oocyte. Epigenetic alterations to the DNA or to the histone packaging proteins are independent of gene sequences. Aberrant epigenetic events may lead to variable gene expression or mitosis and consequent effects on development and phenotype. Although much of the epigenetic marking is reset during embryogenesis and development, the impact of epimutations on progeny remains unexplored.