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First published online 10 January 2007
doi: 10.1242/dev.02787
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Review |
Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology (CDB), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 6500047, Japan. Laboratory for Development and Regenerative Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunokicho, Chuo-ku, Kobe, Hyogo 6500017, Japan.
E-mail: niwa{at}cdb.riken.jp
SUMMARY
Mouse embryonic stem (ES) cells are pluripotent, as they have the ability to differentiate into the various cell types of a vertebrate embryo. Pluripotency is a property of the inner cell mass (ICM), from which mouse ES cells are derived, and of the epiblast of the blastocyst. Recent extensive molecular studies of mouse ES cells have revealed the unique molecular mechanisms that govern pluripotency. These studies show that ES cells continue to self-renew because of a self-organizing network of transcription factors that prevents their differentiation and promotes their proliferation, and because of epigenetic processes that might be under the control of the pluripotent transcription factor network.
Introduction
Mouse embryonic stem (ES) cells, and the cells of the embryonic inner cell
mass (ICM) from which mouse ES cells are derived, are pluripotent. According
to recent consensus, pluripotency describes a cell's ability to give rise to
all of the cells of an embryo and adult
(Solter, 2006
). Studies over
the past few years have revealed the role that transcription factor networks
and epigenetic processes play in the maintenance of ES cell pluripotency
(Niwa et al., 2000;
Mitsui et al., 2003;
Chambers et al., 2003;
Boyer et al., 2005;
Niwa et al., 2005;
Boyer et al., 2006). Among the
findings to have emerged from these studies is that the functions of these
transcription factors depend on the stage of development of a pluripotent
cell, indicating that these factors function in combination with other
processes (Sieweke and Graf,
1998). The activity of these transcription factors also depends on
the accessibility of their target genes, which are made more or less
accessible by the modification of their DNA, histones, or chromatin structure
(Jaenisch and Bird, 2003). In
this review, I discuss new insights into how transcription factor networks
maintain mouse ES cell pluripotency and how these factors interface with
epigenetic processes to control the pluripotency and differentiation of mouse
ES cells.
An overview of mouse ES cell derivation, proliferation and differentiation
Pluripotent embryonic lineages and ES cell derivation
Mouse ES cells are derived mainly from the ICM of the mouse blastocyst
(Evans and Kaufman, 1981;
Martin, 1981) (see
Fig. 1). As the embryo
develops, the ICM gives rise to two distinct cell lineages: the extraembryonic
endoderm, which goes on to form the extraembryonic tissues; and the epiblast,
which gives rise to the primitive ectoderm at the egg-cylinder stage of
embryogenesis, from which the embryo proper arises. The primitive ectoderm is
distinct from the ICM in several ways. It cannot give rise to the
trophectoderm, nor to the primitive endoderm (see
Fig. 1); it also has an
epithelial morphology distinct from that of the ICM
(Gardner and Rossant, 1979).
Importantly, the primitive ectoderm is the only cell lineage in which
pluripotency is maintained at this stage of development, enabling it to give
rise to all three embryonic germ layers and to primordial germ cells (see
Fig. 1). However, as it lacks
the ability to differentiate into the extraembryonic, primitive endodermal and
trophectodermal lineages, the primitive ectoderm is less pluripotent than the
cells of the ICM and possesses `restricted' pluripotency.
Traditionally, pluripotency has often been defined as the ability to
generate all cell types of an embryo apart from the trophectoderm (the
precursor to the bulk of the embryonic part of the placenta)
(Bioani and Schöler,
2006). This is because an earlier analysis of chimeric mouse
embryos, produced by the injection of ICM cells and ES cells into 8-cell
embryos or blastocysts, had shown that ICM cells are excluded from the
trophectoderm lineage (Beddington and
Robertson, 1989). However, it has subsequently been found that the
ICM does still possess the ability to differentiate into the trophectoderm
lineage (Pierce et al., 1988),
as do ES cells under particular culture conditions
(Niwa et al., 2005).
Therefore, in this review, I define pluripotency as the ability to generate
all cell types, including the trophectoderm, without the self-organizing
ability to generate a whole organism [see also Solter
(Solter, 2006) for similar
definitions of these terms].
ES cell proliferation
Pluripotency is maintained during ES cell self-renewal through the
prevention of differentiation and the promotion of proliferation. In fact, ES
cells can self-renew continuously for years if they are cultured under
conditions that prevent their differentiation; for example, in the presence of
leukemia inhibitory factor (Lif), a growth factor that is necessary for
maintaining mouse ES cells in a proliferative, undifferentiated state
(Suda et al., 1987). But how
is pluripotency itself protected via self-renewal at the molecular level? This
question is discussed in more detail below.
ES cell differentiation
Although ES cells are described as being pluripotent, they can only
differentiate directly into three cell types: the primitive ectoderm, the
primitive endoderm and trophectoderm cells, analogous to the differentiation
ability of cells of the ICM.
The differentiation of mouse ES cells can be induced by the ectopic
expression of certain transcription factors. For example, the expression of
the transcription factor Gata6 in ES cells results in their differentiation
into primitive endoderm (Fujikura et al.,
2002). Likewise, the expression of the caudal-type homeobox
transcription factor 2 (Cdx2) induces ES cells to differentiate into
trophectoderm (Niwa et al.,
2005). Therefore, both of these factors have to be tightly
repressed for ES cells to self-renew, as discussed in more detail below.
Self-renewal by preventing differentiation
As mentioned above, ES cell pluripotency is maintained during self-renewal
by the prevention of differentiation and the promotion of proliferation. For
mouse ES cells, Lif is a key factor that prevents differentiation. Lif belongs
to the interleukin-6 cytokine family and binds to a heterodimeric receptor
consisting of the Lif-receptor ß and gp130 (Il6st - Mouse Genome
Informatics). This binding results in the activation of the canonical Jak/Stat
(Janus kinase signal transducer and activator of transcription) pathway. It
has been reported that Stat3 activation is essential and sufficient to
maintain the pluripotency of mouse ES cells
(Niwa et al., 1998;
Matsuda et al., 1999), and
that c-Myc is a candidate target of Stat3
(Cartwright et al., 2005).
|
) that
acts as a gatekeeper to prevent ES cell differentiation. Artificial repression
of Oct3/4 in ES cells induces differentiation along the trophectodermal
lineage; when overexpressed, ES cells differentiate mainly into primitive
endoderm-like cells (see Fig.
2B) (Niwa et al.,
2000).
Oct3/4 has been reported to directly prevent differentiation towards
trophectoderm by interacting with Cdx2 (a trigger for trophectoderm
differentiation; see Fig.
2D,E), to form a repressor complex. This complex interferes with
the autoregulation of these two factors, giving rise to a reciprocal
inhibition system that establishes their mutually exclusive expression
(Niwa et al., 2005). As such,
the downregulation of Oct3/4 results in an upregulation of Cdx2, and vice
versa - a mechanism that might account for the two different pathways that
lead to pluripotent stem cells and to trophectoderm cells.
Both the inhibition of Stat3 activity and the overexpression of Oct3/4
stimulate ES cells to differentiate into primitive endoderm-like cells
(Fig. 2B)
(Niwa et al., 1998;
Niwa et al., 2000). The
existence has been suggested of an unidentified co-factor of Oct3/4 that is
activated by Stat3 (Niwa,
2001). The normal functions of this co-factor could be disrupted
by an excess of Oct3/4, which might disrupt the functions of a ternary complex
(consisting of Oct3/4, its co-factor and a general transcription unit, which
activates target genes) via the saturation of protein interactions. This is
supported by evidence that this `overdose effect' of Oct3/4 on ES cell
differentiation does not require Oct3/4 DNA-binding activity
(Niwa et al., 2002). In such a
model, the target gene(s) of this particular complex would normally prevent ES
cells from differentiating into primitive endoderm by repressing the trigger
factor, Gata6. Nanog is an NK-2 class homeobox transcription factor that is
expressed throughout the pluripotent cells of the ICM. As overexpression of
Nanog in mouse ES cells can maintain them in a pluripotent state in the
absence of Lif, it is a good candidate for this hypothetical Gata6 repressor
(Chambers et al., 2003;
Mitsui et al., 2003). Indeed,
Nanog-null ES cells differentiate into Gata6-positive parietal
endoderm-like cells, which have a morphology that is similar to that of
Gata6-induced cells (Fig. 2)
(Mitsui et al., 2003).
However, although it has been reported that Nanog expression is
partly regulated by Oct3/4 and Sox2, a member of the Sox (SRY-related HMG box)
family (Kuroda et al., 2005;
Rodda et al., 2005), and
although artificial Nanog expression can block the differentiation of
ES cells into primitive endoderm cells [induced by either the withdrawal of
Lif (Chambers et al., 2003) or
the formation of embryoid bodies (EBs: ball-like structures that form when ES
cells are kept in suspension culture and which mimic the egg-cylinder stage of
embryogenesis] (Hamazaki et al.,
2004), no direct evidence for the repression of Gata6 by
Nanog has yet been found.
The gatekeeper function of Nanog might not be restricted to preventing the
differentiation of ES cells into primitive endoderm, as it has been reported
that Nanog also blocks neuronal differentiation induced by the removal of Lif
and bone morphogenetic protein (BMP) from serum-free culture
(Ying et al., 2003). In
addition, Nanog can also reverse mesoderm specification by repressing
brachyury, which encodes the mesoderm-specific T-box transcription factor T.
This factor directly activates Nanog expression, indicating that
negative feedback is involved in the balance between self-renewal and
mesodermal differentiation (Suzuki et
al., 2006a). Thus, Nanog can block primitive endodermal
differentiation, neuronal differentiation and mesodermal differentiation under
different culture conditions
Promoting self-renewal through proliferation
Under optimized culture conditions, in which Lif is essential
(Smith et al., 1988), mouse
ES cells divide symmetrically every 12 hours. During self-renewal, most ES
cells are in the S phase of the cell cycle, with only a few in G1
(Burdon et al., 2002).
Recent findings suggest that the phosphoinositide-3-kinase (PI3K)/Akt
signaling pathway plays a pivotal role in promoting the proliferation,
survival and/or differentiation of mouse ES cells (see
Fig. 3). The deletion of
Pten, which encodes a negative regulator of PI3K, in mouse ES cells
has been reported to increase ES cell viability and proliferation
(Sun et al., 1999), and it
has recently been reported that the artificial activation of Akt is sufficient
to maintain ES cell self-renewal in the absence of Lif
(Watanabe et al., 2006).
|
).
Eras encodes a constitutively active form of a Ras-family small
GTPase that activates PI3K to stimulate ES cell proliferation and
tumorigenicity after ectopic transplantation in vivo
(Takahashi et al., 2003). The
Tcl1 gene product augments Akt activation by forming a stable
heterodimeric complex with Akt (Teitell,
2005). Knockdown of Tcl1 in mouse ES cells impairs
self-renewal by inducing differentiation and/or repressing their proliferation
(Ivanova et al., 2006; Matoba et al.,
2006). However, the molecular mechanisms that direct the
expression of Eras and Tcl1 in ES cells have yet to be
identified.
The transcription factor b-Myb has been reported to be an accelerator of
cell-cycle progression in mouse ES cells. Overexpression of a
dominant-negative form of b-Myb in these cells results in G1 arrest
(Iwai et al., 2001),
indicating that b-Myb is transcriptionally activated in G1 and
promotes the transition to S phase by a complex mechanism
(Joaquin and Watson, 2003).
Moreover, b-Myb-null blastocysts show defective ICM outgrowth in
vitro (Tanaka et al., 1999),
suggesting that b-Myb might play an important role in promoting the cell cycle
in ES cells. However, neither the transcriptional regulation of b-Myb nor its
precise function in regulating the cell cycle in mouse ES cells have yet been
analyzed.
The basic helix-loop-helix transcription factor Myc is a wellknown
accelerator of the cell cycle, acting via the transcriptional activation of
cyclin E expression to promote G1-S transition
(Hooker and Hurlin, 2006).
Recently, Cartwright et al. (Cartwright et
al., 2005) reported that c-Myc is a direct target of
Stat3, and that overexpression of a dominant-active form of c-Myc that has a
greater stability than the wild-type protein renders the self-renewal of mouse
ES cells independent of Lif. By contrast, the overexpression of a
dominant-negative form of c-Myc antagonizes mouse ES cell self-renewal and
promotes differentiation. These findings suggest that the regulation of the
G1-S transition may contribute to the maintenance of pluripotency, which is
promoted by the Lif-Stat3 pathway in mouse ES cells
(Burdon et al., 2002).
|
). Mouse ES cells with reduced expression of Utf1 show
reduced proliferation in vitro and reduced tumorigenicity in vivo
(Nishimoto et al., 2005). Utf1
possesses a stem-cell-specific enhancer that is activated by Oct3/4 and Sox2
(Nishimoto et al., 1999), so
it can be regarded as a link between the pluripotent transcription factor
network and the promotion of proliferation.
Mouse ES cells that lack Sall4, one of the mouse homologs of the
Drosophila homeotic gene spalt that encodes a zinc-finger
transcription factor, were recently reported to show reduced proliferation
ability (Sakaki-Yumoto et al.,
2006). Another study showed that Sall4 interacts with Nanog to
activate Sall4 and Nanog
(Wu et al., 2006). However,
Sall4 expression is not restricted to mouse ES cells, and Nanog is
still expressed in Sall4-null ES cells
(Sakaki-Yumoto et al., 2006),
so the physiological contribution of this positive-feedback loop to the
maintenance of pluripotency remains to be confirmed.
Mechanisms to maintain self-renewal
In order to maintain the stable self-renewal of ES cells, the mechanisms that prevent their differentiation and promote their proliferation must be transmitted to their daughter cells. Thus, the expression levels of the genes that are involved in these mechanisms need to be stably maintained.
A transcription factor network that is stabilized by positive and negative
regulation between its components is a good mechanism for maintaining the
stable gene expression patterns that determine a particular cell phenotype
(von Dassow et al., 2000).
Moreover, the application of systems biological views, such as the Boolean
network models, allows us to explain how small changes to a few components of
a network can trigger the dynamic transition of a transcription factor network
from one state to another (Kauffman,
2004). Random Boolean network models are a way of modeling
networks that are composed of multiple factors which have multiple inputs in
complex systems. They are based on Boolean logic, in which multiple logical
operators, such as AND and OR, are united into expressions about the factor
with binary values such as 1 and 0
(Kauffman, 2004).
Sox2 occupies an important position in the maintenance of the pluripotent
transcription factor network (Fig.
4B). As discussed above, Sox2 is known to co-operate with Oct3/4
in activating Oct3/4 target genes (Yuan
et al., 1995). To date, ES-specific enhancers that contain binding
sites for Oct3/4 and Sox2 have been identified in several genes, including
Fgf4 (Yuan et al.,
1995), osteopontin (Spp1 - Mouse Genome Informatics)
(Botquin et al., 1998),
Utf1 (Nishimoto et al.,
1999), Fbxo15
(Tokuzawa et al., 2003),
Nanog (Kuroda et al.,
2005; Rodda et al.,
2005) and Lefty1
(Nakatake et al., 2006).
Interestingly, both Oct3/4 and Sox2 possess enhancers that
are activated by the Oct3/4-Sox2 complex in a stem-cell-specific manner
(Chew et al., 2005;
Okumura-Nakanishi et al.,
2005; Tomioka et al.,
2002). Sox2-null embryos die immediately after
implantation (Avilion et al.,
2003), and knockdown of Sox2 in mouse ES cells induces
differentiation into multiple lineages, including trophectoderm, indicating
its functional importance in the maintenance of pluripotency
(Ivanova et al., 2006). The
generation of Sox2-null ES cells would 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
neuroepithelium of Gcnf-null embryos
(Fuhrmann et al., 2001).
Oct3/4 repression following the induction of differentiation is also
delayed in Gcnf-null ES cells (Gu
et al., 2005b). Chicken ovalbumin upstream promoter-transcription
factors (Coup-tf) I and II, encoded by Nr2f1 and Nr2f2,
respectively, also function as negative regulators of Oct3/4
expression (Ben-Shushan et al.,
1995). The balance between these positive and negative regulators
might determine the precise level of Oct3/4 expression in response to
extracellular stimuli (Fig.
4A).
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The feedback regulatory circuit that maintains pluripotency interacts with
the feedback loop shown in Fig.
4B, in which Oct3/4, Sox2 and Nanog function to maintain their
expression to promote continuous ES cell self-renewal. This loop determines
the differentiation fate of ES cells by influencing the expression of
transcription factors, such 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 a
model in which two positive-feedback loops are connected by negative-feedback
loops. In such a system, a small quantitative asymmetry in one loop can be
converted into its exclusive expression
(Becskei et al., 2001).
Moreover, as Gcnf, Nr2f1 and Nr2f2 are upregulated after the
induction of either trophectoderm or primitive endoderm differentiation
(Fujikura et al., 2002;
Niwa et al., 2005), these
negative regulators might form the negative-feedback loop that shuts down
Oct3/4 in differentiated cells, and which could then be followed by
epigenetic chromatin modifications that result in the repression of the
Oct3/4 promoter (Feldman et al.,
2006).
The transition of the pluripotent transcription factor network to either
the trophectodermal or extraembryonic-endodermal network is most likely to be
regulated by the presence or absence of extracellular signals, such as the
removal of Lif from mouse ES cells or the formation of EBs. However, the
activation of Cdx2 or the repression of Oct3/4 might occur
in mouse ES cells through the infrequent spontaneous differentiation of these
cells towards trophectoderm, which can occur under standard culture conditions
(Beddington and Robertson,
1989). This tallies with evidence that Oct3/4 and Cdx2 compete
with each other to be expressed during blastocyst formation, and with evidence
that Oct3/4 expression is dominant in the ICM
(Niwa et al., 2005).
Therefore, the gatekeeper function of Nanog, which is an Oct3/4 target and
prevents extraembryonic endoderm differentiation, appears to be more important
in mouse ES cells, as these cells are regulated by extracellular signals.
Indeed, Nanog could be at the hub of these multiple signal transduction
pathways. As mentioned above, Nanog can block primitive endoderm
differentiation (Chambers et al.,
2003), neuronal differentiation
(Ying et al., 2003) and
mesoderm differentiation (Suzuki et al.,
2006a) under different culture conditions. Recent studies have
shown that Nanog interacts with Smad1 to inhibit the expression of brachyury
(Suzuki et al., 2006b) and
with Sall4 to form a positive regulatory loop for Nanog and
Sall4 (Wu et al.,
2006); also, Nanog expression is activated by Foxd3
(Pan et al., 2006) and is
repressed by Tp53 (Trp53 - Mouse Genome Informatics)
(Lin et al., 2005), Gcnf
(Nr6a1 - Mouse Genome Informatics) (Gu et
al., 2005b), Tcf3 (Pereira et
al., 2006) and the Grb2-Mek (Mdk - Mouse Genome Informatics)
pathways (Hamazaki et al.,
2006). However, during mouse development, Nanog
transcription is downregulated in the epiblast and in early primitive ectoderm
(Hart et al., 2004;
Hatano et al., 2005), where
Oct3/4 and Sox2 continue to be expressed
(Avilion et al., 2003;
Rosner et al., 1990). It is
noteworthy that Nanog expression levels in P19 embryonal carcinoma
(EC) cells is much lower than that in ES cells, although both EC and ES cells
express similar levels of Oct3/4 and Sox2
(Chambers et al., 2003). This
suggests that the positive-feedback circuitry in the pluripotent transcription
factor network does not always require Nanog, and that the transcription
factor network can establish a different stable circuit that maintains the
levels of Oct3/4 and Sox2 expression required to maintain pluripotency with or
without Nanog.
Two other factors have recently been reported to be necessary for the
maintenance of ES cell self-renewal: estrogen-related receptor ß (Esrrb)
and T-box transcription factor Tbx3, both identified by functional screening
mediated by RNA interference (Ivanova et
al., 2006). Repression of Esrrb in mouse ES cells results
in their differentiating into a mixture of extraembryonic and embryonic
lineages, whereas knockdown of Tbx3 triggers differentiation into
mainly the embryonic lineages that are derived from the primitive ectoderm.
Since the effect of repressing these genes can be cancelled out by the
overexpression of Nanog, the maintenance of Nanog expression is one
of their functions. The transcriptional regulation of their expression in ES
cells has yet to be analyzed, but multiple binding sites for Oct3/4 and Nanog
have been found in the mouse Esrrb gene
(Loh et al., 2006). In
addition, a recent protein interaction network analysis identified two
transcription factors, the BTB-domain-containing protein Nac1 (Btbd14b - Mouse
Genome Informatics) and the zinc-finger protein Zfp281, which interact with
Nanog and are essential for maintaining the self-renewal of mouse ES cells
(Wang et al., 2006). Further
analyses will be required to integrate these genes into the current
transcription factor network model described in this review.
An epigenetic mechanism for self-renewal
A series of recent studies have revealed that mouse and human ES cells
possess certain novel epigenetic features. Polycomb-group (PcG) complex
proteins mainly act to stabilize a repressive chromatin structure. Polycomb
repressive complex 2 (PRC2), which consists of Ezh2, Eed and Suz12 in ES
cells, functions as a histone methyltransferase on lysine 27 (K27) of histone
H3, resulting in its tri-methylation (H3K27me3), a methylation mark that is
associated with transcriptionally inactive genes
(Cao and Zhang, 2004). In
general, the distribution of this repressive chromatin mark is mutually
exclusive to that of the tri-methylation mark H3K4me3, which is associated
with transcriptionally active regions
(Strahl and Allis, 2000;
Lund and van Lohuizen, 2004).
However, Bernstein et al. reported that in mouse ES cells, these histone marks
co-localize in particular regions, which they named `bivalent domains'
(Bernstein et al., 2006). These
domains, which are composed of short chromatin elements marked by H3K4me3
flanked by larger regions that contain H3K27me3, are associated with genes
that are expressed at low levels (Fig.
5B) (Bernstein et al.,
2006). Interestingly, the bivalent domains map to highly conserved
noncoding elements (HCNEs) that have previously been identified as being
conserved among the genomes of primates and rodents and which contain few
retrotransposons (Bernstein et al.,
2006). Moreover, half of these bivalent domains contain target
sites that are common to Oct3/4, Sox2 and Nanog, as identified by genome-wide
ChIP-on-Chip analysis (Boyer et al.,
2005). Thus, these domains might signify the chromatin structure
of genes that are in a differentiation-ready state, as proposed in the
`Localised Marking Model' by Szutoristz and Dillon
(Szutoristz and Dillon,
2005). According to this model, most tissue-specific genes in ES
cells would be targets for sequence-specific factors that can recruit
histone-modifying enzymes, resulting in the formation of early transcription
competence marks (ETCMs), which are enriched for histone H3 and H4 acetylation
(H3Ac and H4Ac, respectively), and H3K4me3, all of which are histone marks
associated with transcriptionally active regions. In both bivalent domains and
ETCMs, H3K4me3 marks spread as genes near them become transcriptionally
active, whereas H3K27me3 exclusively occupies those genes that are repressed
during the differentiation of a particular cell type. Because the global level
of H3K27me3 in ES cells is lower than that in differentiated cells, the
mechanism by which this repressive mark targets such sites is of interest. Lee
et al. (Lee et al., 2006)
performed ChIP-on-Chip analysis for Suz12, Eed and H3K27me3, and revealed that
Suz12- and Eed-binding sites significantly overlap with each other and with
H3K27me3 marks on the highly evolutionarily-conserved regions of
transcriptionally silent genes, including Gata4 and Cdx2, in
ES cells. The 1800 genes identified as targets of Suz12 included most of the
targets repressed by Oct3/4, Sox2 and Nanog
(Boyer et al., 2005). Boyer et
al. (Boyer et al., 2006) also
identified 512 common target genes of PRC2 and PRC1 by ChIP-on-Chip analysis
and found that they were marked by H3K27me3, and that 87% were upregulated in
the absence of PRC2 in Eed-null ES cells.
These findings suggest that the dynamic repression of developmental
pathways in ES cells by epigenetic processes may be required for the
maintenance of pluripotency; but this conclusion requires, in my view, further
study. This is because observations made in ES cells that are deficient for
members of the PRC2 and PRC1 complexes do not fit easily into this model. For
example, Eed-null ES cells can still self-renew, maintain normal
morphology and express Oct3/4, Sox2 and Nanog normally in the complete absence
of PRC2 and despite a dramatic decrease in H3K27me3. These cells just show a
high rate of spontaneous differentiation
(Boyer et al., 2006;
Azuara et al., 2006). Although
the expression of Gata4 and Gata6, as well as of several
neural-specific genes, are upregulated in the absence of Eed, these ES cells
can still produce all three germ layers on injection into blastocysts
(Montgomery et al., 2005;
Azuara et al., 2006).
Suz12-null ES cells also show features similar to those of
Eed-null ES cells (Lee et al.,
2006). The establishment of Ezh2-null ES cells has not
been reported (O'Carroll et al.,
2001), but it has been shown that Ezh2 protein becomes
undetectable in Eed-null ES cells, and is restored by the
introduction of an Eed transgene
(Montgomery et al., 2005). ES
cells lacking Rnf2/Ring1ß, a component of PRC1, are also viable
and show decreased amounts of histone H2A ubiquitination
(Napoles et al., 2004). These
findings indicate that the PcG proteins and the PRC1 and PRC2 complexes are
not required for the maintenance of pluripotency.
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If all genomic information is utilized at least once during the development
of an organism, all genes should be ready to be expressed when they are
required to execute pluripotency during development and, in general, the
expression of a large number of genes is a common feature of stem cells
(Zipori, 2004). Therefore, in
pluripotent stem cells, many genes might be weakly expressed and, during
differentiation, the expression levels of many might be reduced, whereas those
of others are increased, determining the progeny's phenotype. Indeed,
genome-wide gene expression profiling using microarrays has revealed that a
variety of genes are expressed at low levels in ES cells
(Carter et al., 2005). This
might be a consequence of their chromatin structure being in an open
configuration, allowing the leaky expression of genes by the general
transcription machinery with neither positive nor negative regulation
(Roeder, 2005)
(Fig. 5B).
The leaky expression of a large number of genes characteristic of the ES
cell pluripotent state is likely to be the result of both genetic and
epigenetic mechanisms and processes. Through epigenetic processes, the
pluripotent epigenome keeps the chromatin structure open to allow for rapid
genetic regulation (Fig. 5B)
(Zipori, 2004). The general
abundance of transcriptionally active chromatin marks, such as H3K4me3 and
H4Ac, in ES cells fits with this idea (Lee
et al., 2004; Azuara et al.,
2006). Hyperdynamic chromatin restructuring has been observed in
mouse ES cells during self-renewal as rapid exchanges of histone H1 and
HP1
(Meshorer et al.,
2006), which might contribute to keeping the chromatin structure
of ES cells open. The existence of such a globally relaxed chromatin structure
is supported by the following evidence. Remarkable differences exist in the
distribution and frequency of high electron density areas, which were
originally designated as heterochromatin
(Brown, 1966), between ES and
parietal endoderm cells (Fig.
5B). DNaseI hypersensitive sites, which correlate with
transcriptionally active chromatin
(Weintraub and Groudine,
1976), are frequently detected in genes regardless of their
expression levels in ES cells (Meshorer et
al., 2006). Finally, nuclei in ES cells are about double the
volume of those in differentiated cells
(Faro-Trindade and Cook,
2006). As such, the guidance of cell fates could occur solely via
the action of transcription factors, such as Gata6 and Cdx2, owing to the
unprogrammed state of the pluripotent epigenome, which might allow
transcription factors to freely access their target genes to control
differentiation (Smith,
2005).
By contrast, as shown in Table
1, various epigenetic processes, including PcG/H3K27me3, DNA
methylation, tri-methylation of lysine 9 of histone H3 (H3K9me3) and RNAi, are
not essential for pluripotency. The requirement for H3K4me3 has not been
assessed because a methyltransferase that allows H3K9me3 to be globally marked
in ES cells has not yet been identified. The chromatin remodeling system,
however, might be the exception because it has been reported that the
inactivation of Brg1/Snf2ß, a component of the SWI/SNF and ISWI
complex family involved in ATP-dependent 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.
|
|
). When
Gata4 is ectopically expressed in ES cells, it directs
differentiation into parietal endoderm, as does Gata6
(Fujikura et al., 2002). By
contrast, ectopic 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 endoderm
(a differentiation pathway that is repressed by Nanog, as discussed above)
(Mummery et al., 1990). This
suggests that the genetic function of Gata factors in EC cells is different
from that in ES cells because of the difference in pre-existing transcription
factors in these cell types. However, both the prevention of differentiation
into primitive endoderm and the change in response to the ectopic expression
of Gata4 in P19 EC cells might reflect changes in their epigenetic state,
perhaps owing to changes in the accessibility of their target genes. Since the
phenotype of P19 EC cells is closer to that of primitive ectoderm than to ICM
(Jones-Villeneuve et al.,
1982), a restriction of pluripotency might be mimicked in P19 EC
cells, in which the gatekeeper function of Nanog might be replaced by the
epigenetic repression of its targets. Therefore, the function of Nanog might
be limited to that of a gatekeeper, which blocks ES cells from following
certain differentiation pathways but makes few other contributions to the
state of pluripotency.
How does the transcription factor network determine the pluripotent state
per se? As mentioned above, a combination of positive-feedback loops with
reciprocal inhibitory loops allows continuous input parameters to be converted
into a bimodal probability distribution
(Becskei et al., 2001). This
system was first applied to explain how the ICM and trophectoderm segregate
into mutually exclusive Oct3/4 and Cdx2 expression domains
and could possibly be applied to each differentiation event in development
(Niwa et al., 2005).
Epigenetic mechanisms might follow this process by locking one of the
components that is transcriptionally inactivated by competition into a
repressive state. If this is a general rule in the transition of the
transcription factor networks, by which sequential differentiation events in
development are mediated, what happens if all epigenetic repression is removed
at once? During normal embryonic development, first ectoderm and mesoendoderm
are segregated, and then the latter is separated into mesoderm and endoderm,
in which ectodermal determination is repressed. The system consists of a
combination of positive-feedback loops with reciprocal inhibitory loops, which
work sequentially to choose one fate in these steps. If these systems start to
work at once because of the epigenetic derepression of transcription, the
positive- and negative-feedback loops could end up functioning chaotically and
might result in a disordered state in which none of the transcription factor
networks holds an exclusive position, resulting in there being no
determination of cell phenotype. In addition, a feature of the random Boolean
network is that small changes to a few components can mediate the transition
of the stable condition of the network (`attractor') from one state to
another, but this transition depends strongly on the initial state of the
network. Only a particular change can trigger a transition, and other changes
are cancelled out without any effect on the network, indicating that it might
not be necessary to repress all tissue-specific transcription factor genes to
prevent differentiation in the pluripotent state. This idea is supported by
the fact that the ectopic expression of the tissue-specific transcription
factors merely directs the differentiation of ES cells, and that the
expression of many tissue-specific transcription factors, such as
Pax6 and Pdx1, are detected in ES cells
(Lumelsky et al., 2001;
Okada et al., 2004).
Therefore, the function of the pluripotent transcription factor network might
be limited to the activation of the epigenetic processes that generate the
open chromatin structure required for rapid changes in the transcriptional
status of tissue-specific genes during ES cell differentiation and
development: for example, by activating the enzymes that result in
transcriptionally repressive histone marks being exchanged for those of
actively transcribed genes.
The establishment of pluripotency in vivo
During development, both genetic and epigenetic mechanisms could be
involved in the establishment of the pluripotent state in the cells of the ICM
through the reprogramming of nuclei in fertilized eggs. Such reprogramming
activity is present in the cytoplasm of fertilized eggs, as proven by the
generation of cloned embryos from somatic cell nuclear transfer
(Wilmut et al., 1997).
However, it is still unclear which mechanism contributes to this activity
because the enzymes that modify the epigenetic state, as well as maternally
transcribed and translated transcription factors, are present in fertilized
eggs.
Recently, Takahashi and Yamanaka addressed this question. They reported
that the co-introduction of four transgenes encoding the transcription factors
Oct3/4, Sox2, c-Myc and Klf4 into somatic cells, such as embryonic and adult
tail-tip fibroblasts, resulted in the generation of induced pluripotent stem
(iPS) cells, which gave rise to chimeric embryos following their injection
into mouse blastocysts (Takahashi and
Yamanaka, 2006). The functions of Oct3/4, Sox2 and c-Myc have been
mentioned above. Klf4 is well known as an oncogene
(Rowland and Peeper, 2006),
but overexpression of Klf4 in mouse ES cells reduces the
differentiation ability of EBs (Li et al.,
2005). Klf4 can also bind to the proximal promoters of Oct3/4
target genes, such as Lefty1, and helps to activate Oct3/4 and Sox2
(Nakatake et al., 2006). These
four factors are thought to establish pluripotency in somatic cells as follows
(Fig. 6). First, c-Myc promotes
DNA replication, thereby relaxing chromatin structure, which allows Oct3/4 to
access its target genes. Sox2 and Klf4 also co-operate with Oct3/4 to activate
target genes that encode transcription factors which establish the pluripotent
transcription factor network and which, together with Oct3/4, Sox2 and Klf4,
result in the activation of the epigenetic processes that establish the
pluripotent epigenome. The iPS cells have a similar global gene expression
profile to that of mouse ES cells. Interestingly, Nanog is not
required exogenously to establish pluripotency in iPS cells and its endogenous
expression is not always activated in established pluripotent stem cells by
these four factors, supporting the hypothesis that the function of Nanog in
the maintenance of pluripotency is context dependent.
In iPS cells, the repressive histone marks in the promoter regions of Oct3/4 and Nanog are replaced by active marks, such as H3K4me and H4Ac, although DNA methylation is only partially erased. This suggests that Oct3/4, Sox2, c-Myc and Klf4 are indeed able to alter the epigenetic state of a cell and establish the pluripotent epigenome. This change should be mediated by enzymatic activities that erase the repressive histone marks (such as demethylases for H3K9 and H3K27) and generate active histone marks (such as H3K4 methyltransferase and histone acetyltransferase). Thus, to establish and maintain pluripotency, the genes encoding these enzymes would be activated by the pluripotent transcription factor network. Under such artificial conditions, the transcription factor network could orchestrate all the requirements for pluripotency.
Conclusion
Recent progress in understanding the establishment and maintenance of ES cell pluripotency has revealed the importance and functions of various key transcription factors. By contrast, although several features of the pluripotent epigenome have been discovered, their requirement for and involvement in the maintenance and establishment of pluripotency remain unclear. In the future, it will be necessary to confirm how genetic mechanisms determine the pluripotent epigenome and how the pluripotent epigenome functions to maintain the pluripotent transcription factor network.
ACKNOWLEDGMENTS
I thank Prof. Austin Smith (Cambridge University, UK), Dr Minoru Ko (NIH, USA), Drs Masaki Okano, Jun-ichi Nakayama and Teruhiko Wakayama (RIKEN CDB, Japan), Dr Haruhiko Koseki (RIKEN RCAI, Japan), Prof. Yo-ichi Shinkai (Kyoto University, Japan), Dr Kiyoe Ura (Osaka University, Japan) and Prof. Mitsuyoshi Nakao (Kumamoto University, Japan) for helpful discussion and suggestions. I also thank Dr Shigenobu Yonemura and Ms Naoko Ikue (RIKEN CDB, Japan) for help with the electron-microscopy data.
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