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© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
REVIEW
Chromatin features and the epigenetic regulation of pluripotency
states in ESCs
Wee-Wei Tee* and Danny Reinberg*
In pluripotent stem cells, the interplay between signaling cues, epigenetic
regulators and transcription factors orchestrates developmental
potency. Flexibility in gene expression control is imparted by
molecular changes to the nucleosomes, the building block of
chromatin. Here, we review the current understanding of the role of
chromatin as a plastic and integrative platform to direct gene
expression changes in pluripotent stem cells, giving rise to distinct
pluripotent states. We will further explore the concept of epigenetic
asymmetry, focusing primarily on histone stoichiometry and their
associated modifications, that is apparent at both the nucleosome and
chromosome-wide levels, and discuss the emerging importance of
these asymmetric chromatin configurations in diversifying epigenetic
states and their implications for cell fate control.
KEY WORDS: Nucleosomes, Epigenetic regulators, Chromatin,
Pluripotency
Introduction
Embryonic stem cells (ESCs) possess the remarkable abilities of
self-renewal and differentiation. They have the capacity to generate
differentiated cells comprising all three embryonic germ layers. This
pluripotent capacity makes them an excellent in vitro system with
which to study mammalian development (Gifford et al., 2013; Xie
et al., 2013) and disease modeling (Merkle and Eggan, 2013).
To fulfill this pluripotent capacity, ESCs have acquired a
sophisticated transcriptional circuitry, helmed by the triad of master
transcription factors, OCT4 (POU domain, class 5, transcription factor
1; POU5F1), SOX2 (SRY box 2) and NANOG, which operate in an
interconnected autoregulatory loop (Chambers and Tomlinson, 2009).
Additional transcription factors, signaling effectors and epigenetic
regulators converge on this ‘core pluripotency’ network, to stabilize the
self-renewing state through positive-feedback mechanisms (Young,
2011). Yet to retain multi-lineage differentiation potential, this
self-organizing gene circuitry must remain amenable to perturbation
(s) elicited by extrinsic stimuli, e.g. developmental signaling cues,
to bring about appropriate changes in gene expression programs.
This necessitates a suitably structured chromatin configuration, one
that is highly tractable and tailored for the physiological needs of
pluripotent cells.
In this Review, we will present an updated view of pluripotency
within the context of epigenetic regulation, placing particular emphasis
on the relationship between transcription factors, chromatin regulators
and signaling cascades in shaping pluripotency sub-states. We will
discuss the molecular attributes that underlie the dynamic ESC
chromatin landscape and how breaking the nucleosome symmetry
Howard Hughes Medical Institute, Department of Biochemistry and Molecular
Pharmacology, New York University School of Medicine, New York, NY 10016, USA.
*Authors for correspondence ([email protected];
[email protected])
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adds an additional layer of flexibility in gene regulation that may
contribute to the diversification of cellular states.
Distinct pluripotency states are influenced by extracellular
signaling and epigenetic regulators
Developmental specification is accompanied by a progressive loss
of cellular potential, and is marked by an increase in epigenetic
restriction (Gifford et al., 2013; Skora and Spradling, 2010). As
development commences, the zygote loses its totipotent capacity
and undergoes successive cleavages to give rise to the blastocyst,
which comprises three distinct lineages: the epiblast and primitive
endoderm cells, which are derived from the inner cell mass (ICM),
and the extra-embryonic trophectoderm cells (Rossant and Tam,
2009). The pluripotent epiblast cells in the ICM will give rise to all
somatic lineages of the embryo and the germline. Notably, this
pluripotent capacity persists only transiently for a few days in vivo;
however, when explanted in vitro, further development is halted
and pluripotency can be captured and propagated indefinitely in
the form of ESCs.
Mouse ESCs were first isolated from the ICM compartment of
blastocysts in 1981 (Evans and Kaufman, 1981), and are one of the
earliest and best-studied prototypes of pluripotent cells. The derivation
of human ESCs, also from the ICM of explanted blastocyst prior to
implantation, came almost 20 years later (Thomson et al., 1998).
Since then, different stem cells corresponding to distinct embryonic
precursors and pluripotency sub-states in vivo have been described.
They differ from the conventional ESC pluripotent state in their
developmental origins, transcription factor and signaling requirements,
as well as their epigenetic configurations (Table 1). For example, unlike
mouse ESCs, conventional human ESCs exhibit a pronounced
tendency for X-chromosome inactivation in female cells, and are
generally less amenable to genetic manipulation (Buecker and Geijsen,
2010; Hanna et al., 2010b). In this regard, it was postulated that
conventional human ESCs might bear a greater resemblance to
the mouse epiblast stem cells (EpiSCs), which originate from a
developmentally more advanced post-implantation epiblast that has
undergone X chromosome inactivation and cannot efficiently form
germline chimeras when injected into blastocysts (Brons et al., 2007;
Tesar et al., 2007). This diminished potency may be due to the
differences in transcriptional and chromatin constituency between
mouse EpiSCs and ESCs (Song et al., 2012).
The ability to derive stem cells of different molecular and phenotypic
characteristics at different developmental stages in the mouse embryo
has led to the idea that the pluripotent state is not invariant, but rather a
continuum of states that can be modulated by extrinsic signaling cues,
both in vivo and in vitro (Pera and Tam, 2010). Numerous studies have
delineated the signaling principles central to the establishment and
maintenance of these different pluripotency states (Ng and Surani,
2011). For example, the LIF (leukemia inhibitory factor)/STAT3
(signal transducer and activator of transcription 3) and FGF (fibroblast
growth factor)/ERK (extracellular signal-regulated kinase; MAPK1)
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Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
Table 1. Different states of pluripotency
Teratoma
formation
in mouse
Blastocyst
chimerism in
mouse
ExtraX chromosome status
embryonic
lineages
Mouse
Human
Oct4 distal
enhancer
DNA methylation,
imprinting and
Human H3K27me3
Cell line
Origin
ESC –
conventional
(mouse and
human)
Pre-implantation
epiblast
Yes
Yes (mouse) No
Inefficient
(human)
Both active in
females
Propensity for
inactivation
Active
Inactive Prominent occupancy
For a review, see
on lineage-specifying
Hanna et al., 2010b
genes
ESC –
groundstate
(mouse and
human)
Pre-implantation
epiblast
Yes
Yes
No
Both active in
females
Both active in
females
Active
Active
EpiSC (mouse)
Post-implantation Yes
epiblast
Inefficient
No
One inactivated
NA
Inactive NA
Prominent occupancy
on silenced genes
Brons et al., 2007;
Tesar et al., 2007
EG (mouse)
Primordial germ
cells
Yes
Yes
No
Both active in
females
NA
Active
NA
DNA hypomethylation
in groundstate
condition. Genomic
imprints remain
methylated.
Durcova-Hills et al.,
2006; Leitch et al.,
2010, 2013
Male GSC
(mouse)
Testis
Yes
Yes
No
NA
NA
ND
NA
Male-specific genomic
imprinting pattern
Kanatsu-Shinohara
et al., 2004
Hex+ ESC
(mouse)
ESC in 2i/LIF
Yes
Yes
Yes
ND
NA
ND
NA
ND
Morgani et al., 2013
2C-ESC
(mouse)
ESC in serum/LIF Yes
Yes
Yes
ND
NA
ND
NA
ND
Macfarlan et al., 2012
rESC (mouse)
EpiSC
Yes
reprogrammed
to naïve
pluripotency
Yes
No
Both active in
females
NA
Active
NA
Prominent occupancy
Bao et al., 2009;
on lineage-specifying
Gillich et al., 2012;
genes
Guo et al., 2009;
Hanna et al., 2009
iPSC (mouse
and human)
Somatic cells via Yes
reprogramming
Yes
No
Both active in
females
Both active in
females
Active
Active
Prominent occupancy
For a review, see
on lineage-specifying
Stadtfeld and
genes
Hochedlinger, 2010
Mouse
References
Reduced occupancy
Chan et al., 2013;
on lineage-specifying
Gafni et al., 2013;
genes
Marks et al., 2012;
Ying et al., 2008
Different states of pluripotency can be attributed to different tissues of origin as well as to perturbations in the extrinsic and intrinsic cellular environment. Different pluripotency sub-states
have varying developmental potential and epigenetic status. Conventional refers to serum- and feeder-containing culture, whereas ground state refers to serum- and feeder-free culture
supplemented with chemical inhibitors against specific signaling cascades.
2C-ESC, 2-cell like ESCs; EG, embryonic germ cells; EpiSC, epiblast stem cells; ESC, embryonic stem cells; GSC germline stem cells; Hex-ESC, ESCs expressing the extra-embryonic
marker Hex; iPSC, induced pluripotent stem cells; NA, not applicable; ND, not determined; rESC, reprogrammed epiblast ESC-like cells.
human ESCs were obtained independently of transgene expression,
via the use of various small-molecule inhibitors and cytokines
that include 2i/LIF, among others. Finally, this ‘2i’ inhibition strategy
has also proven to be highly effective in promoting nuclear
reprogramming of somatic cells into induced pluripotent stem cells
(iPSC), in part through conditioning the chromatin for widespread
remodeling (Rais et al., 2013; Silva et al., 2008).
Signaling pathways converge onto the chromatin
Chromatin is the basic regulatory unit of the eukaryotic genetic
material. It comprises repeating arrays of nucleosomes, each
consisting of 147 bp of DNA wrapped around a histone octamer
made up of two molecules of each histone: H2A, H2B, H3 and H4.
Histones are classified as either canonical or variant, depending on
their primary sequence and mode of deposition during the cell
cycle. They are subjected to various post-translational modifications
and demarcate different transcriptional domains in the genome
(Fig. 1). The different combinations in which these modifications
occur can significantly expand the regulatory properties of histone,
beyond that of a structural scaffold upon which the DNA is wrapped
(Kouzarides, 2007). This can occur in at least two ways: first, by
directly altering the biophysical properties of the nucleosome
particle through steric changes in histone-histone (within the same
or neighboring nucleosomes) and/or histone-DNA interactions; and
second, via the recruitment of effector proteins that directly
recognize the modifications (Taverna et al., 2007).
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pathways constitute opposing axes that govern the balance between
self-renewal and differentiation, respectively, in conventional mouse
ESCs (Niwa et al., 2009). However, FGF signaling is crucial for the
maintenance of mouse EpiSCs (Lanner and Rossant, 2010). Notably,
experimental manipulation of these signaling pathways can perturb
the equilibrium between these two pluripotent sub-states (Bao et al.,
2009; Chou et al., 2008). More recently, it has been demonstrated that
blocking WNT signaling in mouse ESCs likewise leads to
destabilization of the self-renewing state and promotes efficient
transition to EpiSCs (ten Berge et al., 2011). Collectively, these
studies highlight the surprisingly facile interconversion between
mouse ESCs and EpiSCs, and the power of extrinsic signals to
surmount epigenetic barriers. Of importance, pioneering work by
Smith and colleagues showed that dual inhibition (‘2i’) of the MEK
(mitogen activated protein kinase)-ERK and GSK3 (glycogen
synthase kinase 3β) signaling pathways in conventional serumcultured mouse ESCs reinforces their capacity for self-renewal,
culminating in the so-called pluripotent ‘groundstate’ (Ying et al.,
2008). This seminal work provided the foundation for subsequent
studies directed at deriving germline-competent ESCs from
previously recalcitrant models, such as the nonobese diabetic mouse
strain, as well as from the rat (Buehr et al., 2008; Hanna et al., 2009; Li
et al., 2008; Nichols et al., 2009). More recently, an equivalent human
groundstate-like naïve pluripotent stem cell has been described (Chan
et al., 2013; Gafni et al., 2013). Unlike previous instances (Buecker
et al., 2010; Hanna et al., 2010a; Wang et al., 2011b), these naïve
Fig. 1. See next page for legend.
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Fig. 1. Distinct transcriptional complexes and chromatin regulators mark
active pluripotent and repressed developmental genes in ESCs.
Epigenetic marks (colored hexagons, see key) are bestowed by specific
chromatin modifying proteins (colored ovals). Same color pairs denote an
interaction, e.g. the H3K4me3 mark (green star) is imposed by MLL2 and SET1
complexes (green ovals). (A) Hallmarks of an active transcriptional state
include the presence of H3K4me3, histone H3/H4 acetylations such as
H3K27ac (imposed by CBP/p300), and the assembly of RNA polymerase II
(RNAPII) and associated regulatory proteins at the core promoter. Serine 5
(Ser 5P) phosphorylation of RNAPII is associated with transcription initiation,
whereas serine 2 phosphorylation (Ser 2P) is associated with transcription
elongation (Adelman and Lis, 2012). Active enhancers are marked by
H3K27ac, H3K4me1, ELL3, RNAPII and enhancer-transcribed RNAs
(eRNAs), whereas cohesin and mediator complexes act to regulate enhancerpromoter looping (Calo and Wysocka, 2013). ELL3 facilitates the assembly of
SEC on the promoters of active genes for robust gene activation (Lin et al.,
2013). Histone variants H2A.Z and H3.3 are enriched at promoters and
enhancers, and the formation of a H2A.Z/H3.3 ‘hybrid’ nucleosome may
contribute to the dynamic nucleosome turnover at these sites. Other chromatin
remodeling complexes such as esBAF, TIP60, CHD1/7 (Young, 2011) and
PADI4 (Christophorou et al., 2014) are also involved in setting up the active
transcriptional state. (B) Developmental gene promoters are marked by the
presence of both H3K4me3 and H3K27me3. NuRD-mediated histone
deacetylations promote H3K27me3 deposition by PRC2, whereas SETDB1, a
H3K9 histone methyltransferase, further represses several developmental
genes in ESCs (Young, 2011). RNAPII-Ser5P is evident on these
developmental promoters, whereas RNAPII-Ser2P is not. Short non-coding
RNAs may be transcribed at the promoter-proximal regions, potentially leading
to the recruitment of PRC2 (Kanhere et al., 2010; Voigt et al., 2013). PRC1
reinforces gene repression through histone H2A ubiquitylation as well as via
other non-enzymatic mechanisms, including nucleosome compaction and
interference with the transcription process (Simon and Kingston, 2013).
Developmental gene enhancers are marked by the presence of H3K27me3
and H3K4me1, but not H3K27ac. Despite the presence of CBP/p300, these
enhancers are incapable of promoting gene activation in pluripotent cells, but
will do so during differentiation, upon the loss of enhancer H3K27me3 marks
(Rada-Iglesias et al., 2011). (C) Silent genes generally show reduced
nucleosome dynamics, meaning they are more ‘compact’, and show less
extensive transcriptional regulator binding. Silent genes are repressed by
chromatin regulators that methylate histone H3K9 and/or H3K27me3. Promoter
DNA sequences may also be methylated by DNA methyltransferases, DNMTs,
giving rise to 5-methyl-cytosine (5meC). CHD1/7, chromodomain helicase
DNA-binding protein 1/7; ELL3, elongation factor RNA polymerase II-like 3;
esBAF, ES cell-specific SWI/SNF-like ATP-dependent chromatin remodeling
complex; HAT, histone acetyltransferase; HMT, histone methyltransferase;
MLL, mixed-lineage leukemia; PAD14, peptidyl arginine deiminase type IV;
PRC1/2, polycomb repressive complex 1/2; SEC, super elongation complex;
SET1A/B, SET domain-containing 1A/B; SETDB1, SET domain, bifurcated 1;
TIP60, K(lysine) acetyltransferase 5; TFs, transcripton factors.
Given the instructive nature of signaling pathways in governing
cell state transitions, it is conceivable that signaling effectors must,
directly or indirectly, converge on chromatin, orchestrating changes
in chromatin structure and gene expression. Indeed, increasing
evidence now shows that signaling effectors can bind directly to
chromatin, leading to gene expression changes (Badeaux and Shi,
2013). For example, in mouse ESCs, the nuclear localization of
tyrosine kinases, JAK1 and JAK2 (Janus kinases 1 and 2), which
are involved in the LIF-STAT3 pathway, promotes phosphorylation
of tyrosine 41 of histone H3 (H3Y41), and interferes with
heterochromatin protein 1α (HP1α) binding. This direct signaling
to chromatin contributes to the expression of pluripotency genes
such as Nanog and Sox2 (Griffiths et al., 2011). Furthermore,
the β-catenin-TCF1/3 complex, a core component of the WNT
signaling pathway, as well as the mitogen-activated protein (MAP)
kinases, can also act directly on chromatin to regulate self-renewal
and differentiation (Cole et al., 2008; Goke et al., 2013; Tiwari et al.,
2012). In the case of the ‘2i-LIF’ mouse ESCs (Table 1), inhibition
Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
of ERK and GSK3 signaling pathways leads to global DNA
hypomethylation and a profound decrease in the repressive histone
modification mark H3K27me3, which is imposed by the polycomb
repressive complex 2 (PRC2) on developmental loci (Ficz et al.,
2013; Habibi et al., 2013; Leitch et al., 2013; Marks et al., 2012).
The loss of DNA methylation is mediated through the TET
enzymes, as well as the increased expression of PRDM14, which
represses the expression of both DNMT3A and DNMT3B DNA
methyltransferases (Grabole et al., 2013; Leitch et al., 2013; Yamaji
et al., 2013). Our own findings also indicate that in mouse ESCs,
ERK directly regulates PRC2 deposition and promotes the
establishment of the poised RNA polymerase II transcriptional
apparatus on developmental promoters (Tee et al., 2014). In
addition, the LIF/STAT3 pathway is also known to cooperate with
epigenetic regulators such as the SWI/SNF chromatin remodeler
complex esBAF to antagonize PRC2 deposition on active genes
(Ho et al., 2011). In this case, esBAF may cooperate with
Topoisomerase 2a to modulate chromatin accessibility, thereby
conditioning the genome for STAT3 binding (Dykhuizen et al.,
2013; Thakurela et al., 2013). Together, the parallel activation of
ERK and STAT3 pathways help to promote the distinctive
chromatin features inherent to the serum-primed pluripotent state.
Expanded states of pluripotency
The reduced level of DNA methylation and altered PRC2
occupancy in the ‘2i-LIF’ mouse ESC model may be indicative of
the epigenetic foundation of groundstate pluripotency in vitro.
These cells are also thought to parallel the early epiblast cells of the
ICM in vivo, wherein the DNA methylation level is lowest following
the development of the zygote to blastocyst (Smith et al., 2012),
concomitant with imprinted X chromosome reactivation in female
embryos (Mak et al., 2004; Okamoto et al., 2004). Interestingly,
a recent finding demonstrates that a small fraction of mouse ESCs
grown under this specific culture regime co-expresses markers of
both embryonic and extra-embryonic lineages (termed Hex+
ESCs), and are able to generate both trophectoderm and epiblast
lineages when reintroduced into embryos (Morgani et al., 2013).
These cells clearly exhibit an expanded cellular potency in a
chimeric setting, when compared with conventional ESCs, which
do not typically contribute towards the trophectoderm lineage.
The possibility that alleviation of particular restrictive epigenetic
modifications by ‘2i-LIF’ may provoke expansion of cellular
plasticity is consistent with the notion that lineage specification is
generally accompanied by stabilizing epigenetic configurations.
However, a second study provided a surprising twist. It was
observed that conventional mouse ESCs grown in the presence of
serum and LIF could transit through a previously undescribed stem
cell state that resembles that of the totipotent 2-cell (2C) blastomeres
in vivo (Macfarlan et al., 2012). The authors reported a striking
upregulation of retrotransposons, such as endogenous retroviruses
(ERVs), that accompanied the expression of 2C-specific proteincoding genes. Interestingly, many of the latter generated chimeric
transcripts linked to the retrotransposons, suggesting that they had
co-opted the regulatory elements of neighboring retroelements for
coordinated expression, a property also likely exploited in the
human ESC transcriptional circuitry (Kunarso et al., 2010). The
expression of ERVs continues to persist in a sub-population of
in vitro cultured ESCs, termed 2-cell like ESCs (2C-ESCs).
Although these 2C-ESCs are rare and transitory in nature, most, if
not all, of the ESCs cultured in vitro are capable of passing through
this peculiar state at some point. These 2C-ESCs have a
transcriptional and epigenetic profile similar to that of the
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totipotent embryonic cells and are lacking in OCT4, SOX2 and
NANOG. In this regard, they are molecularly distinct from the Hex
+ESCs. Contrary to expectation, addition of ‘2i’ is antagonistic to
the emergence of these 2C-ESCs, whereas conditions of high
oxygen promote entry into this privileged state. Importantly,
abrogation of repressive chromatin modifiers such as KAP1
(tripartite motif-containing 28; TRIM28), LSD1 (lysine (K)specific demethylase 1A; KDM1A) and G9a (euchromatic histone
lysine N-methyltransferase 2; EHMT2) were also able to promote
the derivation of these totipotent-like cells, suggesting that this
innate totipotent-like capacity may be epigenetically constrained in
conventional ESCs. Whether this constitutes a bona fide mechanism
to allow ESCs to drift into alternative pluripotent states, or whether
it is simply the result of a compromised epigenetic machinery
remains to be determined. In the case of the Hex+ and 2C-ESCs, this
event may provide a means to alleviate mutational loads acquired
during consecutive DNA replication and cell divisions, perhaps in a
manner analogous to the epigenetic reprogramming events that
occur in the mammalian germline (Hackett et al., 2012; Surani and
Tischler, 2012).
Oxygen and nutrients mediate epigenetic dynamics
It is increasingly evident that environmental conditions such as
oxygen tension and nutrient content can influence the metastability
and fidelity of stem cell states, in part through epigenetic alterations
(Lengner et al., 2010; Macfarlan et al., 2012; Mathieu et al., 2013).
The regulatory importance of oxygen availability in influencing
stem cell phenotypes, beyond that of cellular bioenergetics, may
perhaps be better appreciated when considering the bioavailability
of these elements and conditions with respect to the in vivo contexts
where stem cells reside. Stem and multipotent progenitor cells are
typically localized in highly specialized microenvironments in vivo,
which can either be hypoxic or well oxygenated, depending on the
proximity to blood vessels (Simon and Keith, 2008). Many studies
have uncovered a role for hypoxia in promoting the undifferentiated
progenitor state, through activation of different intracellular
signaling pathways, as well as the hypoxia-inducible transcription
factors (Cipolleschi et al., 1993; Ezashi et al., 2005; Yoshida et al.,
2009). The latter, for example, bind the Oct4 promoter to promote
an ‘open’ configuration (Covello et al., 2006). In this regard, the
atypical response of the 2C-ESCs to high oxygen content may strike
as an apparent oddity, highlighting a fundamental difference in
the prototypic ESC pluripotent state that may relate to their distinct
developmental origins.
Other studies have also established a role of vitamin C in
pluripotency and nuclear reprogramming. This antioxidant promotes
TET enzyme-mediated DNA demethylation and acts as a co-factor for
histone demethylases JHDM1A/1B in ESCs (Blaschke et al., 2013;
Chen et al., 2013b; Esteban et al., 2010; Stadtfeld et al., 2012; Wang
et al., 2011a). This finding is in line with the emerging importance of
metabolic regulation in ESCs (Shyh-Chang et al., 2013a). Of notable
mention is threonine dehydrogenase, an enzyme that is highly
expressed in pluripotent stem cells and modulates the generation of
S-adenosylmethionine, a metabolite used in protein methylation
reactions such as that used by histone-modifying enzymes for the
catalysis of histone methylation. Inhibition of this pathway leads to a
profound loss of histone H3K4 tri-methylation, and compromises
ESC pluripotency (Shyh-Chang et al., 2013b). In summary, these
studies show that extensive signal-to-chromatin-based mechanisms
direct developmental potency, and that differential epigenetic control
in stem cell populations underpins transcriptional fluctuation and
promotes cellular plasticity.
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Epigenetic regulation underlies transcriptional heterogeneity in stem
cells
Epigenetic regulators play a central role in controlling transcriptional
output and variability. These regulators include the chromatin
remodeling complexes, such as SWI/SNF and NuRD, that act to
regulate nucleosome spacing and dynamics, as well as histonemodifying complexes such as histone deacetylases (HDACs) and the
PRCs. Studies using different model organisms have shown that
many of these chromatin regulators are involved in regulating
transcriptional variability (Lehner et al., 2006; Raj et al., 2010). In
mice, haploinsufficiencies in Dnmt3a and Trim28 have been shown
to increase phenotypic variation in inbred littermates (Whitelaw et al.,
2010). In the case of serum-cultured ESCs, heterogeneous expression
of pluripotency factors is postulated to regulate phenotypic plasticity
(Chambers et al., 2007; Hayashi et al., 2008; Toyooka et al., 2008).
However, it is noteworthy that this phenomenon is less pronounced
in the ‘groundstate’ ESCs (Wray et al., 2011). At the molecular level,
heterogenous gene expression must be imparted by differential
modulation of chromatin architecture (Hayashi et al., 2008).
Although unrestrained transcriptional fluctuations are likely to be
deleterious to cell viability, it is conceivable that cells may benefit
from some degree of gene expression heterogeneity as a means to
increase phenotypic variance during lineage specification and for
survival fitness in general (Huang, 2009). In this regard, it is perhaps
of no coincidence that a similar repertoire of epigenetic regulators is
involved in the transition of pluripotent ESCs into the 2C totipotentlike state.
The equilibrium and frequency of sub-state switching enforced by
signaling pathways must invariably feed back into events that occur at
the level of the enhancer and promoter. For example, studies have
shown that the disassembly of promoter nucleosomes is rate limiting
for gene expression (Boeger et al., 2008), and that competition
between nucleosome and promoter proximal occupation of RNA
polymerase II can fine-tune gene expression (Gilchrist et al., 2010).
These and other studies indicate that the assembly of the
transcriptionally competent chromatin state is a highly dynamic
process that can be regulated on several fronts, thus affording various
opportunities for the fine-tuning of gene expression. In serumcultured ESCs, the NuRD complex appears to be especially important
for promoting transcriptional heterogeneity of pluripotency genes
and lineage commitment (Reynolds et al., 2012a). NuRD harbors
both chromatin remodeling and histone deacetylase activities,
mediated through different constituents in the complex (Zhang
et al., 1998, 1999). Situated at the heart of this complex is methylCpG binding protein 3 (MBD3), which serves as a scaffold for the
assembly of other components (Kaji et al., 2006, 2007). Notably, loss
of MBD3, and hence NuRD, function in mouse ESCs leads to
elevated expression of select pluripotency genes that impedes lineage
commitment (Reynolds et al., 2012a). Several of these NuRDrepressed pluripotency genes, e.g. Tbx3 (T-box 3), Klf4 (Kruppel-like
factor 4) and Foxd3 (forkhead box D3) are also targets of the PRCs
(Walker et al., 2010). Notably, in a separate study by Brookes et al.,
these genes are classified as ‘PRC2 active’, based on their apparent
active transcription yet paradoxical presence of PRCs on their
promoters in the context of a heterogenous ESC population (Brookes
et al., 2012). Further analysis revealed an independent association of
PRCs with the elongative form of RNA polymerase II on these genes
(Fig. 1), suggesting that these two components may exist separately
within a cell (i.e. on different alleles) or in distinct cell populations. It
was also postulated that the heterogenous expression of key
pluripotency genes, such as Nanog might impact on this dynamic
(Brookes et al., 2012).
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The ESC epigenetic landscape
Genome-wide charting of DNA and histone modifying enzymes,
along with their associated modifications in stem cells and
differentiating tissues have given rise to chromatin-modification
‘maps’, which may be used to identify DNA elements of regulatory
importance (Gifford et al., 2013; Stergachis et al., 2013; Xie et al.,
2013; Zhu et al., 2013). This wealth of epigenetic information on the
chromatin is further organized spatially, in a three-dimensional
manner in relation to the nuclear structure (Jin et al., 2013; PhillipsCremins et al., 2013). Several epigenetic features have been
extensively described in ESCs (Young, 2011) and some of these
are highlighted in Fig. 1.
Bivalent chromatin domains
The preponderance of the bivalent H3K4me3-H3K27me3 chromatin
domain is arguably one of the more distinctive epigenetic
characteristics described in ESCs and has received considerable
attention (Azuara et al., 2006; Bernstein et al., 2006). Bivalent
domains are historically described as chromatin stretches comprising
oligonucleosomes that harbor both the activating H3K4me3 and the
repressive H3K27me3 marks. At the time of the discovery, no
distinction was made as to whether the marks co-exist on a single
nucleosome particle or on different nucleosomes. In addition, it is
important to note that bivalent domains are not exclusive to stem
cells as they also occur in differentiated tissues, albeit at lower
frequency. While the factors involved in the establishment of this
bivalent state are well defined, the exact function of this chromatin
configuration remains debatable, in part due to a lack of an
appropriate genetic experimental model (reviewed by Voigt et al.,
2013). Towards this end, two groups have recently identified mixedlineage leukemia 2 (MLL2) as the enzyme that directs H3K4me3
deposition on bivalent promoters (Denissov et al., 2014; Hu et al.,
2013a). Whereas loss of MLL2 abolished H3K4me3 specifically on
bivalent promoters, it did not significantly alter the transcriptional
kinetics of these genes upon retinoic acid treatment. This calls into
question the purported role of bivalency in priming gene expression
(Box 1), and also highlights a redundant mechanism involving other
H3K4 methyltransferases, such as MLL1. It was also proposed that
MLL2 engages unmethylated CpG islands and functions primarily
as a pioneer H3K4 methyltransferase to mark bivalent promoters,
with the presence of PRCs somehow blocking the engagement of
SET1 H3K4 methyltransferases known to deposit H3K4me3 at
active genes (Denissov et al., 2014). However, it will be important to
further examine the gene expression kinetics of Mll2-null as well as
Mll1/2 double-knockout ESCs in other physiological settings that
are representative of development in vivo, e.g. in the context of germ
layer differentiation. This is pertinent given that both Mll2 and PRC2
knockout mice are embryonic lethal, with PRC2 mutants showing
defects during gastrulation (reviewed by Voigt et al., 2013), and that
Mll2-null embryoid bodies are defective in select HoxB gene
inductions (Glaser et al., 2006).
Bivalent domains have also been detected in mammalian gametes
(Ng et al., 2013; Sachs et al., 2013), although it remains unclear whether
the marks co-exist on the same nucleosome. Nevertheless, the retention
of residual H3K4me3-K27me3-marked nucleosomes in the sperm
genome and the observation that genes marked by higher levels of
Box 1. Are bivalently marked asymmetric
nucleosome functionally relevant?
The existence of asymmetrically modified nucleosomes may afford
greater flexibility in gene expression regulation. Although inherently
attractive, this hypothesis still requires rigorous testing on several fronts.
Some of these key questions listed below may be addressed using either
in vitro reconstituted transcription assays or genome-editing tools to
engineer a ‘reporter’ for bivalent promoters in vivo. Examples of the latter
include the recently described chromatin in vivo assay (CiA), a powerful
tool that allows for the specific perturbation of histone modification
patterns in a locus-specific manner (Hathaway et al., 2012), as well as
the use of programmable transcription activator-like effector (TALE)
repeat fusions (Maeder et al., 2013; Mendenhall et al., 2013) and/or
CRISPR-mediated editing (Chen et al., 2013a; Cong et al., 2013).
Key questions
How do the kinetics of demethylases differ for (a)symmetrically
modified nucleosomes?
What is the impact of an (a)symmetric configuration on nucleosome
turnover/stability?
Does the spatial inequality of post-translational modifications affect
the binding affinities of effector proteins that can potentially recognize
combinations of post-translational modifications?
Does the distribution and composition of (a)symmetrically modified
nucleosomes change in response to developmental stimuli and/or cell
cycle progression, as in the case of heterotypic H2A-H2A.Z
nucleosomes?
Are (a)symmetrically modified nucleosomes differentially distributed
on regulatory elements, e.g. promoters versus enhancers, and does
this exert a differential impact on gene expression priming?
Does perturbing ‘bivalency’ in vivo affect the kinetics and inheritance
of gene expression patterns during development?
•
•
•
•
•
•
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Mechanistically, NuRD-mediated deacetylation of H3K27 may
facilitate the recruitment of PRC2 to repressed lineage genes,
including these ‘PRC2-active’ genes (Brookes et al., 2012;
Reynolds et al., 2012b). In this instance, it will also be of interest
to assess the impact of NuRD on the global nucleosome occupancy
in ESCs, particularly at enhancers and promoters where NuRD
resides (Reynolds et al., 2012b; Whyte et al., 2012; Yildirim et al.,
2011). This may reveal further insights into how the interplay
between nucleosome remodeling and RNA polymerase II pausing
(Gilchrist et al., 2010) may contribute to the dynamic gene
expression regulation in ESCs (Henikoff, 2008).
The aforementioned group of NuRD-PRC2 co-target genes also
make up part of the extended network of pluripotent transcription
factors that function to stabilize the core OCT4, NANOG and SOX2
regulatory circuitry (Walker et al., 2011). Thus, an important role of
NuRD in ESCs may be to modulate expression of these ‘extended’
pluripotency genes, thereby destabilizing the pluripotency network
and lowering the threshold required for cells to respond to
differentiation cues and exit the self-renewing state. Consistent with
this, depletion of MBD3 during iPSC generation yields unprecedented
reprogramming efficiency, effectively eliminating stochastic effects in
this otherwise inefficient process (Rais et al., 2013); however this effect
may be context-dependent (dos Santos et al., 2014). In addition, UTX
[lysine (K)-specific demethylase 6A; KDM6A], a histone H3K27me3
demethylase, is required during iPSC generation to remove trimethyl
marks from H3K27me3 in the case of early expressed pluripotency
genes (Mansour et al., 2012). Notably, the loss of PRC2 and LIN53, a
component of the NuRD complex, is an absolute pre-requisite for
triggering germ to somatic cell conversion in C. elegans (Patel et al.,
2012; Tursun et al., 2011), which collectively points to an important
role of these transcriptional repressors in safeguarding lineage fidelity.
However, it is interesting to note that the loss of PRC2 apparently
impedes iPSC generation even when using the optimized homogenous
‘secondary’ reprogramming cells (Buganim et al., 2012; Onder et al.,
2012; Pereira et al., 2010), suggesting that loss of PRC2 may result in
additional deleterious effects during nuclear reprogramming.
Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
H3K27me3 at their promoters are more likely to stay repressed during
early embryogenesis (Brykczynska et al., 2010; Erkek et al., 2013;
Hammoud et al., 2009) may argue for a potential role of bivalency
in transgenerational epigenetic inheritance and for development in
general. However, further in vivo functional assessment is necessary to
render support for or to debunk this hypothesis (Box 1).
Chromatin organization in pluripotent cells
In ESCs, the bivalent chromatin domains are embedded in a wider
context of a highly dynamic and accessible chromatin landscape,
which is postulated to constrain PRC2 occupancy and H3K27me3
spreading (Zhu et al., 2013). Indeed, it is well established that
pluripotent cells have a more dynamic chromatin structure compared
with their differentiated counterparts (Gaspar-Maia et al., 2011). For
example, undifferentiated ESCs contain a lower abundance of
constitutive heterochromatin, marked by fewer DAPI-dense foci
(Efroni et al., 2008). Pluripotent chromatin is also marked by
strategic deposition of histone variants at key regulatory elements, as
well as a dynamic binding of structural chromatin proteins such as
HP1 (Meshorer et al., 2006). The regulated displacement of linker
histones H1 at active regulatory regions also contributes towards
chromatin decondensation (Christophorou et al., 2014). These
chromatin features are likely to be important for attaining the
transcriptionally pervasive pluripotent state (Efroni et al., 2008).
Notably, some of these chromatin features are also observed in
pluripotent cells in vivo, such as the ICM (Ahmed et al., 2010), and
are recapitulated during nuclear reprogramming (Fussner et al., 2011;
Mattout et al., 2011).
The plasticity of ESCs is often attributed to hyperdynamic
chromatin features. However, whether permissive chromatin
structure is instructive for pluripotency or merely a consequence
thereof, remains an open question. Emerging evidence suggests it is
likely a combination of both. For example, the extent of
heterochromatin compaction in ESCs and ICM cells inversely
correlates with NANOG expression (Fussner et al., 2011). More
importantly, loss of OCT4 is sufficient to trigger ectopic formation of
silenced heterochromatin domains in the ICM cells (Ahmed et al.,
2010). Using OCT4 as a further example, an additional study showed
that the differential kinetics of OCT4 binding in the early mouse
embryo might contribute toward lineage allocation, and it was
postulated that this differential binding might be regulated by the state
of chromatin accessibility (Plachta et al., 2011). This study thus
implies a role for local chromatin structure in regulating transcription
factor occupancy. Interestingly, a reduced level of OCT4 in mouse
ESCs apparently results in enhanced self-renewal, comparable with
that observed in ‘2i-LIF’ (Karwacki-Neisius et al., 2013). Somewhat
counter-intuitively, this overall reduction in OCT4 level is
accompanied by an increase in OCT4 and NANOG occupancies at
pluripotency-associated enhancers. This phenomenon reflects either
an active redistribution of OCT4 to key regulatory sites and/or an
inherent tighter, or additive association of OCT4 at these specific
genomic regions. On a speculative note, it is plausible that certain
chromatin features may underlie these ‘attractor’ sites, and in the case
of the early embryos, may contribute in part to the differential kinetics
of OCT4-chromatin binding (Plachta et al., 2011). One such
chromatin feature may be the presence of architectural proteins such
as cohesin. It is well documented that transcription factor binding
often occurs in a clustered fashion that is orchestrated by the cohesin
complex. Depletion of cohesin results in reduced DNA accessibility
and transcription factor binding, indicating a causative role for cohesin
in promoting the formation of these transcription factor clusters (Yan
et al., 2013). Cohesin may thus help to confer robustness in gene
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expression by helping to stabilize highly occupied cis-regulatory
modules (Faure et al., 2012) (Fig. 1). This is consistent with the
findings in ESCs that pluripotency factors such as OCT4, NANOG
and KLF4 interact and cooperate with cohesin complex in a highly
coordinated fashion to access and regulate expression of target loci
(Apostolou et al., 2013; Wei et al., 2013). As noted previously, it will
be important to assess transcription factor binding occupancy in the
context of local chromatin features, and how global changes in
nucleosome dynamics correlate with transcription heterogeneity and
developmental specification (Li et al., 2012; Teif et al., 2012).
The binary relationship between transcription factor binding and
open chromatin may be overly simplistic. Recent evidence suggests
that OCT4 itself can function as pioneer factor, readily accessing
regions of closed chromatin structure (Soufi et al., 2012). In fact this
property is not merely restricted to pluripotency factors, but also
exhibited by numerous lineage-specific regulators (Zaret and Carroll,
2011). Although pioneer factors can bind to nucleosomal DNA,
additional determinants are likely in place to direct target specificity in
a context-dependent manner. A recent study aimed at understanding
the role of ASCL1 in somatic cell-neuron transdifferentiation
identified a ‘trivalent’ chromatin feature that may help guide this
pioneer factor to target regions (Wapinski et al., 2013). Notably, this
trivalent signature, comprising H3K9me3, H3K4me1 and H3K27ac
putatively within a single nucleosome, is present only in cell types that
are amenable to neuronal transdifferentiation, perhaps serving as a
means to identify other recipient cells that are permissive for neural
reprogramming. This work raises the exciting possibility that certain
chromatin features can instruct transcription factor-target specificity,
paving the way for future research to assess the universality of such a
‘chromatin-guided’ mechanism.
Breaking nucleosome symmetry and diversifying epigenetic
regulation
Role of histone variants
Given the importance of nucleosome dynamics in gene regulation, what
then are the molecular attributes that affect nucleosome turnover and/or
remodeling? Among many factors, histone variants may be particularly
well poised to fulfill such a role. Unlike canonical histones such as H2A
and H3.1/2, which are primarily expressed during S phase and
deposited in a strict DNA replication-dependent manner, histone
variants such as H2A.Z and H3.3 are expressed and deposited
throughout the cell cycle, uncoupled from DNA synthesis (Campos and
Reinberg, 2009). Histone variants are also subjected to different posttranslational modifications and as such could be selectively deployed to
alter local epigenetic configuration through replacement of canonical
histones at any point during the cell cycle (Hake et al., 2006; Loyola
et al., 2006; McKittrick et al., 2004). When incorporated, histone
variants can alter the overall stability of the nucleosome particle,
regulating nucleosome turnover at key regulatory elements in the
genome (Bonisch and Hake, 2012; Chen et al., 2013c; Henikoff, 2008;
Jin and Felsenfeld, 2007). Some histone variants have also evolved
highly specialized functions, whereas others are expressed in a tissuespecific manner (Talbert and Henikoff, 2010). Interestingly, in certain
cases the regulatory function of histone variants can be compensated by
upregulation of other related histone isoforms, indicative of functional
redundancy (Lin et al., 2000; Montellier et al., 2013). In fact, a recent
report suggested that canonical histones bearing particular posttranslational modification combinations could compensate for the
lack of a particular histone variant, highlighting remarkable plasticity in
the process (Montellier et al., 2013). In this case, depletion of a testisspecific H2B variant, TH2B (histone cluster 1, H2ba; Hist1h2ba),
during spermatogenesis induced compensatory mechanisms via the
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upregulation of canonical H2B, as well as via the additional installation
of specific post-translational modifications on histones H3 and H4, to
drive the completion of histone-protamine exchange. It is likely that
such crosstalk between histone genes is necessary to ensure robust
completion of key biological processes and therefore is presumably
employed in other developmental settings. Along this line, a similar
mechanism may also operate in mouse ESCs depleted of H3.3. Notably,
despite the prevalent deposition of histone H3.3 at key regulatory and
genic sites, only modest transcriptional abnormalities were observed in
H3.3-depleted mutant ESCs that successfully differentiated into all
three germ layers during teratoma formation (Banaszynski et al., 2013).
This highlights the possibility that, as in the case of TH2B, canonical H3
(or post-translational modification changes on other histones) might
rescue, in part, the lack of H3.3 at affected loci (Banaszynski et al.,
2013; Goldberg et al., 2010).
The engagement of histone variants significantly expands the
flexibility of epigenetic control in maintaining pluripotency as well
as during development. Consistent with the crucial importance of
histone variants in modulating gene expression is their frequent
presence at regulatory elements in the genome, coinciding with
promoters and enhancers, many of which are also ‘hotspots’ of
transcription factor binding. For example in mouse ESCs, H2A.Z
occupies both active and inactive regions, including distal
regulatory elements (Hu et al., 2013b; Ku et al., 2012). Notably,
H2A.Z depletion leads to reduced chromatin accessibility,
concomitant with impaired MLL, PRC2 and OCT4 targeting (Hu
et al., 2013b). This has led to the view that H2A.Z may act in a
general manner to modulate local chromatin accessibility, thereby
facilitating efficient targeting of transcriptional activating or
repressing complexes to their respective sites in the genome. As
mentioned previously, H3.3 also maps to both active and inactive
regulatory elements, although, unlike H2A.Z, the loss of H3.3
deposition specifically impacts targeting of the polycomb, but not
that of the trithorax machinery (Banaszynski et al., 2013).
Regions enriched for H2A.Z/H3.3 often coincide with regions of
nucleosome depletion (Chen et al., 2013c; He et al., 2010), indicative
of active nucleosome exchange and/or remodeling (Fig. 1). The
observation that H2A.Z, when in complex with H3.3, constitutes a
highly unstable nucleosome in vivo is relevant here, highlighting that a
key regulatory role for H2A.Z might be to amplify the intrinsic
instability of H3.3-containing nucleosomes (Jin and Felsenfeld, 2007)
(see Fig. 3). This ‘hybrid’ configuration, as initially referred to by the
authors to draw attention to the co-existence of two distinct histone
variants in the same nucleosome particle, maps to nucleosome-free
regions of active promoters and other regulatory regions (Jin et al.,
2009). Although these studies were performed in somatic cells, the
findings very likely also pertain to ESCs, given that both cell types
share common features with respect to genomic regions co-occupied
by H2A.Z and H3.3.
Histone variants and tetramer splitting
In addition to promoting nucleosome turnover, another interesting
feature of H3.3-containing nucleosomes is that a small but significant
proportion of them undergo a higher incidence of (H3.3-H4)2 tetramer
splitting in vivo compared with H3.1/2-containing nucleosomes that
are highly stable (Fig. 2). In yeast, where the single H3 isoform is
homologous to H3.3, (H3-H4)2 tetramer splitting can be observed on
transcriptionally active loci (Katan-Khaykovich and Struhl, 2011).
The same is observed in mammalian cells, although the frequency of
(H3.3-H4)2 splitting appears to correlate more with enhancer
specificity and potentially during DNA replication (Huang et al.,
2013; Xu et al., 2010). Considering that ESCs have a significantly
Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
Fig. 2. Histone variant and nucleosome asymmetry. (A) Canonical H3.1/2containing nucleosomes do not undergo (H3-H4)2 tetramer splitting and hence
are inherited in a conservative manner during DNA replication. This involves
random segregation of intact parental histone octamers to the leading and
lagging strands, coupled with deposition of newly synthesized nucleosomes.
In this model, parental nucleosomes provide the template for chromatinmodifying enzymes to reconstitute full post-translation modification patterns
(marked as ‘stars’) on neighboring ‘new’ nucleosomes. (B) A small proportion
of mononucleosomes containing the histone variant H3.3 can undergo a high
incidence of nucleosome turnover and tetramer splitting in vivo. These events
likely occur on transcriptionally active promoters and enhancers, potentially
coupling nucleosome dynamics with transcription factor occupancy. In theory,
for homotypic H3.3 nucleosomes, the symmetric partitioning of equivalent
tetramers constitutes a semi-conservative mode of mitotic epigenetic
inheritance. In this model, the parental tetramer, rather than the whole
nucleosome, provides a template for chromatin modifying enzymes to
reconstitute post-translational modification patterns on the newly deposited
tetramers. This potentially provides a means for robust propagation of active
transcriptional states of certain key regulatory loci following mitosis. The
canonical H3 histone and the H3.3 histone variant can harbor different
post-translational modifications (Hake et al., 2006; Loyola et al., 2006;
McKittrick et al., 2004). Red, H2B; green, H4.
larger number of epigenetically marked enhancers compared with
differentiated lineages (Stergachis et al., 2013; Whyte et al., 2013), it
will be interesting to examine the frequency and specificity of (H3.3H4)2 tetramer splitting as a function of differentiation, and whether
pluripotency and/or lineage-specific transcription factors, either acting
in isolation or in collaboration with histone chaperones, may play a
role in orchestrating this process. Such analysis is important given the
realization that it is the chromatin state of enhancers, rather than that of
promoters, that better reflects cell type-specific activities (Heintzman
et al., 2009; Hnisz et al., 2013; Rada-Iglesias et al., 2011; Visel et al.,
2009). Additionally, many studies have now shown that epigenetic
patterning of enhancers occurs prior to cell fate specification, and that
this enhancer-priming event permits differential access of signaling
molecules and transcription factors that drive cell type-specific gene
expression programs (Bergsland et al., 2011; Liber et al., 2010; Mullen
et al., 2011; Trompouki et al., 2011). The finding that a given
transcription factor binds first to the enhancer before potentiating
epigenetic changes at the promoter during reprogramming, further
attests to the instructive nature of enhancer marking in directing cell
fate changes (Taberlay et al., 2011). Therefore, understanding the
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Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
Fig. 3. Different permutations of histone stoichiometry
within the 8-histone core of the nucleosome. A homotypic
arrangement refers to the presence of identical histone pairs
for each sister histone pair. A heterotypic arrangement occurs
when one sister histone pair is not identically matched, and
therefore contains both canonical and variant histones. For
simplicity, only select H2A-H2A.Z and H3-H3.3 combinations
are shown, although pairings involving other histone variants
are possible. Theoretically, different homo/heterotypic
nucleosomal states can either progress through a step-wise
cyclical fashion (black arrows) or via direct conversion (red
arrows). Red, H2B; green, H4.
Nucleosome stoichiometry: homotypic versus heterotypic
The aforementioned studies demonstrating the impact of nucleosome
composition in gene expression are an appropriate reminder of the
fundamental importance of nucleosome stoichiometry in gene
regulation, a feature perhaps overshadowed by the overwhelming
attention on histone tail post-translational modifications. A
prevailing view is that the octameric nucleosome particle is
symmetrical, comprising identical pairs of each of the four core
histone proteins. This type of nucleosome composition is referred to
as homotypic. However, the observation that the nucleosome core
particle can actually accommodate a non-identical H2A-H2A.Z
pair, a composition referred to as heterotypic, has challenged this
view (Fig. 3). In the case of H2A-H2A.Z heterotypic nucleosomes,
although initial structural studies did not support the feasibility of
such a conformation (Suto et al., 2000), their presence has been
detected in vivo, in organisms spanning from yeast, fly and mouse to
human (Luk et al., 2010; Nekrasov et al., 2012; Viens et al., 2006;
Weber et al., 2010). Importantly, as shown in mouse trophoblast
stem cells, the composition of the H2A.Z-H2A nucleosome particle
changes dynamically throughout the cell cycle, with heterotypic
and homotypic nucleosomes localizing to different regions of
the genome and assuming different functions (Nekrasov et al.,
2012). Thus, heterotypic nucleosomes represent one spectrum of
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nucleosome asymmetry wherein the two copies of sister histones are
clearly non-equivalent. It is conceivable that other histone proteins,
in particular H3-H3.3, may be similarly engaged given their overall
similarity in amino acid composition and identical interaction
surface within the tetramer (Luger et al., 1997) (Fig. 3). Importantly,
the presence of a mixture of homotypic and heterotypic H3
nucleosome populations can potentially influence the choice
between two different modes of nucleosome deposition (Ahmad
and Henikoff, 2002) and how active transcriptional states, e.g.
primed enhancers, are epigenetically inherited. Understanding
the precise mechanisms of how histone chaperones regulate the
deposition and/or exchange of histones may reveal further insights
into how the different permutations of histone stoichiometry are
established (Fig. 3). Given our previous discussion on the regulatory
roles of ‘hybrid’ H2A.Z/H3.3 nucleosomes, it will be interesting to
examine the full histone stoichiometry in this case, to assess whether
sister histones are homotypic or heterotypic. If different ‘hybridheterotypic’ permutations were possible, then this would greatly
expand the number of epigenetic conformations, in addition to
the epigenetic diversity made possible by various combinations of
post-translational modifications (Fig. 3).
Asymmetrically modified mononucleosomes
Theoretically, histone variant-imposed nucleosomal asymmetry can
be further amplified by the presence of non-identical post-translational
modifications carried by each sister histone (Fig. 4). Indeed, as shown
by Voigt et al., asymmetrically modified nucleosomes exist in vivo,
in both pluripotent and differentiated cells (Voigt et al., 2012).
By using an affinity-purification-based mass spectrometry (MS)
methodology, the authors were able to assess in a quantitative
manner the distribution of post-translational modifications on H3
DEVELOPMENT
dynamics of tetramer splitting and identifying the players that regulate
this process will reveal novel insights into how enhancers participate in
cell fate specification. Indeed, if (H3.3-H4)2 tetramer splitting is
restricted to active enhancers during DNA replication, it may provide a
semi-conservative means for active propagation of these instructive
regulatory elements through cell divisions, an exciting possibility that
warrants further testing.
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Development (2014) 141, 2376-2390 doi:10.1242/dev.096982
Fig. 4. An asymmetrically modified mononucleosome may function as an additional epigenetic regulatory layer. Asymmetric, but not symmetric,
presentation of H3K4me3 permits methylation of H3K27 (and vice versa) on the opposite sister histone. (A) An asymmetrically modified nucleosome. In this
example, H3K4me3 (green star) and H3K27me3 (red star) modifications reside on opposing sister H3 histones in a mutually exclusive fashion. This constitutes
the bivalently marked mononucleosome that demarcates developmental gene promoters and/or poised enhancers in vivo. This asymmetric configuration can
impact on the kinetics of PRC2 and MLL/SET1 complexes, as evident by the reciprocal cis-inhibition of H3K27 and H3K4 methylations. (B) Symmetrically
modified nucleosomes. Prior deposition of either one of the two modifications on both sister H3 histones will antagonize the placement of the other mark on the
same histone tail. Canonical H3.1/2 histones are depicted in blue and H3.3 variants in purple. The latter is known to be preferentially enriched for active
modifications such as H3K4me3, and is depicted as such. MLL, mixed-lineage leukemia; PRC, polycomb repressive complex 2; SET1, SET domain-containing 1.
binding of PRC2 as being potentially promiscuous in the genome
(Kaneko et al., 2013), whereas occupancy of H3K27me3 is largely
restricted to repressed developmental genes and repetitive elements
in ESCs. In summary, the study by Voigt et al. demonstrates that
sister nucleosomes can be asymmetrically modified. How this may
be integrated with histone variant imposed ‘asymmetry’ to regulate
gene expression in vivo remains to be fully elucidated.
Epigenetic asymmetry: moving beyond the mononucleosome
ESCs largely divide in a symmetrical fashion with equal partitioning
of genetic and epigenetic materials, giving rise to functionally
equivalent daughter cells. However, a recent report documents that
altering the cell polarity in ESCs can perturb this symmetry (Habib
et al., 2013). When subjecting mouse ESCs to localized WNT3A
signaling, the authors observed that the ESCs underwent asymmetric
division instead. Remarkably, daughter cells proximal to the source
of WNT3A had higher expression levels of pluripotency genes,
whereas distal cells acquired differentiation hallmarks. This is
reminiscent of adult stem cells in vivo that mostly undergo
asymmetric cell divisions to generate a self-renewed progenitor and
a daughter cell destined for differentiation (Clevers, 2005). A central
question relating to this process is how is the parental epigenetic
information maintained in the progenitor stem cell, but altered in the
differentiating cell? Studies using Drosophila male germline stem
cells (GSCs) as a model system have revealed some fascinating
insights into this process. Male GSCs undergo asymmetric cell
division, giving rise to a self-renewed GSC and a daughter cell
gonialblast that undergoes differentiation. By using a dual-color
labeling strategy to distinguish between ‘old’ and ‘new’ histones,
Tran et al. discovered that pre-existing old canonical H3, but not
H3.3, histones are specifically retained in the GSCs, whereas newly
synthesized H3 histones are enriched in the differentiating gonialblast
cells. Notably, this asymmetric mode of H3 distribution is lost when
GSCs are experimentally manipulated to divide symmetrically (Tran
et al., 2012). Mechanistically, this asymmetric distribution of
histones can occur if (1) pre-existing H3 and newly synthesized H3
are already differentially localized onto the two sets of sister
chromatids during S phase prior to mitosis, and (2) the mitotic
machinery can somehow distinguish between these epigenetically
distinct sister chromatids. It is unclear at this moment how the
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and H4 sister histones within a mononucleosome. The study
revealed that a significant population of mononucleosomes harbors
H3K4me3 exclusively on one sister H3 histone, and H3K27me3 on
the other. This may represent the strictest embodiment of chromatin
‘bivalency’, as both marks are present not only on the same stretch
of chromatin, but on the very same mononucleosome. Importantly,
this observation corroborates the inability to detect H3K4me3 and
H3K27me3 on the same histone tail peptide at a population level
using MS approaches (Voigt et al., 2012; Young et al., 2009).
The presence of asymmetrically marked H3K4me3-H3K27me3
mononucleosomes can further inform us of the modus operandi of
the trithorax and PRC2 machineries in vivo. As Voigt and
colleagues further show, the activity of PRC2 is inhibited by the
presence of active modifications (Voigt et al., 2012). For example,
pre-installation of H3K4me3 on both sister histones impedes PRC2
methylation of H3K27, whereas asymmetric presentation of
H3K4me3 is ineffectual (Voigt et al., 2012). This is consistent
with previous studies in which it was found that H3K36me3,
another active marker that is associated with transcription
elongation, inhibits PRC2 activity (Schmitges et al., 2011; Yuan
et al., 2011). The inverse relationship has also been observed
whereby the presence of H3K27me3 impacts the activity of SET1
H3K4 methyltransferases (Kim et al., 2013) (Fig. 4). These findings
thus provide a molecular insight into how PRC2 and trithorax
complexes function in ESCs. We and others have recently proposed
a regulatory logic for how PRC2 may be targeted to various sites in
the genome, in a manner that is dependent on transcriptional
activity (Brookes et al., 2012; Davidovich et al., 2013; Kaneko
et al., 2013; Tee et al., 2014). We envisage a hierarchical series
of events whereby PRC2 constantly scans the genome for
transcriptional activity, directly assessing the chromatin and
transcriptional configurations at promoters, prior to engagement
and H3K27 methylation. In this case, even though PRC2 may
readily gain access to the promoter regions of highly transcribed
loci, perhaps through contact with nascent RNA, the symmetric
presence of either H3K4me3 and/or H3K36me3 will curtail PRC2mediated H3K27 methylation. Indeed, in vitro experiments suggest
that the presence of symmetric active modifications does not affect
PRC2 binding to the nucleosomes, but rather regulates its activity
(Voigt et al., 2012). This interpretation accounts for the observed
REVIEW
Concluding remarks
In this Review, we have discussed the emerging importance of
epigenetic regulators in governing lineage fidelity and explored how
the manipulation of chromatin factors can perturb the equilibrium
between discrete pluripotent states. The increasing ease in genome
editing and the means to engineer lineage-specific reporters in human
ESCs in a high-throughput fashion have provided exciting inroads into
our understanding of how epigenetic barriers are established during
distinct phases of mammalian development, and how they are
overcome during nuclear reprogramming (Cong et al., 2013; Poser
et al., 2008; Wang et al., 2013). Mapping of histone post-translational
modifications as a function of lineage specification has proven to be
extremely informative, and has been the primary method of choice to
assess genome-wide chromatin states. However, most studies
performed to date do not provide information about the exact
histone composition within the nucleosomes or distinguish between
modifications on sister histones.
The view that a nucleosome is symmetrical must be revised in light
of observations demonstrating the functional differences between
heterotypic and homotypic nucleosomes, and how asymmetric
presentation of post-translational modifications on sister histones
can impact the kinetics of histone-modifying enzymes. Clearly, the
extent to which these nonequivalent nucleosomal states exist in vivo
remains to be investigated, but they may indeed be the norm rather than
the exception. The continual discovery of new histone variants,
e.g. through alternative splicing, adds to the growing list of possible
nucleosome conformations (Bonisch et al., 2012; Rasmussen et al.,
1999). Last but not least, an additional layer of complexity that
warrants attention is the finding that histone modifications, as well as
nucleosomes, can often be asymmetrically positioned around discrete
transcription factor binding sites, with skewed enrichment on one side
2386
of the binding site, but not on the opposite end (Kundaje et al., 2012).
It is unclear how this directionality impacts on gene regulation, but a
clue to this may be gleaned from the recent description that a class of
pioneer transcription factors apparently exerts a stronger effect in
chromatin opening on one side of their motif than on the other
(Sherwood et al., 2014). In conclusion, epigenetic asymmetry is
manifested in different forms, from within the most fundamental
nucleosome particle, to larger regulatory transcription factor binding
units, as well as the inheritance of epigenetically distinct sister
chromatids, collectively attesting to the remarkably complex realm of
gene expression regulation.
Acknowledgements
We thank Drs Eric Campos, Matthias Stadtfeld, Maria Elena Torres-Padilla and
Lynne Vales for critical reading of the manuscript.
Competing interests
The authors declare no competing financial interests.
Funding
Work in the Reinberg laboratory is supported by grants from the National Institutes of
Health (NIH), New York State Stem Cell Science (NYSTEM) and the Howard
Hughes Medical Institute. W.-W.T. is a New York Stem Cell FoundationDruckenmiller Fellow. This research was supported by The New York Stem Cell
Foundation. Deposited in PMC for release after 12 months.
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