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- The Company of Biologists
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- © 2021 The Company of Biologists. All rights reserved.
- ISSN
- 0950-1991
- eISSN
- 0950-1991
- DOI
- 10.1242/dev.02787
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Abstract
REVIEW 635 Development 134, 635-646 (2007) doi:10.1242/dev.02787 Hitoshi Niwa Mouse embryonic stem (ES) cells are pluripotent, as they have Rossant, 1979). Importantly, the primitive ectoderm is the only cell the ability to differentiate into the various cell types of a lineage in which pluripotency is maintained at this stage of vertebrate embryo. Pluripotency is a property of the inner cell development, enabling it to give rise to all three embryonic germ mass (ICM), from which mouse ES cells are derived, and of the layers and to primordial germ cells (see Fig. 1). However, as it lacks epiblast of the blastocyst. Recent extensive molecular studies of the ability to differentiate into the extraembryonic, primitive mouse ES cells have revealed the unique molecular mechanisms endodermal and trophectodermal lineages, the primitive ectoderm that govern pluripotency. These studies show that ES cells is less pluripotent than the cells of the ICM and possesses ‘restricted’ continue to self-renew because of a self-organizing network of pluripotency. transcription factors that prevents their differentiation and Traditionally, pluripotency has often been defined as the ability to promotes their proliferation, and because of epigenetic generate all cell types of an embryo apart from the trophectoderm processes that might be under the control of the pluripotent (the precursor to the bulk of the embryonic part of the placenta) transcription factor network. (Bioani and Schöler, 2006). This is because an earlier analysis of chimeric mouse embryos, produced by the injection of ICM cells Introduction and ES cells into 8-cell embryos or blastocysts, had shown that ICM Mouse embryonic stem (ES) cells, and the cells of the embryonic cells are excluded from the trophectoderm lineage (Beddington and inner cell mass (ICM) from which mouse ES cells are derived, are Robertson, 1989). However, it has subsequently been found that the pluripotent. According to recent consensus, pluripotency describes ICM does still possess the ability to differentiate into the a cell’s ability to give rise to all of the cells of an embryo and adult trophectoderm lineage (Pierce et al., 1988), as do ES cells under (Solter, 2006). Studies over the past few years have revealed the role particular culture conditions (Niwa et al., 2005). Therefore, in this that transcription factor networks and epigenetic processes play in review, I define pluripotency as the ability to generate all cell types, the maintenance of ES cell pluripotency (Niwa et al., 2000; Mitsui including the trophectoderm, without the self-organizing ability to et al., 2003; Chambers et al., 2003; Boyer et al., 2005; Niwa et al., generate a whole organism [see also Solter (Solter, 2006) for similar 2005; Boyer et al., 2006). Among the findings to have emerged from definitions of these terms]. these studies is that the functions of these transcription factors depend on the stage of development of a pluripotent cell, indicating ES cell proliferation that these factors function in combination with other processes Pluripotency is maintained during ES cell self-renewal through the (Sieweke and Graf, 1998). The activity of these transcription factors prevention of differentiation and the promotion of proliferation. In also depends on the accessibility of their target genes, which are fact, ES cells can self-renew continuously for years if they are made more or less accessible by the modification of their DNA, cultured under conditions that prevent their differentiation; for histones, or chromatin structure (Jaenisch and Bird, 2003). In this example, in the presence of leukemia inhibitory factor (Lif), a review, I discuss new insights into how transcription factor networks growth factor that is necessary for maintaining mouse ES cells in a maintain mouse ES cell pluripotency and how these factors interface proliferative, undifferentiated state (Suda et al., 1987). But how is with epigenetic processes to control the pluripotency and pluripotency itself protected via self-renewal at the molecular level? differentiation of mouse ES cells. This question is discussed in more detail below. An overview of mouse ES cell derivation, ES cell differentiation proliferation and differentiation Although ES cells are described as being pluripotent, they can only Pluripotent embryonic lineages and ES cell derivation differentiate directly into three cell types: the primitive ectoderm, Mouse ES cells are derived mainly from the ICM of the mouse the primitive endoderm and trophectoderm cells, analogous to the blastocyst (Evans and Kaufman, 1981; Martin, 1981) (see Fig. 1). As differentiation ability of cells of the ICM. the embryo develops, the ICM gives rise to two distinct cell lineages: The differentiation of mouse ES cells can be induced by the the extraembryonic endoderm, which goes on to form the ectopic expression of certain transcription factors. For example, the extraembryonic tissues; and the epiblast, which gives rise to the expression of the transcription factor Gata6 in ES cells results in primitive ectoderm at the egg-cylinder stage of embryogenesis, from their differentiation into primitive endoderm (Fujikura et al., 2002). which the embryo proper arises. The primitive ectoderm is distinct Likewise, the expression of the caudal-type homeobox transcription from the ICM in several ways. It cannot give rise to the factor 2 (Cdx2) induces ES cells to differentiate into trophectoderm trophectoderm, nor to the primitive endoderm (see Fig. 1); it also has (Niwa et al., 2005). Therefore, both of these factors have to be tightly an epithelial morphology distinct from that of the ICM (Gardner and repressed for ES cells to self-renew, as discussed in more detail below. Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 6500047, Japan. Laboratory Self-renewal by preventing differentiation for Development and Regenerative Medicine, Kobe University Graduate School of As mentioned above, ES cell pluripotency is maintained during self- Medicine, 7-5-1 Kusunokicho, Chuo-ku, Kobe, Hyogo 6500017, Japan. renewal by the prevention of differentiation and the promotion of E-mail: [email protected] proliferation. For mouse ES cells, Lif is a key factor that prevents DEVELOPMENT 636 REVIEW Development 134 (4) Fig. 1. Pluripotent lineages in the mouse embryo. A schematic view of mouse preimplantation development. (A) Pluripotent stem cells (green) are imaged in a morula as the inner cells, which (B) then form the inner cell mass (ICM) of the blastocyst. (C) After giving rise to the primitive endoderm on the surface of the ICM, pluripotent stem cells then form the epiblast and start to proliferate rapidly after implantation. (D) They then form the primitive ectoderm, a monolayer epithelium that has restricted pluripotency which goes on to give rise to the germ cell lineage and to the somatic lineages of the embryo. Certain key transcription factors (blue) are required for the differentiation of the various embryonic lineages. differentiation. Lif belongs to the interleukin-6 cytokine family and on ES cell differentiation does not require Oct3/4 DNA-binding binds to a heterodimeric receptor consisting of the Lif-receptor activity (Niwa et al., 2002). In such a model, the target gene(s) of and gp130 (Il6st – Mouse Genome Informatics). This binding results this particular complex would normally prevent ES cells from in the activation of the canonical Jak/Stat (Janus kinase signal differentiating into primitive endoderm by repressing the trigger transducer and activator of transcription) pathway. It has been factor, Gata6. Nanog is an NK-2 class homeobox transcription reported that Stat3 activation is essential and sufficient to maintain factor that is expressed throughout the pluripotent cells of the ICM. the pluripotency of mouse ES cells (Niwa et al., 1998; Matsuda et As overexpression of Nanog in mouse ES cells can maintain them al., 1999), and that c-Myc is a candidate target of Stat3 (Cartwright in a pluripotent state in the absence of Lif, it is a good candidate et al., 2005). for this hypothetical Gata6 repressor (Chambers et al., 2003; The POU family transcription factor Oct3/4, which is encoded by Mitsui et al., 2003). Indeed, Nanog-null ES cells differentiate into Pou5f1, is also a pivotal regulator of pluripotency (Nichols et al., Gata6-positive parietal endoderm-like cells, which have a 1998) that acts as a gatekeeper to prevent ES cell differentiation. morphology that is similar to that of Gata6-induced cells (Fig. 2) Artificial repression of Oct3/4 in ES cells induces differentiation (Mitsui et al., 2003). However, although it has been reported that along the trophectodermal lineage; when overexpressed, ES cells Nanog expression is partly regulated by Oct3/4 and Sox2, a differentiate mainly into primitive endoderm-like cells (see Fig. 2B) member of the Sox (SRY-related HMG box) family (Kuroda et al., (Niwa et al., 2000). 2005; Rodda et al., 2005), and although artificial Nanog Oct3/4 has been reported to directly prevent differentiation expression can block the differentiation of ES cells into primitive towards trophectoderm by interacting with Cdx2 (a trigger for endoderm cells [induced by either the withdrawal of Lif trophectoderm differentiation; see Fig. 2D,E), to form a repressor (Chambers et al., 2003) or the formation of embryoid bodies complex. This complex interferes with the autoregulation of these (EBs: ball-like structures that form when ES cells are kept in two factors, giving rise to a reciprocal inhibition system that suspension culture and which mimic the egg-cylinder stage of establishes their mutually exclusive expression (Niwa et al., 2005). embryogenesis] (Hamazaki et al., 2004), no direct evidence for As such, the downregulation of Oct3/4 results in an upregulation of the repression of Gata6 by Nanog has yet been found. Cdx2, and vice versa – a mechanism that might account for the two The gatekeeper function of Nanog might not be restricted to different pathways that lead to pluripotent stem cells and to preventing the differentiation of ES cells into primitive endoderm, trophectoderm cells. as it has been reported that Nanog also blocks neuronal Both the inhibition of Stat3 activity and the overexpression of differentiation induced by the removal of Lif and bone Oct3/4 stimulate ES cells to differentiate into primitive endoderm- morphogenetic protein (BMP) from serum-free culture (Ying et al., like cells (Fig. 2B) (Niwa et al., 1998; Niwa et al., 2000). The 2003). In addition, Nanog can also reverse mesoderm specification existence has been suggested of an unidentified co-factor of Oct3/4 by repressing brachyury, which encodes the mesoderm-specific T- that is activated by Stat3 (Niwa, 2001). The normal functions of box transcription factor T. This factor directly activates Nanog this co-factor could be disrupted by an excess of Oct3/4, which expression, indicating that negative feedback is involved in the might disrupt the functions of a ternary complex (consisting of balance between self-renewal and mesodermal differentiation Oct3/4, its co-factor and a general transcription unit, which (Suzuki et al., 2006a). Thus, Nanog can block primitive endodermal activates target genes) via the saturation of protein interactions. differentiation, neuronal differentiation and mesodermal This is supported by evidence that this ‘overdose effect’ of Oct3/4 differentiation under different culture conditions DEVELOPMENT Development 134 (4) REVIEW 637 Fig. 2. Differentiation of mouse ES cells. (A) Mouse ES cells differentiate into three cell types – primitive endoderm, trophectoderm (TE) and primitive ectoderm – mimicking the differentiation potential of pluripotent stem cells in preimplantation embryos. (B-E) Different culture conditions can induce ES cells to differentiate into certain lineages. (B) In the absence of Lif and in the presence of an excess of Oct3/4, ES cells differentiate into primitive endoderm-like cells, whereas (C) in the absence of Nanog and in the presence of Gata6, they differentiate into parietal endoderm-like cells. (D,E) Removing Oct3/4 from, and adding Cdx2 to, ES cell culture induces TE-like differentiation. MEFc, mouse embryonic fibroblast conditioned medium. Promoting self-renewal through proliferation inducing differentiation and/or repressing their proliferation Under optimized culture conditions, in which Lif is essential (Smith (Ivanova et al., 2006; Matoba et al., 2006). However, the molecular et al., 1988), mouse ES cells divide symmetrically every 12 hours. mechanisms that direct the expression of Eras and Tcl1 in ES cells During self-renewal, most ES cells are in the S phase of the cell have yet to be identified. cycle, with only a few in G1 (Burdon et al., 2002). The transcription factor b-Myb has been reported to be an Recent findings suggest that the phosphoinositide-3-kinase accelerator of cell-cycle progression in mouse ES cells. (PI3K)/Akt signaling pathway plays a pivotal role in promoting the Overexpression of a dominant-negative form of b-Myb in these cells proliferation, survival and/or differentiation of mouse ES cells (see results in G1 arrest (Iwai et al., 2001), indicating that b-Myb is Fig. 3). The deletion of Pten, which encodes a negative regulator of transcriptionally activated in G1 and promotes the transition to S phase PI3K, in mouse ES cells has been reported to increase ES cell by a complex mechanism (Joaquin and Watson, 2003). Moreover, b- viability and proliferation (Sun et al., 1999), and it has recently been Myb-null blastocysts show defective ICM outgrowth in vitro (Tanaka reported that the artificial activation of Akt is sufficient to maintain et al., 1999), suggesting that b-Myb might play an important role in ES cell self-renewal in the absence of Lif (Watanabe et al., 2006). promoting the cell cycle in ES cells. However, neither the Two modulators of the PI3K/Akt pathway are specifically transcriptional regulation of b-Myb nor its precise function in expressed in ES cells, Eras and Tcl1 (Fig. 3) (Takahashi et al., 2005). regulating the cell cycle in mouse ES cells have yet been analyzed. Eras encodes a constitutively active form of a Ras-family small The basic helix-loop-helix transcription factor Myc is a well- GTPase that activates PI3K to stimulate ES cell proliferation and known accelerator of the cell cycle, acting via the transcriptional tumorigenicity after ectopic transplantation in vivo (Takahashi et al., activation of cyclin E expression to promote G1-S transition (Hooker 2003). The Tcl1 gene product augments Akt activation by forming and Hurlin, 2006). Recently, Cartwright et al. (Cartwright et al., a stable heterodimeric complex with Akt (Teitell, 2005). 2005) reported that c-Myc is a direct target of Stat3, and that Knockdown of Tcl1 in mouse ES cells impairs self-renewal by overexpression of a dominant-active form of c-Myc that has a DEVELOPMENT 638 REVIEW Development 134 (4) Fig. 3. Regulation of proliferation of mouse ES cells. (A) Pluripotent transcription factors activate the expression of (B) certain effectors that drive ES cell proliferation. Among these, Eras and Tcl1 stimulate the (C) phosphoinositide-3-kinase (PI3K)/Akt signaling pathway to promote the cell cycle, whereas b-Myb and c-Myc activate the progression of the cell cycle directly. How Utf1 and Sall4 affect ES cell proliferation remains unknown. greater stability than the wild-type protein renders the self-renewal A transcription factor network that is stabilized by positive and of mouse ES cells independent of Lif. By contrast, the negative regulation between its components is a good mechanism overexpression of a dominant-negative form of c-Myc antagonizes for maintaining the stable gene expression patterns that determine a mouse ES cell self-renewal and promotes differentiation. These particular cell phenotype (von Dassow et al., 2000). Moreover, the findings suggest that the regulation of the G1-S transition may application of systems biological views, such as the Boolean contribute to the maintenance of pluripotency, which is promoted by network models, allows us to explain how small changes to a few the Lif-Stat3 pathway in mouse ES cells (Burdon et al., 2002). components of a network can trigger the dynamic transition of a Undifferentiated embryonic cell transcription factor 1 (Utf1) was transcription factor network from one state to another (Kauffman, first identified as a transcriptional co-factor that is expressed in mouse 2004). Random Boolean network models are a way of modeling ES cells in a stem-cell-specific manner (Okuda et al., 1998). Mouse networks that are composed of multiple factors which have multiple ES cells with reduced expression of Utf1 show reduced proliferation inputs in complex systems. They are based on Boolean logic, in in vitro and reduced tumorigenicity in vivo (Nishimoto et al., 2005). which multiple logical operators, such as AND and OR, are united Utf1 possesses a stem-cell-specific enhancer that is activated by into expressions about the factor with binary values such as 1 and 0 Oct3/4 and Sox2 (Nishimoto et al., 1999), so it can be regarded as a (Kauffman, 2004). link between the pluripotent transcription factor network and the Sox2 occupies an important position in the maintenance of the promotion of proliferation. pluripotent transcription factor network (Fig. 4B). As discussed Mouse ES cells that lack Sall4, one of the mouse homologs of the above, Sox2 is known to co-operate with Oct3/4 in activating Drosophila homeotic gene spalt that encodes a zinc-finger transcription Oct3/4 target genes (Yuan et al., 1995). To date, ES-specific factor, were recently reported to show reduced proliferation ability enhancers that contain binding sites for Oct3/4 and Sox2 have (Sakaki-Yumoto et al., 2006). Another study showed that Sall4 been identified in several genes, including Fgf4 (Yuan et al., interacts with Nanog to activate Sall4 and Nanog (Wu et al., 2006). 1995), osteopontin (Spp1 – Mouse Genome Informatics) (Botquin However, Sall4 expression is not restricted to mouse ES cells, and et al., 1998), Utf1 (Nishimoto et al., 1999), Fbxo15 (Tokuzawa et Nanog is still expressed in Sall4-null ES cells (Sakaki-Yumoto et al., al., 2003), Nanog (Kuroda et al., 2005; Rodda et al., 2005) and 2006), so the physiological contribution of this positive-feedback loop Lefty1 (Nakatake et al., 2006). Interestingly, both Oct3/4 and Sox2 to the maintenance of pluripotency remains to be confirmed. possess enhancers that are activated by the Oct3/4-Sox2 complex in a stem-cell-specific manner (Chew et al., 2005; Okumura- Mechanisms to maintain self-renewal Nakanishi et al., 2005; Tomioka et al., 2002). Sox2-null embryos In order to maintain the stable self-renewal of ES cells, the die immediately after implantation (Avilion et al., 2003), and mechanisms that prevent their differentiation and promote their knockdown of Sox2 in mouse ES cells induces differentiation into proliferation must be transmitted to their daughter cells. Thus, the multiple lineages, including trophectoderm, indicating its expression levels of the genes that are involved in these mechanisms functional importance in the maintenance of pluripotency need to be stably maintained. (Ivanova et al., 2006). The generation of Sox2-null ES cells would DEVELOPMENT Development 134 (4) REVIEW 639 help to elucidate the precise function of Sox2 and the identification of its target genes, as would also be the case for Oct3/4. The identification of common target sites in the regulatory elements of Oct3/4, Sox2 and Nanog by recent studies using chromatin immunoprecipitation (ChIP) together with genome-wide location techniques has suggested that Oct3/4, Sox2 and Nanog might form a regulatory feedback circuit that maintains pluripotency in human and mouse ES cells; in this circuit, all three transcription factors regulate themselves, as well as each other (Boyer et al., 2005; Loh et al., 2006). Although this feedback model has not been confirmed in ES cells, a positive-feedback loop alone would be incapable of allowing the transcription factor network to maintain pluripotency because pluripotency is extremely sensitive to the expression levels of Oct3/4 (Niwa et al., 2000). Since even a slight overdose of Oct3/4 triggers differentiation, the network requires a negative-feedback loop in order to tightly regulate Oct3/4 expression levels. An experimental model in prokaryotic cells has revealed that a simple negative-feedback loop can dramatically stabilize the expression level of a gene (Becskei and Serrano, 2000). Therefore, a direct or indirect negative-feedback loop could be sufficient to regulate the quantitative expression of Oct3/4 within the range required to maintain pluripotency. To date, two regulatory elements, a distal and a proximal enhancer, have been identified as stem-cell-specific enhancers of Oct3/4 (Yeom et al., 1996), to which many positive and negative regulators are recruited (Fig. 4A). Among them, members of the orphan nuclear receptor superfamily, which can bind to the proximal enhancer, are known to influence Oct3/4 expression. Liver receptor homolog 1 (Lrh1, also known as Nr5a2) is a putative positive regulator of Oct3/4,as Oct3/4 expression is lost in the epiblast of Lrh1-null embryos and is quickly downregulated after the induction of differentiation in Lrh1-null ES cells (Gu et al., 2005a). By contrast, germ cell nuclear factor (Gcnf, or Nr6a1) is a potential Oct3/4 negative regulator, as the expression domain of Oct3/4 is enlarged and its expression prolonged in the Fig. 4. A transcription factor network to control ES cell self- renewal and differentiation. (A) Transcriptional regulation of the neuroepithelium of Gcnf-null embryos (Fuhrmann et al., 2001). mouse Oct3/4 gene. There are four evolutionally conserved regions Oct3/4 repression following the induction of differentiation is also (CR1-4) that contain multiple transcription factor (TF) binding sites. The delayed in Gcnf-null ES cells (Gu et al., 2005b). Chicken ovalbumin TFs that bind to these sites are shown above and either activate (red) or upstream promoter-transcription factors (Coup-tf) I and II, encoded repress (blue) transcription. DE, distal enhancer; PE, proximal enhancer; by Nr2f1 and Nr2f2, respectively, also function as negative PP, proximal promoter. (B) Transcription factor networks for pluripotent regulators of Oct3/4 expression (Ben-Shushan et al., 1995). The stem cells (green), trophectoderm (yellow) and primitive balance between these positive and negative regulators might (extraembryonic) endoderm (blue). Positive-feedback loops between determine the precise level of Oct3/4 expression in response to Oct3/4, Sox2 and Nanog maintain their expression to promote extracellular stimuli (Fig. 4A). continuous ES cell self-renewal. Cdx2 is autoregulated and forms a reciprocal inhibitory loop with Oct3/4, which acts to establish their mutually exclusive expression patterns. A similar regulatory loop, not A transcription factor network for self-renewal yet confirmed, might exist for Nanog and Gata6. A combination of The feedback regulatory circuit that maintains pluripotency interacts positive-feedback loops and reciprocal inhibitory loops converts with the feedback loop shown in Fig. 4B, in which Oct3/4, Sox2 and continuous input parameters into a bimodal probability distribution, Nanog function to maintain their expression to promote continuous resulting in a clear segregation of these cell lineages (see text for ES cell self-renewal. This loop determines the differentiation fate of details). Coup-tfs and Gcnf act as a negative-feedback system to ES cells by influencing the expression of transcription factors, such repress Oct3/4 completely. as Cdx2 (which promotes trophectodermal differentiation) and Gata6 (which promotes primitive endoderm differentiation). Rapid transitions between the pluripotent state and one of these differentiation states have been theoretically confirmed to occur in shuts down Oct3/4 in differentiated cells, and which could then be a model in which two positive-feedback loops are connected by followed by epigenetic chromatin modifications that result in the negative-feedback loops. In such a system, a small quantitative repression of the Oct3/4 promoter (Feldman et al., 2006). asymmetry in one loop can be converted into its exclusive expression The transition of the pluripotent transcription factor network to (Becskei et al., 2001). Moreover, as Gcnf, Nr2f1 and Nr2f2 are either the trophectodermal or extraembryonic-endodermal network is upregulated after the induction of either trophectoderm or primitive most likely to be regulated by the presence or absence of extracellular endoderm differentiation (Fujikura et al., 2002; Niwa et al., 2005), signals, such as the removal of Lif from mouse ES cells or the these negative regulators might form the negative-feedback loop that formation of EBs. However, the activation of Cdx2 or the repression DEVELOPMENT 640 REVIEW Development 134 (4) of Oct3/4 might occur in mouse ES cells through the infrequent chromatin structure. Polycomb repressive complex 2 (PRC2), which spontaneous differentiation of these cells towards trophectoderm, consists of Ezh2, Eed and Suz12 in ES cells, functions as a histone which can occur under standard culture conditions (Beddington and methyltransferase on lysine 27 (K27) of histone H3, resulting in its Robertson, 1989). This tallies with evidence that Oct3/4 and Cdx2 tri-methylation (H3K27me3), a methylation mark that is associated compete with each other to be expressed during blastocyst formation, with transcriptionally inactive genes (Cao and Zhang, 2004). In and with evidence that Oct3/4 expression is dominant in the ICM general, the distribution of this repressive chromatin mark is (Niwa et al., 2005). Therefore, the gatekeeper function of Nanog, mutually exclusive to that of the tri-methylation mark H3K4me3, which is an Oct3/4 target and prevents extraembryonic endoderm which is associated with transcriptionally active regions (Strahl and differentiation, appears to be more important in mouse ES cells, as Allis, 2000; Lund and van Lohuizen, 2004). However, Bernstein et these cells are regulated by extracellular signals. al. reported that in mouse ES cells, these histone marks co-localize Indeed, Nanog could be at the hub of these multiple signal in particular regions, which they named ‘bivalent domains’ transduction pathways. As mentioned above, Nanog can block (Bernstein et al., 2006). These domains, which are composed of primitive endoderm differentiation (Chambers et al., 2003), neuronal short chromatin elements marked by H3K4me3 flanked by larger differentiation (Ying et al., 2003) and mesoderm differentiation regions that contain H3K27me3, are associated with genes that are (Suzuki et al., 2006a) under different culture conditions. Recent expressed at low levels (Fig. 5B) (Bernstein et al., 2006). studies have shown that Nanog interacts with Smad1 to inhibit the Interestingly, the bivalent domains map to highly conserved non- expression of brachyury (Suzuki et al., 2006b) and with Sall4 to coding elements (HCNEs) that have previously been identified as form a positive regulatory loop for Nanog and Sall4 (Wu et al., being conserved among the genomes of primates and rodents and 2006); also, Nanog expression is activated by Foxd3 (Pan et al., which contain few retrotransposons (Bernstein et al., 2006). 2006) and is repressed by Tp53 (Trp53 – Mouse Genome Moreover, half of these bivalent domains contain target sites that are Informatics) (Lin et al., 2005), Gcnf (Nr6a1 – Mouse Genome common to Oct3/4, Sox2 and Nanog, as identified by genome-wide Informatics) (Gu et al., 2005b), Tcf3 (Pereira et al., 2006) and the ChIP-on-Chip analysis (Boyer et al., 2005). Thus, these domains Grb2-Mek (Mdk – Mouse Genome Informatics) pathways might signify the chromatin structure of genes that are in a (Hamazaki et al., 2006). However, during mouse development, differentiation-ready state, as proposed in the ‘Localised Marking Nanog transcription is downregulated in the epiblast and in early Model’ by Szutoristz and Dillon (Szutoristz and Dillon, 2005). primitive ectoderm (Hart et al., 2004; Hatano et al., 2005), where According to this model, most tissue-specific genes in ES cells Oct3/4 and Sox2 continue to be expressed (Avilion et al., 2003; would be targets for sequence-specific factors that can recruit Rosner et al., 1990). It is noteworthy that Nanog expression levels histone-modifying enzymes, resulting in the formation of early in P19 embryonal carcinoma (EC) cells is much lower than that in transcription competence marks (ETCMs), which are enriched for ES cells, although both EC and ES cells express similar levels of histone H3 and H4 acetylation (H3Ac and H4Ac, respectively), and Oct3/4 and Sox2 (Chambers et al., 2003). This suggests that the H3K4me3, all of which are histone marks associated with positive-feedback circuitry in the pluripotent transcription factor transcriptionally active regions. In both bivalent domains and network does not always require Nanog, and that the transcription ETCMs, H3K4me3 marks spread as genes near them become factor network can establish a different stable circuit that maintains transcriptionally active, whereas H3K27me3 exclusively occupies the levels of Oct3/4 and Sox2 expression required to maintain those genes that are repressed during the differentiation of a pluripotency with or without Nanog. particular cell type. Because the global level of H3K27me3 in ES Two other factors have recently been reported to be necessary for cells is lower than that in differentiated cells, the mechanism by the maintenance of ES cell self-renewal: estrogen-related receptor which this repressive mark targets such sites is of interest. Lee et al. (Esrrb) and T-box transcription factor Tbx3, both identified by (Lee et al., 2006) performed ChIP-on-Chip analysis for Suz12, Eed functional screening mediated by RNA interference (Ivanova et al., and H3K27me3, and revealed that Suz12- and Eed-binding sites 2006). Repression of Esrrb in mouse ES cells results in their significantly overlap with each other and with H3K27me3 marks on differentiating into a mixture of extraembryonic and embryonic the highly evolutionarily-conserved regions of transcriptionally lineages, whereas knockdown of Tbx3 triggers differentiation into silent genes, including Gata4 and Cdx2, in ES cells. The 1800 genes mainly the embryonic lineages that are derived from the primitive identified as targets of Suz12 included most of the targets repressed ectoderm. Since the effect of repressing these genes can be cancelled by Oct3/4, Sox2 and Nanog (Boyer et al., 2005). Boyer et al. (Boyer out by the overexpression of Nanog, the maintenance of Nanog et al., 2006) also identified 512 common target genes of PRC2 and expression is one of their functions. The transcriptional regulation PRC1 by ChIP-on-Chip analysis and found that they were marked of their expression in ES cells has yet to be analyzed, but multiple by H3K27me3, and that 87% were upregulated in the absence of binding sites for Oct3/4 and Nanog have been found in the mouse PRC2 in Eed-null ES cells. Esrrb gene (Loh et al., 2006). In addition, a recent protein interaction These findings suggest that the dynamic repression of network analysis identified two transcription factors, the BTB- developmental pathways in ES cells by epigenetic processes may be domain-containing protein Nac1 (Btbd14b – Mouse Genome required for the maintenance of pluripotency; but this conclusion Informatics) and the zinc-finger protein Zfp281, which interact with requires, in my view, further study. This is because observations Nanog and are essential for maintaining the self-renewal of mouse made in ES cells that are deficient for members of the PRC2 and ES cells (Wang et al., 2006). Further analyses will be required to PRC1 complexes do not fit easily into this model. For example, Eed- integrate these genes into the current transcription factor network null ES cells can still self-renew, maintain normal morphology and model described in this review. express Oct3/4, Sox2 and Nanog normally in the complete absence of PRC2 and despite a dramatic decrease in H3K27me3. These cells An epigenetic mechanism for self-renewal just show a high rate of spontaneous differentiation (Boyer et al., A series of recent studies have revealed that mouse and human ES 2006; Azuara et al., 2006). Although the expression of Gata4 and cells possess certain novel epigenetic features. Polycomb-group Gata6, as well as of several neural-specific genes, are upregulated (PcG) complex proteins mainly act to stabilize a repressive in the absence of Eed, these ES cells can still produce all three germ DEVELOPMENT Development 134 (4) REVIEW 641 Fig. 5. Characteristics of the pluripotent epigenome. (A) Nuclei of undifferentiated (left) and differentiated (right) ES cells. The nucleus shrinks and the distribution of electron- dense areas, mainly heterochromatin, changes dramatically when ES cells are induced to differentiate into primitive endoderm by the ectopic expression of Gata6. (Electron micrographs courtesy of Naoko Ikue and Shigenobu Yonehara.) (B) Epigenetic features of the pluripotent cell nucleus. The volume of the nucleus is larger than that of a differentiated cell as a result of the relaxed chromatin structure. Small regions of perinuclear heterochromatin exist, but most of the chromatin exists as euchromatin, bearing histone marks associated with transcriptional activity. The hyperdynamics of chromatin proteins (green) might contribute to the maintenance of euchromatin. Bivalent domains are also a feature of the pluripotent epigenome, in which active histone marks (such as H3K4me) are flanked by transcriptionally repressive histone marks (such as H3K9me). layers on injection into blastocysts (Montgomery et al., 2005; microarrays has revealed that a variety of genes are expressed at low Azuara et al., 2006). Suz12-null ES cells also show features similar levels in ES cells (Carter et al., 2005). This might be a consequence of to those of Eed-null ES cells (Lee et al., 2006). The establishment of their chromatin structure being in an open configuration, allowing the Ezh2-null ES cells has not been reported (O’Carroll et al., 2001), but leaky expression of genes by the general transcription machinery with it has been shown that Ezh2 protein becomes undetectable in Eed- neither positive nor negative regulation (Roeder, 2005) (Fig. 5B). null ES cells, and is restored by the introduction of an Eed transgene The leaky expression of a large number of genes characteristic (Montgomery et al., 2005). ES cells lacking Rnf2/Ring1,a of the ES cell pluripotent state is likely to be the result of both component of PRC1, are also viable and show decreased amounts genetic and epigenetic mechanisms and processes. Through of histone H2A ubiquitination (Napoles et al., 2004). These findings epigenetic processes, the pluripotent epigenome keeps the indicate that the PcG proteins and the PRC1 and PRC2 complexes chromatin structure open to allow for rapid genetic regulation (Fig. are not required for the maintenance of pluripotency. 5B) (Zipori, 2004). The general abundance of transcriptionally active chromatin marks, such as H3K4me3 and H4Ac, in ES cells Molecular mechanisms that determine fits with this idea (Lee et al., 2004; Azuara et al., 2006). pluripotency Hyperdynamic chromatin restructuring has been observed in If all genomic information is utilized at least once during the mouse ES cells during self-renewal as rapid exchanges of histone development of an organism, all genes should be ready to be expressed H1 and HP1 (Meshorer et al., 2006), which might contribute to when they are required to execute pluripotency during development keeping the chromatin structure of ES cells open. The existence of and, in general, the expression of a large number of genes is a common such a globally relaxed chromatin structure is supported by the feature of stem cells (Zipori, 2004). Therefore, in pluripotent stem following evidence. Remarkable differences exist in the cells, many genes might be weakly expressed and, during distribution and frequency of high electron density areas, which differentiation, the expression levels of many might be reduced, were originally designated as heterochromatin (Brown, 1966), whereas those of others are increased, determining the progeny’s between ES and parietal endoderm cells (Fig. 5B). DNaseI phenotype. Indeed, genome-wide gene expression profiling using hypersensitive sites, which correlate with transcriptionally active DEVELOPMENT 642 REVIEW Development 134 (4) Table 1. Functions of epigenetic machineries in pluripotent stem cells K KO embryos KO ES cells ICM Marker Restore by Gene Phenotype outgrowth Proliferation expression Differentiation transgene Reference H3K9HMTases Suv39h1/h2 Viable NT Normal Normal Normal NT Peters et al., 2001; Lehnertz et al., 2003 G9a (Ehmt2) Die at NT Normal Normal Defective Restored Tachibana et al., 2002 E9.5 Glp (Ehmt1) Die at NT Normal Normal Defective Restored Tachibana et al., 2005 E9.5 Eset (Setdb1) Die at Defective NT NT NT NT Dodge et al., 2004 E3.5-E5.5 PRC2 (H3K27HMTase) Ezh2 Die at Defective NT NT NT NT O’Carroll et al., 2001 E3.5-E5.5 Eed Die at Normal Normal Normal Defective Restored Faust et al., 1998; E8.5 (mildly) Montgomery et al., 2005 Suz12 Die at Normal Normal Normal NT NT Pasini et al., 2004; Lee et E8.5 al., 2006 PRC1 Rnf2 (Ring1b) Die at Normal Normal Normal NT NT Voncken et al., 2003; E7.5 Napoles et al., 2004 DNA methylation Dnmt1 Die at NT Normal Normal Defective Restored Lei et al., 1996; Gaudet E9.5 et al., 1998 Dnmt3a/3b Die at NT Normal Normal Defective Restored Okano et al., 1999; Chen E11.5 et al., 2003 Dnmt1/3a/3b NT NT Normal Normal Defective Restored Tsumura et al., 2006 Dnmt3l Viable Normal Normal Normal Normal NT Hata et al., 2002 Cgbp (Cxxc1) Die at Normal Normal Normal Defective Restored Carlone and Skalnik, E6.0 2001; Carlone et al., RNAi Dicer1 Die at Defective Retarded/ Normal Defective Restored Bernstein et al., 2003; E7.5 compensated Kanellopoulou et al., 2005; Murchison et al., Chromatin remodeling/Histone exchange Snf2b (Brg1, Die at Defective Not viable NT NT NT Bultman et al., 2000; Smarca4) E4.5-6.0 (F9 EC cells) Sumi-Ichinose et al., Snf2h (Smarca5) Die at Defective NT NT NT NT Stopka and Skoultchi, E4.5-6.0 2003 Snf5 (Smarcb1) Die at Defective NT NT NT NT Klochendler-Yeivin et E4.5-6.0 al., 2000 Srg3 (Smarcc1) Die at Defective NT NT NT NT Kim et al., 2001 E4.5-6.0 Mbd3 Die at Defective Retarded Normal Defective Restored Hendrich et al., 2001; E8.5 Kaji et al., 2006 HirA Die at NT Normal Normal Accelerated NT Roberts et al., 2002; E9.5 Meshorer et al., 2006 NT, not tested. chromatin (Weintraub and Groudine, 1976), are frequently By contrast, as shown in Table 1, various epigenetic processes, detected in genes regardless of their expression levels in ES cells including PcG/H3K27me3, DNA methylation, tri-methylation of (Meshorer et al., 2006). Finally, nuclei in ES cells are about double lysine 9 of histone H3 (H3K9me3) and RNAi, are not essential for the volume of those in differentiated cells (Faro-Trindade and pluripotency. The requirement for H3K4me3 has not been assessed Cook, 2006). As such, the guidance of cell fates could occur solely because a methyltransferase that allows H3K9me3 to be globally via the action of transcription factors, such as Gata6 and Cdx2, marked in ES cells has not yet been identified. The chromatin owing to the unprogrammed state of the pluripotent epigenome, remodeling system, however, might be the exception because it has which might allow transcription factors to freely access their been reported that the inactivation of Brg1/Snf2, a component of target genes to control differentiation (Smith, 2005). the SWI/SNF and ISWI complex family involved in ATP-dependent DEVELOPMENT Development 134 (4) REVIEW 643 chromatin remodeling, affects the viability of F9 EC cells (Sumi- Ichinose et al., 1997), although its specific involvement in the maintenance of pluripotency has not yet been confirmed. Conversely, we can conclude that epigenetic processes are required for proper ES cell differentiation. However, the inability of ES cells to differentiate in response to signals such as the withdrawal of Lif or the addition of retinoic acids, can be restored by the reactivation of the deleted epigenetic genes, indicating that pluripotency is maintained in the absence of these epigenetic mechanisms (Table 1). I propose, therefore, that epigenetic processes are likely to be responsible for the ‘execution’ of the pluripotent program, which is itself established by the transcription factor network, rather than for the ‘maintenance’ of pluripotency per se. A comparison of ES and EC cells might shed light on the function of such epigenetic mechanisms in pluripotent stem cells. The ectopic Fig. 6. Establishment of pluripotency in somatic cell nuclei. In a expression of Gata4, a transcription factor related to Gata6, has recent study (Takahashi and Yamanaka, 2006), four transcription different effects in ES and EC cells. During mouse development, factors, Oct3/4, Sox2, Klf4 and c-Myc, were found to be sufficient to Gata4 is expressed in the primitive endoderm and its derivatives, and establish pluripotency in the nuclei of fibroblasts. Oct3/4, Sox2 and Klf4 then in cardiac precursors (Kelley et al., 1993). When Gata4 is might function together to activate target genes to establish the stable pluripotent transcription factor network, as well as the pluripotent ectopically expressed in ES cells, it directs differentiation into parietal epigenome, whereas c-Myc might enhance the accessibility of target endoderm, as does Gata6 (Fujikura et al., 2002). By contrast, ectopic genes by stimulating DNA replication. expression of Gata4 in P19 EC cells enhances their differentiation into cardiomyocytes (Grepin et al., 1997). As mentioned above, P19 EC cells lack almost any expression of Nanog (Chambers et al., 2003) but nonetheless exhibit a poor capacity to differentiate into primitive negative-feedback loops could end up functioning chaotically and endoderm (a differentiation pathway that is repressed by Nanog, as might result in a disordered state in which none of the transcription discussed above) (Mummery et al., 1990). This suggests that the factor networks holds an exclusive position, resulting in there being genetic function of Gata factors in EC cells is different from that in no determination of cell phenotype. In addition, a feature of the ES cells because of the difference in pre-existing transcription factors random Boolean network is that small changes to a few components in these cell types. However, both the prevention of differentiation can mediate the transition of the stable condition of the network into primitive endoderm and the change in response to the ectopic (‘attractor’) from one state to another, but this transition depends expression of Gata4 in P19 EC cells might reflect changes in their strongly on the initial state of the network. Only a particular change epigenetic state, perhaps owing to changes in the accessibility of their can trigger a transition, and other changes are cancelled out without target genes. Since the phenotype of P19 EC cells is closer to that of any effect on the network, indicating that it might not be necessary primitive ectoderm than to ICM (Jones-Villeneuve et al., 1982), a to repress all tissue-specific transcription factor genes to prevent restriction of pluripotency might be mimicked in P19 EC cells, in differentiation in the pluripotent state. This idea is supported by the which the gatekeeper function of Nanog might be replaced by the fact that the ectopic expression of the tissue-specific transcription epigenetic repression of its targets. Therefore, the function of Nanog factors merely directs the differentiation of ES cells, and that the might be limited to that of a gatekeeper, which blocks ES cells from expression of many tissue-specific transcription factors, such as following certain differentiation pathways but makes few other Pax6 and Pdx1, are detected in ES cells (Lumelsky et al., 2001; contributions to the state of pluripotency. Okada et al., 2004). Therefore, the function of the pluripotent How does the transcription factor network determine the transcription factor network might be limited to the activation of the pluripotent state per se? As mentioned above, a combination of epigenetic processes that generate the open chromatin structure positive-feedback loops with reciprocal inhibitory loops allows required for rapid changes in the transcriptional status of tissue- continuous input parameters to be converted into a bimodal specific genes during ES cell differentiation and development: for probability distribution (Becskei et al., 2001). This system was first example, by activating the enzymes that result in transcriptionally applied to explain how the ICM and trophectoderm segregate into repressive histone marks being exchanged for those of actively mutually exclusive Oct3/4 and Cdx2 expression domains and could transcribed genes. possibly be applied to each differentiation event in development (Niwa et al., 2005). Epigenetic mechanisms might follow this The establishment of pluripotency in vivo process by locking one of the components that is transcriptionally During development, both genetic and epigenetic mechanisms could inactivated by competition into a repressive state. If this is a general be involved in the establishment of the pluripotent state in the cells rule in the transition of the transcription factor networks, by which of the ICM through the reprogramming of nuclei in fertilized eggs. sequential differentiation events in development are mediated, what Such reprogramming activity is present in the cytoplasm of fertilized happens if all epigenetic repression is removed at once? During eggs, as proven by the generation of cloned embryos from somatic normal embryonic development, first ectoderm and mesoendoderm cell nuclear transfer (Wilmut et al., 1997). However, it is still unclear are segregated, and then the latter is separated into mesoderm and which mechanism contributes to this activity because the enzymes endoderm, in which ectodermal determination is repressed. The that modify the epigenetic state, as well as maternally transcribed system consists of a combination of positive-feedback loops with and translated transcription factors, are present in fertilized eggs. reciprocal inhibitory loops, which work sequentially to choose one Recently, Takahashi and Yamanaka addressed this question. They fate in these steps. If these systems start to work at once because of reported that the co-introduction of four transgenes encoding the the epigenetic derepression of transcription, the positive- and transcription factors Oct3/4, Sox2, c-Myc and Klf4 into somatic DEVELOPMENT 644 REVIEW Development 134 (4) cells, such as embryonic and adult tail-tip fibroblasts, resulted in the Becskei, A. and Serrano, L. (2000). Engineering stability in gene networks by autoregulation. Nature 405, 590-593. generation of induced pluripotent stem (iPS) cells, which gave rise Becskei, A., Seraphin, B. and Serrano, L. (2001). Positive feedback in eukaryotic to chimeric embryos following their injection into mouse blastocysts gene networks: cell differentiation by graded to binary response conversion. (Takahashi and Yamanaka, 2006). The functions of Oct3/4, Sox2 EMBO J. 20, 2528-2535. Beddington, R. S. and Robertson, E. J. (1989). An assessment of the and c-Myc have been mentioned above. Klf4 is well known as an developmental potential of embryonic stem cells in the midgestation mouse oncogene (Rowland and Peeper, 2006), but overexpression of Klf4 embryo. Development 105, 733-737. in mouse ES cells reduces the differentiation ability of EBs (Li et al., Ben-Shushan, E., Sharir, H., Pikarsky, E. and Bergman, Y. (1995). A dynamic balance between ARP-1/COUP-TFII, EAR-3/COUP-TFI, and retinoic acid 2005). Klf4 can also bind to the proximal promoters of Oct3/4 target receptor:retinoid X receptor heterodimers regulates Oct-3/4 expression in genes, such as Lefty1, and helps to activate Oct3/4 and Sox2 embryonal carcinoma cells. Mol. Cell. Biol. 15, 1034-1048. (Nakatake et al., 2006). These four factors are thought to establish Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., pluripotency in somatic cells as follows (Fig. 6). First, c-Myc Fry, B., Meissner, A., Wernig, M., Plath, K. et al. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, promotes DNA replication, thereby relaxing chromatin structure, 315-326. which allows Oct3/4 to access its target genes. Sox2 and Klf4 also Bernstein, E., Kim, S. Y., Carmell, M. A., Murchison, E. P., Alcorn, H., Li, M. Z., co-operate with Oct3/4 to activate target genes that encode Mills, A. A., Elledge, S. J., Anderson, K. V. and Hannon, G. J. (2003). Dicer is essential for mouse development. Nat. Genet. 35, 215-217. transcription factors which establish the pluripotent transcription Bioani, M. and Schöler, H. R. (2006). Regulatory networks in embryo-derived factor network and which, together with Oct3/4, Sox2 and Klf4, pluripotent stem cells. Nat. Rev. Mol. Cell Biol. 6, 872-884. result in the activation of the epigenetic processes that establish the Botquin, V., Hess, H., Fuhrmann, G., Anastassiadis, C., Gross, M. K., Vriend, G. and Schöler, H. R. (1998). New POU dimer configuration mediates pluripotent epigenome. The iPS cells have a similar global gene antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and expression profile to that of mouse ES cells. Interestingly, Nanog is Sox-2. Genes Dev. 12, 2073-2090. not required exogenously to establish pluripotency in iPS cells and Boyer, L., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K. et al. (2006). Polycomb its endogenous expression is not always activated in established complexes repress developmental regulators in murine embryonic stem cells. pluripotent stem cells by these four factors, supporting the Nature 441, 349-353. hypothesis that the function of Nanog in the maintenance of Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., pluripotency is context dependent. Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G. et al. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, In iPS cells, the repressive histone marks in the promoter regions 947-956. of Oct3/4 and Nanog are replaced by active marks, such as H3K4me Brown, S. W. (1966). Heterochromatin. Science 151, 417-425. and H4Ac, although DNA methylation is only partially erased. This Bultman, S., Gebuhr, T., Yee, D., Mantia, C. L., Nicholson, J., Gilliam, A., Randazzo, F., Metzger, D., Chambon, P., Crabtree, G. et al. (2000). A Brg1 suggests that Oct3/4, Sox2, c-Myc and Klf4 are indeed able to alter null mutation in the mouse reveals functional differences among mammalian the epigenetic state of a cell and establish the pluripotent epigenome. SWI/SNF complexes. Mol. Cell 6, 1287-1295. This change should be mediated by enzymatic activities that erase Burdon, T., Smith, A. and Savatier, P. (2002). Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12, 432-438. the repressive histone marks (such as demethylases for H3K9 and Cao, R. and Zhang, Y. (2004). The functions of E(Z)/EZH2-mediated methylation H3K27) and generate active histone marks (such as H3K4 of lysine 27 in histone H3. Curr. Opin. Genet. Dev. 14, 155-164. methyltransferase and histone acetyltransferase). Thus, to establish Carlone, D. L. and Skalnik, D. G. (2001). CpG binding protein is crucial for early and maintain pluripotency, the genes encoding these enzymes would embryonic development. Mol. Cell. Biol. 21, 7601-7606. Carlone, D. L., Lee, J. H., Young, S. R. L., Dobrota, E., Butler, J. S., Ruiz, J. and be activated by the pluripotent transcription factor network. Under Skalnik, D. G. (2005). Reduced genomic cytosine methylation and defective such artificial conditions, the transcription factor network could cellular differentiation in embryonic stem cells lacking CpG binding protein. Mol. orchestrate all the requirements for pluripotency. Cell. Biol. 25, 4881-4891. Carter, M. G., Sharov, A. A., VanBuren, V., Dudekula, D. B., Carmack, C. E., Nelson, C. and Ko, M. S. (2005). Transcript copy number estimation using a Conclusion mouse whole-genome oligonucleotide microarray. Genome Biol. 6, R61. Recent progress in understanding the establishment and Cartwright, P., McLean, C., Sheppard, A., Rivett, D., Jones, K. and Dalton, S. (2005). LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc- maintenance of ES cell pluripotency has revealed the importance dependent mechanism. Development 132, 885-896. and functions of various key transcription factors. By contrast, Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and although several features of the pluripotent epigenome have been Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643-655. discovered, their requirement for and involvement in the Chen, T., Ueda, Y., Dodge, J. E., Wang, Z. and Li, E. (2003). Establishment and maintenance and establishment of pluripotency remain unclear. In maintenance of genomic methylation patterns in mouse embryonic stem cells by the future, it will be necessary to confirm how genetic mechanisms Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594-5605. determine the pluripotent epigenome and how the pluripotent Chew, J. L., Loh, Y. H., Zhang, W., Chen, X., Tam, W. L., Yeap, L. S., Li, P., Ang, Y. S., Lim, B., Robson, P. et al. (2005). Reciprocal transcriptional regulation of epigenome functions to maintain the pluripotent transcription factor Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol. Cell. network. Biol. 25, 6031-6046. Dodge, J. E., Kang, Y. K., Beppu, H., Lei, H. and Li, E. (2004). Histone H3-K9 I thank Prof. Austin Smith (Cambridge University, UK), Dr Minoru Ko (NIH, methyltransferase ESET is essential for early development. Mol. Cell. Biol. 24, USA), Drs Masaki Okano, Jun-ichi Nakayama and Teruhiko Wakayama (RIKEN 2478-2486. CDB, Japan), Dr Haruhiko Koseki (RIKEN RCAI, Japan), Prof. Yo-ichi Shinkai Evans, M. J. and Kaufman, M. H. (1981). Establishment in culture of (Kyoto University, Japan), Dr Kiyoe Ura (Osaka University, Japan) and Prof. pluripotential cells from mouse embryos. Nature 292, 154-156. Mitsuyoshi Nakao (Kumamoto University, Japan) for helpful discussion and Faro-Trindade, I. and Cook, P. P. (2006). A conserved organization of transcription during embryonic stem cell differentiation and in cells with high C suggestions. I also thank Dr Shigenobu Yonemura and Ms Naoko Ikue (RIKEN value. Mol. Biol. Cell 17, 2910-2920. CDB, Japan) for help with the electron-microscopy data. Faust, C., Lawson, K. A., Schork, N. J., Thiel, B. and Magnuson, T. (1998). The References Polycomb-group gene eed is required for normal morphogenetic movements Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N. and Lovell- during gastrulation in the mouse embryo. Development 125, 4495-4506. Badge, R. (2003). Multipotent cell lineages in early mouse development depend Feldman, N., Gerson, A., Fang, J., Li, E., Zhang, Y., Shinkai, Y., Cedar, H. and on SOX2 function. Genes Dev. 17, 126-140. Bergman, Y. (2006). G9a-mediated irreversible epigenetic inactivation of Oct- Azuara, V., Perry, P., Sauer, S., Spivakov, M., Jorgensen, H. F., John, R. M., 3/4 during early embryogenesis. Nat. Cell Biol. 8, 188-194. Gouti, M., Casanova, M., Warnes, G., Merkenschlager, M. et al. (2006). Fuhrmann, G., Chung, A. C., Jackson, K. J., Hummelke, G., Baniahmad, A., Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532-538. Sutter, J., Sylvester, I., Scholer, H. R. and Cooney, A. J. (2001). Mouse DEVELOPMENT Development 134 (4) REVIEW 645 germline restriction of Oct4 expression by germ cell nuclear factor. Dev. Cell 1, Lee, J. H., Hart, S. R. L. and Skalnik, D. G. (2004). Histone deacetylase activity is 377-387. required for embryonic stem cell differentiation. Genesis 38, 32-38. Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S., Nakao, K., Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G., Levine, S. S., Kumar, R. Miyazaki, J.-i. and Niwa, H. (2002). Differentiation of embryonic stem cells is M., Chevalier, B., Johnstone, S. E., Cole, M. F., Isono, K. et al. (2006). induced by GATA factors. Genes Dev. 16, 784-789. Control of developmental regulators by polycomb in human embryonic stem Gardner, R. L. and Rossant, J. (1979). Investigation of the fate of 4.5 day post- cells. Cell 125, 301-313. coitum mouse inner cell mass cells by blastocyst injection. J. Embryol. Exp. Lehnertz, B., Ueda, Y., Derijck, A. A. H. A., Braunschweig, U., Perez-Burgos, Morphol. 52, 141-152. L., Kubicek, S., Chen, T., Li, E., Jenuwein, T. and Peters, A. H. F. M. (2003). Gaudet, F., Talbot, D., Leonhardt, H. and Jaenisch, R. (1998). A short DNA Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to methyltransferase isoform restores methylation in vivo. J. Biol. Chem. 273, major satellite repeats at pericentric heterochromatin. Curr. Biol. 13, 1192-1200. 32725-32729. Lei, H., Oh, S. P., Okano, M., Juttermann, R., Goss, K. A., Jaenisch, R. and Li, Grepin, C., Nemer, G. and Nemer, M. (1997). Enhanced cardiogenesis in E. (1996). De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells overexpressing the GATA-4 transcription factor. embryonic stem cells. Development 122, 3195-3205. Development 124, 2387-2395. Li, Y., McClintick, J., Zhong, L., Edenberg, H. J., Yoder, M. C. and Chan, R. J. Gu, P., Goodwin, B., Chung, A. C., Xu, X., Wheeler, D. A., Price, R. R., Galardi, (2005). Murine embryonic stem cell differentiation is promoted by SOCS-3 and C., Peng, L., Latour, A. M., Koller, B. H. et al. (2005a). Orphan nuclear inhibited by the zinc finger transcription factor Klf4. Blood 105, 635-637. receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of Lin, T., Chao, C., Saito, S., Mazur, S. J., Murphy, M. E., Appella, E. and Xu, Y. embryonic development. Mol. Cell. Biol. 25, 3492-3505. (2005). p53 induces differentiation of mouse embryonic stem cells by Gu, P., LeMenuet, D., Chung, A. C., Mancini, M., Wheeler, D. A. and Cooney, suppressing Nanog expression. Nat. Cell Biol. 7, 165-171. A. J. (2005b). Orphan nuclear receptor GCNF is required for the repression of Loh, Y. H., Wu, Q., Chew, J. L., Vega, V. B., Zhang, W., Chen, X., Bourque, G., pluripotency genes during retinoic acid-induced embryonic stem cell George, J., Leong, B., Liu, J. et al. (2006). The Oct4 and Nanog transcription differentiation. Mol. Cell. Biol. 25, 8507-8519. network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, Hamazaki, T., Oka, M., Yamanaka, S. and Terada, N. (2004). Aggregation of 431-440. embryonic stem cells induces Nanog repression and primitive endoderm Lumelsky, N., Blondel, O., Laeng, P., Velasco, I., Ravin, R. and McKay, R. differentiation. J. Cell Sci. 117, 5681-5686. (2001). Differentiation of embryonic stem cells to insulin-secreting structures Hamazaki, T., Kehoe, S. M., Nakano, T. and Terada, N. (2006). The Grb2/Mek similar to pancreatic islets. Science 292, 1389-1394. pathway represses Nanog in murine embryonic stem cells. Mol. Cell. Biol. 26, Lund, A. H. and van Lohuizen, M. (2004). Epigenetics and cancer. Genes Dev. 7539-7549. 18, 2315-2335. Hart, A. H., Hartley, L., Ibrahim, M. and Robb, L. (2004). Identification, cloning Martin, G. R. (1981). Isolation of a pluripotent cell line from early mouse embryos and expression analysis of the pluripotency promoting Nanog genes in mouse cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. and human. Dev. Dyn. 230, 187-198. Sci. USA 78, 7634-7638. Hata, K., Okano, M., Lei, H. and Li, E. (2002). Dnmt3L cooperates with the Matoba, R., Niwa, H., Masui, S., Ohtsuka, S., Carter, M. G., Sharov, A. A. and Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints Ko, M. S. H. (2006). Dissecting Oct3/4-regulated gene networks in embryonic in mice. Development 129, 1983-1993. stem cells by expression profiling. PLos One 1, e26. Hatano, S. Y., Tada, M., Kimura, H., Yamaguchi, S., Kono, T., Nakano, T., Matsuda, T., Nakamura, T., Nakao, K., Arai, K., Katsuki, M., Heike, T. and Suemori, H., Nakatsuji, N. and Tada, T. (2005). Pluripotential competence of Yokota, T. (1999). STAT3 activation is sufficient to maintain an undifferentiated cells associated with Nanog activity. Mech. Dev. 122, 67-79. state of mouse embryonic stem cells. EMBO J. 18, 4261-4269. Hendrich, B., Guy, J., Ramsahoye, B., Wilson, V. A. and Bird, A. (2001). Closely Meshorer, E., Yellajoshula, D., George, E., Scambler, P. J., Brown, D. T. and related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse Misteli, T. (2006). Hyperdynamic plasticity of chromatin proteins in pluripotent development. Genes Dev. 15, 710-723. embryonic stem cells. Dev. Cell 10, 105-116. Hooker, C. W. and Hurlin, P. J. (2006). Of Myc and Mnt. J. Cell Sci. 119, 208- Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., 216. Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein Ivanova, N., Dobrin, R., Lu, R., Kotenko, I., Levorse, J., DeCoste, C., Schafer, Nanog is required for maintenance of pluripotency in mouse epiblast and ES X., Lun, Y. and Lemischka, I. R. (2006). Dissecting self-renewal in stem cells cells. Cell 113, 631-642. with RNA interference. Nature 442, 533-538. Montgomery, N. D., Yee, D., Chen, A., Kalantry, S., Chamberlain, S. J., Otte, Iwai, N., Kitajima, K., Sakai, K., Kimura, T. and Nakano, T. (2001). Alteration A. P. and Magnuson, T. (2005). The murine polycomb group protein Eed is of cell adhesion and cell cycle properties of ES cells by an inducible dominant required for global histone H3 Lysine-27 methylation. Curr. Biol. 15, 942-947. interfering Myb mutant. Oncogene 20, 1425-1434. Mummery, C. L., Feyen, A., Freund, E. and Shen, S. (1990). Characteristics of Jaenisch, R. and Bird, A. (2003). Epigenetic regulation of gene expression: how embryonic stem cell differentiation: a comparison with two embryonal the genome integrates intrinsic and environmental signals. Nat. Genet. 33, carcinoma cell lines. Cell Differ. Dev. 30, 195-206. S245-S254. Murchison, E. P., Partridge, J. F., Tam, O. H., Cheloufi, S. and Hannon, G. J. Joaquin, M. and Watson, R. J. (2003). Cell cycle regulation by the B-Myb (2005). Characterization of Dicer-deficient murine embryonic stem cells. Proc. transcription factor. Cell Mol. Life Sci. 60, 2389-2401. Natl. Acad. Sci. USA 102, 12135-12140. Jones-Villeneuve, E. M., McBurney, M. W., Rogers, K. A. and Kalnins, V. I. Nakatake, Y., Fukui, N., Iwamatsu, Y., Masui, S., Takahashi, K., Yagi, R., Yagi, (1982). Retinoic acid induces embryonal carcinoma cells to differentiate into K., Miyazaki, J. I., Matoba, R., Ko, M. S. et al. (2006). Klf4 cooperates with neurons and glial cells. J. Cell Biol. 94, 253-262. Oct3/4 and Sox2 to activate the Lefty1 core promoter in embryonic stem cells. Kaji, K., Caballero, I. M., MacLeod, R., Nichols, J., Wilson, V. A. and Mol. Cell. Biol. 26, 7772-7782. Hendrich, B. (2006). The NuRD component Mbd3 is required for pluripotency Napoles, M., Mermoud, J. E., Wakao, R., Tang, Y. A., Endoh, M., Appanah, of embryonic stem cells. Nat. Cell Biol. 8, 285-292. R., Nesterova, T. B., Silva, J., Otte, A. P., Vidal, M. et al. (2004). Polycomb Kanellopoulou, C., Muljo, S. A., Kung, A. L., Ganesan, S., Drapkin, R., group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene Jenuwein, T., Livingston, D. M. and Rajewsky, K. (2005). Dicer-deficient silencing and X inactivation. Dev. Cell 7, 663-676. mouse embryonic stem cells are defective in differentiation and centromeric Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., silencing. Genes Dev. 19, 489-501. Chambers, I., Scholer, H. and Smith, A. (1998). Formation of pluripotent stem Kauffman, S. (2004). A proposal for using the ensemble approach to understand cells in the mammalian embryo depends on the POU transcription factor Oct4. genetic regulatory networks. J. Theor. Biol. 230, 581-590. Cell 95, 379-391. Kelley, C., Blumberg, H., Zon, L. I. and Evans, T. (1993). GATA-4 is a novel Nishimoto, M., Fukushima, A., Okuda, A. and Muramatsu, M. (1999). The transcription factor expressed in endocardium of the developing heart. gene for the embryonic stem cell coactivator UTF1 carries a regulatory element Development 118, 817-827. which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol. Kim, J. K., Huh, S.-O., Choi, H., Lee, K.-S., Shin, D., Lee, C., Nam, J.-S., Kim, Cell. Biol. 19, 5453-5465. H., Chung, H., Lee, H. W. et al. (2001). Srg3, a mouse homolog of yeast SWI3, Nishimoto, M., Miyagi, S., Yamagishi, T., Sakaguchi, T., Niwa, H., is essential for early embryogenesis and involved in brain development. Mol. Muramatsu, M. and Okuda, A. (2005). Oct-3/4 maintains the proliferative Cell. Biol. 21, 7787-7795. embryonic stem cell state via specific binding to a variant octamer sequence in Klochendler-Yeivin, A., Fiette, L., Barra, J., Muchardt, C., Babinet, C. and the regulatory region of the UTF1 locus. Mol. Cell. Biol. 25, 5084-5094. Yaniv, M. (2000). The murine SNF5/INI1 chromatin remodeling factor is essential Niwa, H. (2001). Molecular mechanism to maintain stem cell renewal of ES cells. for embryonic development and tumor suppression. EMBO Rep. 1, 500-506. Cell Struct. Funct. 26, 137-148. Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S. Y., Suemori, H., Niwa, H., Burdon, T., Chambers, I. and Smith, A. (1998). Self-renewal of Nakatsuji, N. and Tada, T. (2005). Octamer and Sox elements are required for pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. transcriptional cis regulation of Nanog gene expression. Mol. Cell. Biol. 25, 12, 2048-2060. 2475-2485. Niwa, H., Miyazaki, J. and Smith, A. G. (2000). Quantitative expression of Oct- DEVELOPMENT 646 REVIEW Development 134 (4) 3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Genet. 24, 372-376. Akt/protein kinase B signaling pathway. Proc. Natl. Acad. Sci. USA 96, 6199- Niwa, H., Masui, S., Chambers, I., Smith, A. G. and Miyazaki, J. (2002). 6204. Phenotypic complementation establishes requirements for specific POU domain Suzuki, A., Raya, A., Kawakami, Y., Morita, M., Matsui, T., Nakashima, K., and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol. Gage, F. H., Rodriguez-Esteban, C. and Belmonte, J. C. I. (2006a). Cell. Biol. 22, 1526-1536. Maintenance of embryonic stem cell pluripotency by Nanog-mediated reversal of Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R. mesoderm specification. Nat. Clin. Pract. Cardiovasc. Med. 3, S114-S122. and Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines Suzuki, A., Raya, A., Kawakami, Y., Morita, M., Matsui, T., Nakashima, K., trophectoderm differentiation. Cell 123, 917-929. Gage, F. H., Rodriguez-Esteban, C. and Belmonte, J. C. I. (2006b). Nanog O’Carroll, D., Erhardt, S., Pagani, M., Barton, S. C., Surani, M. A. and binds to Smad1 and blocks bone morphogenetic protein-induced differentiation Jenuwein, T. (2001). The polycomb-group gene Ezh2 is required for early of embryonic stem cells. Proc. Natl. Acad. Sci. USA 103, 10294-10299. mouse development. Mol. Cell. Biol. 21, 4330-4336. Szutoristz, H. and Dillon, N. (2005). The epigenetic basis for embryonic stem cell Okada, Y., Shimazaki, T., Sobue, G. and Okano, H. (2004). Retinoic-acid- pluripotency. BioEssays 27, 1286-1293. concentration-dependent acquisition of neural cell identity during in vitro Tachibana, M., Sugimoto, K., Nozaki, M., Ueda, J., Ohta, T., Ohki, M., differentiation of mouse embryonic stem cells. Dev. Biol. 275, 124-142. Fukuda, M., Takeda, N., Niida, H., Kato, H. et al. (2002). G9a histone Okano, M., Bell, D. W., Haber, D. A. and Li, E. (1999). DNA methyltransferases methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian methylation and is essential for early embryogenesis. Genes Dev. 16, 1779- development. Cell 99, 247-257. 1791. Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Tachibana, M., Ueda, J., Fukuda, M., Takeda, N., Ohta, T., Iwanari, H., Nabeshima, Y., Kuro-o, M., Boon, K., Keaveney, M., Stunnenberg, H. G. et Sakihama, T., Kodama, T., Hamakubo, T. and Shinkai, Y. (2005). Histone al. (1998). UTF1, a novel transcriptional coactivator expressed in pluripotent methyltransferases G9a and GLP form heteromeric complexes and are both embryonic stem cells and extra-embryonic cells. EMBO J. 17, 2019-2032. crucial for methylation of euchromatin at H3-K9. Genes Dev. 19, 815-826. Okumura-Nakanishi, S., Saito, M., Niwa, H. and Ishikawa, F. (2005). Oct-3/4 Takahashi, K. and Yamanaka, S. (2006). Induction of pluripotent stem cells from and Sox2 regulate Oct-3/4 gene in embryonic stem cells. J. Biol. Chem. 280, mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663- 5307-5317. 676. Pan, G., Li, J., Zhou, Y., Zheng, H. and Pei, D. (2006). A negative feedback loop Takahashi, K., Mitsui, K. and Yamanaka, S. (2003). Role of ERas in promoting of transcription factors that controls stem cell pluripotency and self-renewal. tumour-like properties in mouse embryonic stem cells. Nature 423, 541-545. FASEB J. 20, 1730-1732. Takahashi, K., Murakami, M. and Yamanaka, S. (2005). Role of the Pasini, D., Bracken, A. P., Jensen, M. R., Denchi, E. and Helin, K. (2004). Suz12 phosphoinositide 3-kinase pathway in mouse embryonic stem (ES) cells. is essential for mouse development and for EZH2 histone methyltransferase Biochem. Soc. Trans. 33, 1522-1525. activity. EMBO J. 23, 4061-4071. Tanaka, Y., Patestos, N. P., Maekawa, T. and Ishii, S. (1999). B-myb is required Pereira, L., Yi, F. and Merrill, B. J. (2006). Repression of Nanog gene for inner cell mass formation at an early stage of development. J. Biol. Chem. transcription by Tcf3 limits embryonic stem cell self-renewal. Mol. Cell. Biol. 26, 274, 28067-28070. 7479-7491. Teitell, M. A. (2005). The TCL1 family of oncoproteins: co-activators of Peters, A. H., O’Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., transformation. Nat. Rev. Cancer 5, 640-648. Weipoltshammer, K., Pagani, M., Lachner, M., Kohlmaier, A. et al. (2001). Tokuzawa, Y., Kaiho, E., Maruyama, M., Takahashi, K., Mitsui, K., Maeda, Loss of the Suv39h histone methyltransferases impairs mammalian M., Niwa, H. and Yamanaka, S. (2003). Fbx 15 is a novel target of Oct3/4 but heterochromatin and genome stability. Cell 107, 323-337. dispensable for ES cell self-renewal and mouse development. Mol. Cell. Biol. 23, Pierce, G. B., Arechaga, J., Muro, C. and Wells, R. S. (1988). Differentiation of 2699-2708. ICM cells into trophectoderm. Am. J. Pathol. 132, 356-364. Tomioka, M., Nishimoto, M., Miyagi, S., Katayanagi, T., Fukui, N., Niwa, H., Roberts, C., Sutherland, H. F., Farmer, H., Kimber, W., Halford, S., Carey, A., Muramatsu, M. and Okuda, A. (2002). Identification of Sox-2 regulatory Brickman, J. M., Wynshaw-Boris, A. and Scambler, P. J. (2002). Targeted region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res. mutagenesis of the Hira gene results in gastrulation defects and patterning 30, 3202-3213. abnormalities of mesoendodermal derivatives prior to early embryonic lethality. Tsumura, A., Hayakawa, T., Kumaki, Y., Takebayashi, S., Sakaue, M., Mol. Cell. Biol. 22, 2318-2328. Mstsuoka, C., Shimotohno, K., Ishikawa, F., Li, E., Ueda, H. R. et al. (2006). Rodda, D. J., Chew, J. L., Lim, L. H., Loh, Y. H., Wang, B., Ng, H. H. and Maintenance of self-renewal ability of mouse embryonic stem cells in the Robson, P. (2005). Transcriptional regulation of nanog by OCT4 and SOX2. J. absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells Biol. Chem. 280, 24731-24737. 11, 805-814. Roeder, R. G. (2005). Transcriptional regulation and the role of diverse coactivators von Dassow, G., Meir, E., Munro, E. M. and Odell, G. M. (2000). The segment in animal cells. FEBS Lett. 579, 909-915. polarity network is a robust developmental module. Nature 406, 188-192. Rosner, M. H., Vigano, M. A., Ozato, K., Timmons, P. M., Poirier, F., Rigby, P. Voncken, J. W., Roelen, B. A. J., Roefs, M., Vries, S., Verhoeven, E., Marino, W. and Staudt, L. M. (1990). A POU-domain transcription factor in early stem S., Deschamps, J. and Lohuizen, M. (2003). Rnf2 (Ring1b) deficiency causes cells and germ cells of the mammalian embryo. Nature 345, 686-692. gastrulation arrest and cell cycle inhibition. Proc. Natl. Acad. Sci. USA 100, 2468- Rowland, B. D. and Peeper, D. S. (2006). KLF4, p21 and context-dependent 2473. opposing forces in cancer. Nat. Rev. Cancer 6, 11-23. Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D. N., Theunissen, T. W. and Sakaki-Yumoto, M., Kobayashi, C., Sato, A., Fujimura, S., Matsumoto, Y., Orkin, S. H. (2006). A protein interaction network for pluripotency of Takasato, M., Kodama, T., Aburatani, H., Asashima, M., Yoshida, N. et embryonic stem cells. Nature 444, 364-368. al. (2006). The murine homolog of SALL4, a causative gene in Okihiro Watanabe, S., Umehara, H., Murayama, K., Okabe, M., Kimura, T. and syndrome, is essential for embryonic stem cell proliferation, and cooperates Nakano, T. (2006). Activation of Akt signaling is sufficient to maintain with Sall1 in anorectal, heart, brain and kidney development. Development pluripotency in mouse and primate embryonic stem cells. Oncogene 25, 2697- 133, 3005-3013. 2707. Sieweke, M. H. and Graf, T. (1998). A transcription factor party during blood cell Weintraub, H. and Groudine, M. (1976). Chromosomal subunits in active genes differentiation. Curr. Opin. Genet. Dev. 8, 545-551. gave an altered conformation. Science 193, 848-856. Smith, A. (2005). The battlefield of pluripotency. Cell 123, 757-760. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J. and Campbell, K. H. (1997). Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G., Moreau, J., Stahl, Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810- M. and Rogers, D. (1988). Inhibition of pluripotential embryonic stem cell 813. differentiation by purified polypeptides. Nature 336, 688-690. Wu, Q., Chen, X., Zhang, J., Loh, Y. H., Low, T. Y., Zhang, W., Sze, S. K., Lim, Solter, D. (2006). From teratocarcinomas to embryonic stem cells and beyond: a B. and Ng, H. H. (2006). Sall4 interacts with Nanog and co-occupies Nanog history of embryonic stem cell research. Nat. Rev. Genet. 7, 319-327. genomic sites in embryonic stem cells. J. Biol. Chem. 281, 24090-24094. Stopka, T. and Skoultchi, A. I. (2003). The ISWI ATPase Snf2h is required for early Yeom, Y. I., Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K., Gross, M., mouse development. Proc. Natl. Acad. Sci. USA 100, 14097-14102. Hubner, K. and Scholer, H. R. (1996). Germline regulatory element of Oct-4 Strahl, B. D. and Allis, C. D. (2000). The language of covalent histone specific for the totipotent cycle of embryonal cells. Development 122, 881-894. modifications. Nature 403, 41-45. Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003). BMP induction of Id Suda, Y., Suzuki, M., Ikawa, Y. and Aizawa, S. (1987). Mouse embryonic stem proteins suppresses differentiation and sustains embryonic stem cell self-renewal cells exhibit indefinite proliferative potential. J. Cell Physiol. 133, 197-201. in collaboration with STAT3. Cell 115, 281-292. Sumi-Ichinose, C., Ichinose, H., Metsger, D. and Chambon, P. (1997). SNF2- Yuan, H., Corbi, N., Basilico, C. and Dailey, L. (1995). Developmental-specific BRG1 is essential for the viability of F9 murine embryonal carcinoma cells. Mol. activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Cell. Biol. 17, 5976-5986. Genes Dev. 9, 2635-2645. Sun, H., Lesche, R., Li, D. M., Liliental, J., Zhang, H., Gao, J., Gavrilova, N., Zipori, D. (2004). The nature of stem cells: state rather than entity. Nat. Rev. Mueller, B., Liu, X. and Wu, H. (1999). PTEN modulates cell cycle progression Genet. 5, 873-878. DEVELOPMENT
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Development – The Company of Biologists
Published: Feb 15, 2007