Polycomb group genes as the key regulators in gene silencing

The Polycomb group （PcG） genes repress gene expression mainly through chromatin modifications and regulation of chromatin structure. At present, at/east four protein complexes of PcG proteins are identified, including Polycomb repressive complex 1 （PRC1）, Polycomb repressive complex 2 （PRC2）, PHO-repressive complex （PhoRC） and Polycomb repressive deubiquitinase （PR-DUB）. In this review, the recent discoveries of the composition of the above complexes, as well as their roles in regulating histone modifications and gene silencing are discussed. We mainly focus on the composition of PRC1 and PRC2 complex and recruitment of PcG to target genes and mechanisms of PRC1 and PRC2-mediated gene silencing. Although much progress has been made in understanding gene silencing mediated by PcG proteins, we also discuss several important questions that still remained unanswered, such as the inheritance of histone modifications during cell division.     

The Polycomb complex PRC2 and its mark in life

Polycomb group proteins maintain the gene-expression pattern of different cells that is set during early development by regulating chromatin structure. In mammals, two main Polycomb group complexes exist - Polycomb repressive complex 1 (PRC1) and 2 (PRC2). PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A. PRC2 also contributes to chromatin compaction, and catalyses the methylation of histone H3 at lysine 27. PRC2 is involved in various biological processes, including differentiation, maintaining cell identity and proliferation, and stem-cell plasticity. Recent studies of PRC2 have expanded our perspectives on its function and regulation, and uncovered a role for non-coding RNA in the recruitment of PRC2 to target genes.     

The Polycomb group protein EZH2 directly controls DNA methylation

The establishment and maintenance of epigenetic gene silencing is fundamental to cell determination and function. The essential epigenetic systems involved in heritable repression of gene activity are the Polycomb group (PcG) proteins and the DNA methylation systems. Here we show that the corresponding silencing pathways are mechanistically linked. We find that the PcG protein EZH2 (Enhancer of Zeste homolog 2) interacts-within the context of the Polycomb repressive complexes 2 and 3 (PRC2/3)-with DNA methyltransferases (DNMTs) and associates with DNMT activity in vivo. Chromatin immunoprecipitations indicate that binding of DNMTs to several EZH2-repressed genes depends on the presence of EZH2. Furthermore, we show by bisulphite genomic sequencing that EZH2 is required for DNA methylation of EZH2-target promoters. Our results suggest that EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems.     

Nanog promotes transfer of pluripotency after cell fusion

Through cell fusion, embryonic stem (ES) cells can erase the developmental programming of differentiated cell nuclei and impose pluripotency. Molecules that mediate this conversion should be identifiable in ES cells. One candidate is the variant homeodomain protein Nanog, which has the capacity to entrain undifferentiated ES cell propagation. Here we report that in fusions between ES cells and neural stem (NS) cells, increased levels of Nanog stimulate pluripotent gene activation from the somatic cell genome and enable an up to 200-fold increase in the recovery of hybrid colonies, all of which show ES cell characteristics. Nanog also improves hybrid yield when thymocytes or fibroblasts are fused to ES cells; however, fewer colonies are obtained than from ES x NS cell fusions, consistent with a hierarchical susceptibility to reprogramming among somatic cell types. Notably, for NS x ES cell fusions elevated Nanog enables primary hybrids to develop into ES cell colonies with identical frequency to homotypic ES x ES fusion products. This means that in hybrids, increased Nanog is sufficient for the NS cell epigenome to be reset completely to a state of pluripotency. We conclude that Nanog can orchestrate ES cell machinery to instate pluripotency with an efficiency of up to 100% depending on the differentiation status of the somatic cell.     

人生长激素基因转化的小鼠胚胎干细胞特性的研究

报道了携带人生长激素基因（ｈＧＨ）的逆转录病毒载体ｐＩＮＳ－ＧＨ导入小鼠胚胎干细胞（ＥＳ细胞）ＣＣＥ后，虽然用放射免疫法未检测到ｈＧＨ基因的表达，但是，Ｓｏｕｔｈｅｒｎ杂交的结果表明，ｈＧＨ基因的确已经整合到细胞基因组中。对转化的ＥＳ细胞克隆进行了体内外分化能力及嵌合能力的检验，结果表明，经过一系列体外操作的ＥＳ细胞，仍具有分化成多种细胞类型的能力。转化的ＥＳ细胞通过显微注射注入囊胚后，能参与受体     

The molecular mechanism of embryonic stem cell pluripotency maintenance

In vitro cultured embryonic stem （ES） cells are derived from the inner cell mass （ICM） of pre-implantation embryos, and are capable of giving rise to all cell and tissue types of the three germ layers upon being injected back into blastocysts. These ceils are therefore said to possess pluripotency that can be maintained infinitely in culture under optimal conditions. Such pluripotency maintenance is believed to be due to the symmetrical cleavage of the cells in an undifferentiated state. The pluripotency of ES cells is the basis for their various practical and potential applications. ES cells can be used as donor cells to generate knockout or transgenic animals, as in vitro models of mammalian development, and as cell resources for cell therapy in regenerative medicine. The further success in these applications, particularly in the last two, is dependent on the establishment of a culture system with components in the medium clearly defined and the subsequent procedures for controlled differentiation of the cells into specific lineages. In turn, elucidating the molecular mechanism for pluripotency maintenance of ES cells is the prerequisite. This paper summarizes the recent progresses in this area, focusing mainly on the LIF/STAT3, BMPs/Smads, canonical Wnt, TGFβ/activin/nodal, PI3K and FGF signaling pathways and the genes such as oct4, nanog that are crucial in ES cell pluripotency maintenance. The regulatory systems of pluripotency maintenance in both mouse and human ES cells are also discussed. We believe that the cross-talkings between these signaling pathways, as well as the regulatory system underlying pluripotency maintenance will be the main focus in the area of ES cell researches in the future.     

Derivation of androgenetic embryonic stem cells from m-carboxycinnamic acid bishydroxamide (CBHA) treated androgenetic embryos

The androgenetic embyronic stem (aES) cells are useful models in studying the effects of imprinted genes on pluripotency maintaining and embryo development. The expression patterns of imprinted genes are significantly different between uniparental derived aES cells and zygote-derived embryonic stem (ES) cells, therefore, the imprinting related cell pluripotency needs further exploitation. Several approaches have been applied in generation of androgenetic embryos and derivation of aES cell lines. Here, we describe a method to generate androgenetic embryos by injecting two mature sperms into one enucleated oocyte. Then these androgenetic embryos were treated with a histone deacetylase inhibitor: m-carboxycinnamic acid bishydroxamide (CBHA). Further, aES cell lines were successfully derived from these treated androgenetic embryos at blastocyst stage. The CBHA could improve not only the quality of androgenetic embryos, but also the efficiencies of aES (CaES) cells derivation and chimeric mice generation. The imprinted gene expression pattern in the CBHA treated embryo-derived aES (CaES) cells was also highly similar to that of zygote-derived ES cells.     

Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs

During the regeneration of Drosophila imaginal discs, cellular identities can switch fate in a process known as transdetermination. For leg-to-wing transdetermination, the underlying mechanism involves morphogens such as Wingless that, when activated outside their normal context, induce ectopic expression of the wing-specific selector gene vestigial. Polycomb group (PcG) proteins maintain cellular fates by controlling the expression patterns of homeotic genes and other developmental regulators. Here we report that transdetermination events are coupled to PcG regulation. We show that the frequency of transdetermination is enhanced in PcG mutant flies. Downregulation of PcG function, as monitored by the reactivation of a silent PcG-regulated reporter gene, is observed in transdetermined cells. This downregulation is directly controlled by the Jun amino-terminal kinase (JNK) signalling pathway, which is activated in cells undergoing regeneration. Accordingly, transdetermination frequency is reduced in a JNK mutant background. This regulatory interaction also occurs in mammalian cells, indicating that the role of this signalling cascade in remodelling cellular fates may be conserved.     

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.     

Derivation of pluripotent epiblast stem cells from mammalian embryos

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.