Open Chromatin in Pluripotency and Reprogramming

REVIEWS
Open chromatin in pluripotency

and reprogramming
Alexandre Gaspar‑Maia*§||, Adi Alajem‡||, Eran Meshorer‡ and
Miguel Ramalho‑Santos*
Abstract | Pluripotent stem cells can be derived from embryos or induced from adult cells by
reprogramming. They are unique among stem cells in that they can give rise to all cell types
of the body. Recent findings indicate that a particularly ‘open’ chromatin state contributes to
maintenance of pluripotency. Two principles are emerging: specific factors maintain a globally
open chromatin state that is accessible for transcriptional activation; and other chromatin
regulators contribute locally to the silencing of lineage-specific genes until differentiation is
triggered. These same principles may apply during reacquisition of an open chromatin state
upon reprogramming to pluripotency, and during de-differentiation in cancer.
Embryonic stem (ES) cells are the prototypical pluripo- simplicity and broad applicability. Through ectopic
tent stem cell1–3: they have the capacity to generate dif- expression of genes that are over-represented in ES
ferentiated progeny from all three embryonic germ cells, a set of four transcription factors (OCT4 (also
layers (endoderm, mesoderm and ectoderm), as well as the known as POU5F1), Sry-box containing gene 2 (SOX2),
germline4. ES cells also have a very high self-renewing myelocytomatosis oncogene (MYC) and Krüppel-like
capacity and can be expanded essentially indefinitely factor 4 (KLF4)) was shown to reprogramme differen-
in culture. In contrast to ES cells, adult stem cells such tiated mouse cells (both embryonic and adult somatic
as neural stem cells5 or haematopoietic stem cells6 have cells) into induced pluripotent stem (iPS) cells that are
*Departments of Ob/Gyn and a more restricted differentiation capacity: they usually very similar to ES cells. The surprising ability of only
Pathology, Eli and Edythe generate cells of the tissue in which they reside and are, four factors to induce such a dramatic change in cell
Broad Center of Regeneration therefore, called multipotent. fate initiated a whole new field of research. Importantly,
Medicine and Stem Cell
Research, Center for
In recent years, there has been an increased inter- human cells14–17 can also be converted into iPS cells using
Reproductive Sciences and est in pluripotent stem cells because of their promise either the same four factors as in mouse cells or a dif-
Diabetes Center, University of as models for the study of development and disease ferent combination of factors: OCT4, SOX2, LIN28 and
California, San Francisco, in vitro (for examples, see refs 7,8). However, the deriva- NANOG17. Therefore, somatic cell reprogramming, in
513 Parnassus Ave, San
tion of ES cells from early embryos raises technical and particular the induction of pluripotency, greatly expands
Francisco, California
94143‑0525, USA. ethical limitations to their use in research and the clinic. the options for basic research and potential clinical
‡
Department of Genetics, Pluripotent stem cells can also be derived from both the applications of pluripotent stem cells. Understanding
Institute of Life Sciences, fetal and adult germlines9–11, and by somatic cell repro- the molecular regulation of pluripotency is fundamen-
The Hebrew University of gramming. Three major routes have been described for tally important and will facilitate the safe and efficient
Jerusalem, Jerusalem 91904,
Israel.
somatic cell reprogramming to pluripotency: nuclear application of pluripotent stem cells in the clinic.
§
Present address: Mount transfer from a somatic cell to an enucleated oocyte; The pluripotent stem cell state is under the con-
Sinai School of Medicine, fusion of a somatic cell with an ES cell; and induction of trol of a transcriptional circuitry that includes the
Department of Oncological pluripotency in somatic cells by overexpression of key reprogramming factors mentioned above (reviewed
Sciences, 1425 Madison Ave
transcription factors (BOX 1). All of these reprogramming in ref.12). Recent studies indicate that this transcrip-
Rm15‑52, New York City,
New York 10029‑1075, USA. methods are likely to remain useful and informative tional programme is implemented in the context of an
||
These authors contributed in the years ahead. The relative advantages and dis- ‘open’ chromatin state, and it has been proposed that
equally to this work. advantages of each reprogramming method have been this state allows transcriptional programmes to switch
Correspondence to E.M. reviewed elsewhere12 and are not discussed here. rapidly upon induction of differentiation18. This may be
and M.R.S. e‑mails:
[email protected];
Major excitement has surrounded the process by particularly important in pluripotent stem cells, where
[email protected] which pluripotency is induced in somatic cells in the 4 a broad spectrum of differentiation options needs to be
doi:10.1038/nrm3036 years since it was described13, because of its technical available.
36 | jANUARY 2011 | VOLUME 12 www.nature.com/reviews/molcellbio
© 2011 Macmillan Publishers Limited. All rights reserved

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Box 1 | Pluripotent stem cells can be derived from several sources compaction in interphase nuclei21: heterochromatin
represented the more densely stained, compacted areas,
')EGNNU whereas euchromatin represented the sparsely stained
chromatin.
I25EGNNU On the basis of predominantly histological evidence,
2)%U 5RGTOCVQIQPKCN many stem and progenitor cells, from neoblast cells in
'5EGNNU UVGOEGNNU planaria22 to haematopoietic stem cells in mammals23,
'ODT[Q
's have been classically described as having a typical
+PPGTEGNN
OCUU open chromatin conformation that is mostly devoid of
/QWUG heterochromatin. In such studies, histological analysis
$NCUVQE[UV
'
CFWNVQTPGQPCVCN of the nucleus was sufficient to suggest a significant
difference in chromatin structure between these
progenitor cells and their differentiated progeny.
2NWTKRQVGPVUVGOEGNNU
5QOCVKEEGNN Open chromatin in pluripotent stem cells. The idea of
*[DTKFEGNN 5QOCVKEEGNN
P open chromatin is supported by more than histological
51: -.(
P
1%6 /;% %GNNHWUKQP examinations and, in the past several years, the chrom-
K25EGNNU atin state of pluripotent stem cells has attracted consider-
$NCUVQE[UV '5EGNN
P
able attention owing to its distinct features24. Indeed,
chromatin in pluripotent stem cells is increasingly being
recognized as open when compared with chromatin in
'PWENGCVGF 5QOCVKE somatic cells, implying that its overall structure is less
QQE[VG FQPQTEGNN
condensed and that the ratio between euchromatin and
06'5EGNNU
heterochromatin is higher than in differentiating cells.
There are three sources of pluripotent stem cells in vivo (see the figure, top half).
The first line of evidence came from visualizing chro-
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[
Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst, before
embryo implantation1–3. Embryonic germ (EG) cells are derived from primordial germ
matin in ES cells using electron microscopy: hetero-
cells (PGCs) during mid-gestation (embryonic days 8.5–12.5 in the mouse)9,10 and chromatin was prevalent in differentiated cells but much
germline-derived pluripotent stem (gPS) cells are derived from spermatogonial stem less so in undifferentiated ES cells25. Similarly, electron
cells of neonatal and adult testes11. spectroscopic imaging (ESI) demonstrated that the majority
In addition, three major routes for somatic cell reprogramming to pluripotency have of chromatin in ES cells is homogeneously spread and
been described12 (see the figure, bottom half): fusion between a somatic cell and an largely devoid of compact heterochromatin blocks,
ES cell giving rise to reprogrammed hybrid cells; the generation of nuclear transfer whereas in differentiated cells chromatin appeared
embryonic stem (NT-ES) cells, produced by reprogramming of a somatic nucleus by an heterogeneous with distinct blocks of compaction 26.
enucleated oocyte, which is then cultured to the blastocyst stage to allow derivation of Importantly, this pattern of chromatin organization was
ES cells; and the production of induced pluripotent stem (iPS) cells, derived by somatic
recently found in vivo: cells in the inner cell mass (ICM)
cell overexpression of reprogramming transcription factors, most commonly OCT4
(also known as POU5F1), Sry-box containing gene 2 (SOX2), myelocytomatosis oncogene
of the mouse blastocyst at day 3.5, which are the source of
(MYC) and Krüppel-like factor 4 (KLF4)13. ES cells, share the same open chromatin conformation
as ES cells27. ICM cells have highly dispersed chromatin,
with a significantly lower number of condensed clusters
Here, we discuss how chromatin organization is relative to lineage-committed cells. Analysis of global
regulated in pluripotent stem cells. We begin by giving a chromatin compaction using nucleases such as DNase I
historical perspective of how the concept of open chro- and micrococcal nuclease (MNase) also indicates that
matin has evolved and how it has been associated with chromatin becomes less accessible, and thus less sensi-
pluripotency. We then review recent insights into the tive to nuclease digestion, upon differentiation of ES cells
Endoderm
action of chromatin-remodelling factors that maintain to embryoid bodies (EBs) (A.A. and E.M., unpublished
The innermost of the three
germ layers that are formed a globally open chromatin state in pluripotent stem cells. observations, and K. Ura, personal communication) or
during embryonic Finally, we discuss the implications of these insights for induction of differentiation with retinoic acid28.
development. Prominent our understanding of cellular reprogramming, and point The relatively low abundance of heterochromatin also
examples of endodermal out recent parallels found between open chromatin supports the idea of chromatin being in an open confor-
tissues include the epithelia
of the gastrointestinal and
and cancer. mation. Western blot and immunofluorescence analyses
respiratory tracts, thyroid, liver of histone post-translational modifications (PTMs), such
and pancreas, as well as of the Open chromatin and pluripotency as histone H3 tri-methylation on Lys9 (H3K9me3), that
auditory and urinary systems. Defining open chromatin. The term chromatin was are enriched in heterochromatin (BOX 2) , suggest
coined by Walther Flemming in 1882, after he developed that ES cells have considerably less heterochromatin
Mesoderm
The middle of the three germ novel histological staining methods that enabled him to than differentiated cells29. Subsequently, ChIP–chip assays
layers that are formed during observe a unique fibrous structure in the nucleus. This for H3K9me2, which forms ‘large organized chroma-
embryonic development. structure was readily stained and was therefore named tin K9 modifications’ (LOCKs), showed that these
Prominent examples of chromatin (‘stainable material’)19,20. Almost 50 years later, domains spread considerably during differentiation30.
mesodermal tissues include
bone, cartilage, blood, muscle,
in 1928, the distinction between heterochromatin and Furthermore, ChIP–seq analyses showed that H3K9me3
heart, connective tissue and euchromatin was made by Emil Heitz. He distinguished and H3K27me3 expand from around 4% genome
kidney. these two chromatin components based on differential coverage in ES cells to 12% and 16%, respectively, in
NATURE REVIEWS | Molecular cell Biology VOLUME 12 | jANUARY 2011 | 37

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Box 2 | Chromatin and epigenetic patterns histone PTMs, or histone marks, has been of great use
in defining the epigenetic patterns (BOX 2) that may regu-
Chromatin is a complex assembly of DNA, histone proteins and other non-histone late pluripotency 30,31,35,36. In addition, several chromatin-
protein components. Histone proteins form chromatin building blocks, the nucleosomes, modifying enzymes, such as DNA methyltransferases
around which DNA is wrapped. Each nucleosome consists of an octamer of the
(DNMTs), histone methyltransferases (HMTs), his-
canonical core histones H2A, H2B, H3 and H4 and, between two nucleosomes, the
tone demethylases (HDMs), histone acetyltransferases
histone H1 acts as a linker. Alterations to the chromatin structure that do not affect the
genomic sequence are defined as epigenetic modifications. These epigenetic patterns (HATs), histone deacetylases (HDACs) and chromatin-
include methylation of DNA, post-translational modifications (PTMs) of histones (also remodelling proteins, have recently been shown to have
called histone marks) and histone variants that are incorporated into nucleosomes. important roles in ES cells, and these are described
The amino-terminal tails of histones are subject to various PTMs with either below. Interplay between chromatin regulation and the
activating or inhibiting effects on transcription, including acetylation, methylation, transcriptional network that governs pluripotency 37 is
phosphorylation, ubiquitylation, sumoylation, poly-ADP ribosylation and proline also critical and has been reviewed elsewhere38.
isomerization. The most commonly studied are: methylation, in which histone
methyltransferases (HMTs) add a methyl group and histone demethylases (HDMs) Chromatin poised for differentiation. ES cells have a
remove this group; and acetylation, in which the addition and removal of an acetyl
globally open chromatin structure with abundant levels
group is regulated by histone acetyltransferases (HATs) and histone deacetylases
of epigenetic marks that are indicative of active trans-
(HDACs), respectively. Typically, the tri-methylation of Lys 4 in H3 (H3K4me3), together
with histone acetylation, signals binding of RNA polymerase II and transcriptional cription, such as histone H3K4me3 and acetylation of
activation. H3K27me and H3K9me3 signal a repressive transcriptional state, although histones H3 and H4 (refs 29,32,39). However, there must
through recruitment of distinct silencing factors. Chromatin-remodelling complexes be countering mechanisms that silence developmental
also often include regulators of PTMs and may mediate incorporation of histone regulatory genes and prevent premature differentia-
variants (such as H3.3 and H2AZ or macroH2A), which can be associated with either tion. It is thought that these developmental regulators
inactive or active chromatin58. are silenced but poised for activation by the presence of
Modification of the DNA itself is also important. Cytosine DNA methylation on CpG both the activating mark (H3K4me3) and the repres-
islands is mediated by DNA methyltransferases (DNMTs) and is usually repressive. sive mark (H3K27me3)35,36,39. These ‘bivalent’ domains,
DNA methylation is typically a more stable and inheritable epigenetic pattern that
although not strictly specific to ES cells, may lead to the
can persist for several cell generations. However, DNA methylation can be lost
rapid activation of lineage-specific genes through loss of
passively by a lack of methylation after replication, and there also appear to be factors
that can actively de-methylate DNA58. See fIG. 2 for schematic details of these histone H3K27me3 when differentiation is induced.
and DNA modifications. The repressive H3K27 methylation mark is regulated
by the polycomb group (PcG) proteins. PcG proteins
include the polycomb repressive complex 2 (PRC2),
which is involved in the addition of the histone mark,
differentiated cells31. On the other hand, histone acetyla- and PRC1, which recognizes this mark. Genome-wide
tion, a general mark of open chromatin, has been shown analyses of several PcG proteins in human and mouse
to be increased in undifferentiated human ES cells, ES cells revealed their local enrichment in silenced
particularly at the H3K9 residue32. developmental regulatory genes40,41. Moreover, the target
There is also indirect evidence that supports the genes of PcG proteins tend to be co-occupied by the
Ectoderm concept of a preferentially open chromatin state in pluri- transcription factors OCT4, SOX2 and NANOG, which
The outermost of the potent stem cells. In ES cells, fluorescence recovery after are critical regulators of the pluripotent state. However,
three germ layers that are photobleaching experiments have indicated that chro- PcG proteins are not essential for ES cell self-renewal:
formed during embryonic matin contains a fraction of loosely bound architectural in the absence of PcG proteins such as embryonic ecto-
development. Prominent
examples of ectodermal tissues
chromatin proteins, such as core33 and linker histones dermal development (EED)40,42, Suppressor of zeste 12
include the nervous system, and heterochromatin protein 1 (HP1)29; this fraction is homologue (SUZ12)41 and Enhancer of zeste homo-
hair, skin, nails and eyes, as not observed in differentiating cells29,33. In addition, the logue 2 (EZH2)43, ES cells can still be propagated in the
well as the various derivatives ES cell genome is transcriptionally hyperactive: it tran- undifferentiated state. However, these PcG-deficient
of the neural crest, including
scribes normally silenced repetitive elements as well as ES cells cannot silence several lineage-specific markers
bones of the head and
peripheral nerves. coding and non-coding regions, resulting in increased and have differentiation defects. PcG proteins are
levels of total RNA and mRNA26 (fIG. 1). One way to recruited to target DNA by the cofactor jARID2
Heterochromatin counteract this pervasive transcription in ES cells may (jumonji/ARID domain-containing 2)44. jARID2 also
Highly compacted chromatin be by proteasome-mediated degradation of pre-initiation seems to inhibit the enzymatic methyltransferase activity
that is transcriptionally
inactive. Includes structural
transcription assemblies that form at specific regulatory of PRC2, and may therefore regulate both targeting
regions of the chromosome, genes primed for transcription34. and fine-tuning of PRC2 activity in ES cells and during
such as centromeres, that Taken together, these data indicate that chromatin in differentiation44–47.
lack genes (‘constitutive’ ES cells is globally decondensed compared with differen-
heterochromatin) and regions
tiated cells, and that a smaller fraction of the genome in Heterochromatin regulation in ES cells. Another histone
in which genes are silenced in
a given cell type (‘facultative’ ES cells is organized as repressive heterochromatin. mark that is commonly associated with gene repression
heterochromatin). is methylation at H3K9, which increases with differen-
Control of the chromatin landscape tiation of ES cells. One enzyme that is responsible for
Euchromatin Chromatin in ES cells is characterized by a distinct set H3K9 methylation is the HMT G9a (also known as
A form of chromatin that is
relatively decondensed and
of features, and a better knowledge of the enzymes that EHMT2). Interestingly, G9a is required for the silenc-
often transcriptionally active modify this structure has provided insights into the con- ing of OCT4 upon differentiation48. G9a binds directly to
during interphase. trol of chromatin state. Genome-wide mapping of core the promoter of OCT4 and leads to H3K9 methylation,

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C1RGPEJTQOCVKPKP
'5EGNNU
40#
VTCPUETKRV
Electron spectroscopic
imaging
(esI). energy-filtered &0# *
transmission electron 0WENGQUQOG
microscopy, in which the image
*2 &KȭGTGPVKCVKQP 4GRTQITCOOKPI
is formed only by electrons
transmitted within a certain
energy window. It allows
direct quantitative imaging of D%NQUGFEJTQOCVKPKP
elements within the specimen. FKȭGTGPVKCVGFEGNNU
Embryoid body
(eB). A cellular aggregate that
is produced when es cells are
induced to differentiate in
non-adherent conditions that
Figure 1 | chromatin in pluripotent stem cells versus differentiated cells. The structure of chromatin differs
mimic the early stages of between undifferentiated embryonic stem (ES) cells (a) and differentiated cells (b) in several ways. Chromatin structure
embryogenesis. becomes more condensed upon differentiation and more open upon reprogramming. In ES cells, chromatin is globally
decondensed; there are fewer heterochromatin foci and they are larger and more dispersed compared with those of
ChIP–chip differentiated cells. Architectural chromatin proteins, represented here by the histone H1 and heterochromatin protein 1
Chromatin immunoprecipita- (HP1), are loosely bound to chromatin in ES cells and are bound more tightly to chromatin in differentiated cells. In ES
tion (ChIP) followed by cells, chromatin, including heterochromatin, is transcriptionally hyperactive, shown here by high levels of RNA transcripts.
microarray. ChIP is a method
that allows isolation of DNA
sequences that are bound to
a protein of interest using which is followed by recruitment of DNMTs to signal a NANOG, SOX2 and PcG revealed little overlap 52.
specific antibodies. DNA more definite repressive state. G9a may have a dual role Moreover, ES cells show a significant enrichment of
isolated by ChIP is denatured of methylating H3K9 (as a known HMT) and recruit- methylation outside CpG islands, a feature that seems
and hybridized to a tiling array, ing DNMTs — an example of how several layers of to be unique to these cells53. These observations sug-
which typically includes probes
covering the entire genome.
regulation accomplish proper silencing of a particular gest that DNA methylation may represent a unique
Paired probes indicate that gene 49. Therefore, the increase in heterochromatin epigenetic layer that complements other mechanisms
the protein of interest was that occurs upon ES cell differentiation may directly of gene repression and contributes to tight regulation of
bound to that particular contribute to the silencing of regulators of self-renewal the transcriptional programmes that are activated upon
region of DNA.
and pluripotency. G9a is also required for the estab- differentiation.
ChIP–seq lishment of domains of H3K9me2 (LOCKs) in differ-
Chromatin immunoprecipita- entiated cells30, suggesting a more global role for G9a in Chromatin remodelling in ES cells
tion (ChIP) followed by differentiation-induced heterochromatinization. The addition or removal of histone marks or DNA
sequencing. refers to The low level of H3K9 methylation in undifferenti- methylation is only one way in which the chromatin
high-throughput sequencing
of ChIP-isolated DNA, and
ated ES cells is maintained by the histone H3K9 HDMs state can affect the transcriptional programme and thus
provides genome-wide jMjD1A (jumonji domain-containing 1A; also known as pluripotency in stem cells. The structure of chromatin
information of the DNA binding KDM3A) and jMjD2C (also known as KDM4C). These itself, and the positions of nucleosomes, can be altered
sites of the protein of interest. regulate global levels of the repressive marks H3K9me2 both globally and at the level of specific genetic loci by
and H3K9me3, respectively, and maintain the ES cell chromatin-remodelling proteins that alter the histone–
Heterochromatin protein 1
(HP1). A heterochromatin- state by directly demethylating H3K9 at the promoter DNA contacts using the energy of ATP hydrolysis54.
binding protein that recognizes regions of core ES cell factors, allowing their expres- The disruption of the histone–DNA contact itself is
and binds to histone H3 sion50. Interestingly, the genes encoding jMjD1A and poorly understood, but the consequences are that
tri-methylated on Lys9. It jMjD2C are regulated by OCT4, representing a posi- DNA becomes exposed to regulatory proteins, and
includes three isoforms (α, β
and γ), which are encoded by
tive feedback-loop that integrates the action of trans- nucleosomes and the histones become more actively
three different genes (CBX5, cription factors and histone modifiers to maintain the mobile55.
CBX1 and CBX3, respectively). undifferentiated ES cell state. Chromatin-remodelling proteins can be divided into
A different layer of epigenetic regulation in ES cells four families: SWI/SNF (switch/sucrose nonfermenta-
Proteasome
is the DNA methylation of CpG islands. DNMTs are ble), CHD (chromodomain helicase DNA-binding), ISWI
A large multisubunit protein
complex that degrades responsible for this repressive mark, which is correlated (imitation switch) and INO80 (inositol-requiring 80).
proteins. Undesired proteins with specific histone marks51: methylated CpG islands Chromatin remodellers usually form a complex that
are labelled for degradation are present mainly at promoter regions of repressed contains a catalytic subunit with a SWI2/SNF2 ATPase
by the addition of a chain of genes, usually correlated with unmethylated H3K4 domain, a subunit that recognizes chromatin, and addi-
the small protein ubiquitin;
a process that is mediated by
and H3K9me3, and represent around 30% of genes in tional regulatory subunits that mediate interactions
a family of enzymes called ES cells52. However, cross-referencing genomic regions with other proteins and with chromatin itself 56. At least
ubiquitin ligases. with methylation patterns and binding of OCT4, one member of each of these four families is essential

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for mouse embryogenesis (TABLe 1), demonstrating the CHD family. Four subunits from the CHD family of
central role that chromatin remodellers have in devel- chromatin-remodelling enzymes — CHD1, CHD3,
opment. Recent studies have begun to shed light on CHD4 and CHD7 — are implicated in ES cell identity
the specific roles that chromatin remodellers have in and function, although their mechanisms of action differ.
ES cells. CHD1 and CHD7 have not yet been clearly associated
with a known complex (TABLe 1), but the latter binds multi-
SWI/SNF family. The SWI/SNF family is composed of ple subunits of the PBAF complex in neural crest cells
two major complexes: BRG- or BRM-associated factor derived from human ES cells. In these neural crest cells67
(BAF) and polybromo BAF (PBAF)57 (TABLe 1). There is and mouse ES cells68, CHD7 was enriched at enhancer
some heterogeneity in the composition of the BAF and regions, together with H3K4me1, suggesting that CHD7
PBAF complexes in different cell types and tissues58. ES may maintain transcriptional competence in both
cells have a specialized subunit composition termed undifferentiated and differentiating ES cells.
esBAF, which is dynamically regulated during differen- CHD1 binds globally to active euchromatin and
tiation59, and it is not yet clear whether two distinct com- colocalizes with RNA polymerase II (RNAPII) in
plexes (esBAF and esPBAF) exist in ES cells or whether ES cells69. ES cells in which CHD1 has been depleted
the different subunits combine to form a single esBAF. by RNA interference accumulate high levels of hetero-
BRG1 (also known as SMARCA4) is the catalytic chromatin and, although they can be propagated in the
subunit of the esBAF complex. It is downregulated upon undifferentiated state, they cannot differentiate nor-
differentiation and seems to be gradually replaced by mally. These results indicate that CHD1 establishes a
a different catalytic subunit, BRM59,60. Brg1-null mice balance between euchromatin and heterochromatin in
die at the peri-implantation stage61, and knockdown ES cells, which may be critical for the maintenance of
experiments in ES cells resulted in aberrant morphology, pluripotency.
decreased proliferation rate and reduced differentiation CHD3 and CHD4 constitute the catalytic subunit
capacity 26,59,62,63. Furthermore, genome-wide ChIP–chip of the nucleosome-remodelling (NuRD) complex
and ChIP–seq experiments revealed enrichment of BRG1 (TABLe 1), which has been implicated in regulation of
at promoter regions of genes that are also occupied by the ES cells. For example, ES cells lacking the NuRD sub-
pluripotency regulators OCT4, SOX2 and NANOG63,64. unit methyl-CpG-binding domain 3 (MBD3) retain
Intriguingly, BRG1 inhibition in ES cells leads to upregu- their OCT4 expression when induced to differentiate,
lation of both developmental genes and ES cell-specific and show aberrant differentiation potential 70,71 .
genes. These results suggest that BRG1 may not only MBD3-knockdown ES cells also express trophecto-
contribute to the repression of developmental genes but dermal markers, which are not usually detected in
may also fine-tune the expression level of ES cell-specific ES cells. Deletion of another subunit, encoded by Hdac1,
genes, such as Oct4 and Sox2 (refs 63,64). also results in aberrant differentiation of mouse ES cells,
An additional member of the BAF complex that has a leading to spontaneous generation of mesodermal and
role in ES cells is BAF250 (also known as ARID1), which ectodermal lineages at the expense of endoderm 72.
includes two related subunits, BAF250A and BAF250B. Importantly, knockout of Hdac1 (but not Hdac2) leads
BAF250A incorporation into the BAF complex is to mouse embryonic lethality 73–76. NuRD therefore
most prominent in undifferentiated ES cells, whereas seems to have a dual role in silencing differentiation
BAF250B is mostly incorporated after differentiation59. genes in ES cells as well as ES cell-specific genes dur-
Baf250a-deficient mouse ES cells fail to maintain the ing differentiation. Finally, NuRD subunits MBD3
CpG island
expression of stem cell markers and instead activate and metastasis-associated 2 (MTA2) interact with the
A genomic region which genes with known roles in early development and orga- SWI/SNF component BRG1 specifically in ES cells but
contains a high content of nogenesis65. Furthermore, Baf250a–/– ES cells are prone not in differentiating cells59, implying that there may be
cytosine (C) and guanine (G) to differentiation but they seem to lose the ability to crosstalk between chromatin-remodelling complexes
dinucleotides (the ‘p’ refers
form cells of the mesodermal lineage, which is in agree- in pluripotent cells.
to the phosphodiester bond
linking the two bases). CpG ment with the absence of detectable mesoderm in early
islands are found in many mouse Baf250a–/– embryos65. Unlike Baf250a–/– ES cells, ISWI family. The ISWI family of remodellers can form
mammalian promoters, Baf250b–/– ES cells give rise to all three germ layers66, but three distinct complexes — nucleosome-remodelling fac-
and unlike scattered CpGs disruption of Baf250b results in reduced self-renewal tor (NURF), chromatin accessibility complex (CHRAC)
throughout the genome, which
are usually hypermethylated,
ability and accelerated ES cell differentiation66. and ATP-utilizing chromatin assembly and remodelling
promoter CpG islands are There are mixed reports as to the role of BAF155 (also factor (ACF) — of which, the NURF complex seems to
normally hypomethylated. known as SMARCC1) in ES cells. It is highly expressed have the most prominent role in ES cells. Bromodomain
in ES cells59,28 and its reduction leads to aberrant colony PHD finger transcription factor (BPTF), a member of
Helicase
morphology 62 and decreased OCT4 expression64 in the NURF complex, is required for ES cell differentiation
A protein that can unwind
DNA or rNA. undifferentiated ES cells. However, in differentiating both in vivo and in vitro. Bptf-knockout ES cells cannot
ES cells, loss of BAF155 results in perturbed chromatin form teratomas, and Bptf-knockout EBs exhibit severely
Teratoma condensation and increased OCT4 expression28. Based defective expression of all three germ layer markers.
A confined tumour, originating on these results, it can be speculated that the stoichio- In line with this, Bptf-knockout mouse embryos are
from pluripotent cells, that
includes tissues of the three
metry of different BAF subunits, and not their actual defective in the establishment of the anterior–posterior
germ layers, endoderm, levels, determines their function, perhaps reconciling axis during the earliest stages of development and are
mesoderm and ectoderm. these studies. embryonic lethal at day 8.5 (ref. 77) (TABLe 1).

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Table 1 | Chromatin remodellers in ES cells

complex Protein subunits effect on eS cells embryonic lethality
Morphology Proliferation Differentiation
SWI/SNF family
BAF β-actin N/A N/A N/A E9.5 (ref. 135)
BAF47 N/A N/A N/A Peri-implantation134
BAF53A, BAF57, N/A N/A N/A N/A
BAF60A
BAF155 Yes62 Yes59 N/A Post-implantation133
BAF250A Yes 62,65
Yes 65
Yes 65
E6.5 (ref. 65)
BAF250B No66 Yes66 Yes66 N/A
BRG1* Yes 59,62,63
Yes 26,59
Yes 26,59,63
Peri-implantation61
PBAF β-actin N/A N/A N/A E9.5 (ref. 135)
BAF47 N/A N/A N/A Peri-implantation134
BAF53A, BAF57, BAF60A, N/A N/A N/A N/A
BAF180, BAF200
BAF155 Yes62 Yes59 N/A Post-implantation133
BRG1* Yes59,62,63 Yes26,59 Yes26,59,63 Peri-implantation61
CHD family
N/A CHD1* No69 Yes69 Yes69 N/A
N/A CHD2* N/A N/A N/A Perinatal136
N/A CHD7* N/A N/A N/A E10.5 (ref. 137)
N/A CHD8* N/A N/A N/A E8.5 (ref. 138 )
NuRD CHD3*, CHD4*, N/A N/A N/A N/A
GATAD2B, MTA1, MTA2,
MTA3, RBBP4, RBBP7
GATAD2A N/A N/A N/A E10 (ref. 139)
HDAC1 N/A N/A Yes 72
E9.5 (ref. 74)
HDAC2 N/A No72 No72 Perinatal76
MBD3 Yes 71
Yes 70,71
Yes 70,71
N/A
ISWI family
NURF BPTF N/A Yes77 Yes77 E8.5 (ref. 77)
RBBP4, RBBP7, SNF2L* N/A N/A N/A N/A
INO80 family
TIP60 β-actin N/A N/A N/A E9.5 (ref. 135)
BAF53A, BRD8, EPC1, N/A N/A N/A N/A
EPC-like, MEAF6, MRGBP,
MRGX, VPS72
DMAP1 Yes62 Yes62 Yes62 N/A
MRG15 N/A N/A N/A E14.5 (ref. 141)
p400* Yes 62
Yes 62
Yes 62
N/A
RUVBL1, RUVBL2 Yes62 Yes62 N/A N/A
TIP60 Yes 62
Yes 62
Yes 62
~E3.5 (ref. 140)
TRRAP Yes62 Yes62 N/A Peri-implantation142
YEATS4 Yes 62
Yes 62
N/A N/A
BAF, BRG- or BRM-associated factor; BPTF, bromodomain PHD finger transcription factor; BRD8, bromodomain-containing 8;
CHD, chromodomain helicase DNA-binding; DMAP1, DNA methyltransferase 1-associated 1; E, embryonic day; EPC, enhancer of
polycomb; ES, embryonic stem; GATAD, GATA zinc finger domain-containing; HDAC, histone deacetylase; INO80, inositol-requiring
80; ISWI, imitation switch; MBD3, methyl-CpG-binding domain 3; MEAF6, MYST/ESA1-associated factor 6; MRG, MORF-related
gene; MRGBP, MRG-binding protein; MTA, metastasis-associated; N/A, data not available; NuRD, nucleosome-remodelling;
NURF, nucleosome-remodelling factor; PBAF, polybromo BAF; RBBP, retinoblastoma-binding protein; RUVBL1, RuvB-like 1; SWI/SNF,
switch/sucrose nonfermentable; TIP60, TAT-interacting protein of 60 kDa (also known as KAT5); TRRAP, transformation/transcription
domain-associated protein; VPS72, vacuolar protein sorting-associated 72. *Catalytically active.

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Box 3 | The actions of chromatin-remodelling factors CHD members26. It is possible that integrating high lev-
els of active histone marks with the high expression of
particular chromatin remodellers globally orchestrates
an open chromatin state.
'LGEVKQP 5NKFKPI The chromatin remodeller CHD1 may repress
formation of heterochromatin in ES cells69. However,
the mechanisms that orchestrate this opening of chro-
+PUGTVKQP &KOGTGZEJCPIG
matin, tilting the balance between euchromatin and
heterochromatin towards the former, remain unknown
(fIG. 2). Such global ‘anti-silencing’ mechanisms have
Chromatin remodellers are ATP-dependent machines that act to alter the local structure
0CVWTG4GXKGYU^/QNGEWNCT%GNN$KQNQI[ been studied in other species, such as budding and fis-
of chromatin by repositioning (or ‘sliding’), ejecting or incorporating nucleosomes. sion yeast, and such studies may help us understand the
During DNA replication, for example, a group of chromatin remodellers act to insert principles that govern this battle between heterochro-
nucleosomes into the newly forming chromatin fibre (see the figure, bottom left), but matin and euchromatin. In yeast, silent information
other groups of remodellers are active throughout the cell cycle to modify the local
regulator (SIR) proteins bind preferentially to telomeric
structure of chromatin, thereby regulating gene expression. For example, chromatin-
regions and promote the formation of heterochromatin.
remodelling factors such as SWI/SNF (switch/sucrose nonfermentable) and CHD
(chromodomain helicase DNA-binding) family proteins can trigger ejection of a Two redundant mechanisms prevent the spreading of
nucleosome (top left). Other chromatin-remodelling factors, such as ISWI (imitation SIR proteins and heterochromatin: the incorporation
switch) family proteins, can slide a nucleosome (top right). The INO80 (inositol-requiring of the histone variant H2AZ and the methylation of
80) family proteins exchange histone dimers (bottom right), which can introduce histone H3K4, mediated by the methyltransferase SET domain-
variants or modified histones, and have a local impact on chromatin activity56. containing 1 (Set1). Thus, incorporation of specific
histone variants or a modification of canonical histones
prevents binding of SIR proteins79. Another important
anti-silencing mechanism is histone hyperacetylation,
INO80 family. The INO80 family members can form which also prevents SIR proteins from binding80. The
three distinct complexes, INO80, SNF2-related CBP local silencing mediated by the SIR family protein Sir3
activator protein (SRCAP) and TAT-interacting protein requires a complex interaction between the HAT Sas2,
of 60 kDa (TIP60; also known as KAT5)–p400, but only the HMTs disrupter of telomere silencing 1 (Dot1) and
the last has been shown to be important in ES cells so Set1, and the HDM jhd2 (ref. 81), which determine
far. The TIP60–p400 complex facilitates transcription the dynamic balance of silencing versus activation by
by combining nucleosome remodelling with histone directing a competing addition and removal of methyl
acetylase activity. ES cells depleted in different sub- groups at H3K4 and H3K79. Therefore, not only can
units of the TIP60–p400 complex show strikingly simi- different types of histone modifications (acetylation or
lar phenotypes, including altered colony morphology, methylation) interact to regulate silencing but also there
decreased proliferation rates, reduced pluripotency and is a dynamic balance between the opposing actions
overall reduced viability 62, which seem to be a pheno- of histone-modifying enzymes to regulate formation of
type specific to ES cells78. TIP60–p400 probably acts to euchromatin or heterochromatin.
maintain the undifferentiated state of ES cells by binding Extrapolating on the telomere studies from yeast,
to the H3K4me3 mark, an interaction that is facilitated one possible mechanism by which an open chromatin
by NANOG. In addition, TIP60–p400 promotes histone state is maintained in ES cells may be through deposi-
H4 acetylation at both active and repressed genes 62, tion of specific histone variants. For example, H3.3 has
which is also likely to support the stem cell state. been generally associated with active genes and is less
Together, these studies highlight the importance of prone to H3K9 methylation82,83. H3.3 is incorporated
chromatin-remodelling complexes for integrating the in a replication-independent manner by the chaperone
transcriptional programme for pluripotency with epi- HIRA84, and typically colocalizes with regions enriched
genetic information and for silencing this pluripotency in methylation of H3K4 (refs 85,86). This is thought to
programme upon differentiation. In addition, chroma- be a mechanism by which cells may maintain a trans-
tin remodelling may potentially have a broader role in criptional memory; for example, lineage-specific genes
the global maintenance of the open chromatin state of marked by H3.3 are still expressed after reprogramming
ES cells. in Xenopus laevis 87. Interestingly, CHD1 is required in
Telomeric region the Drosophila melanogaster oocyte for incorporation of
A region of repetitive DNA at Maintaining open chromatin in ES cells H3.3 into sperm chromatin: CHD1-mutant oocytes can-
the ends of chromosomes that In addition to being affected by enriched active his- not incorporate H3.3 into the male pronucleus, which
protects the chromosomes
tone marks, open chromatin may also be actively renders the male genome incapable of contributing to
from premature deterioration,
rearrangements and maintained in ES cells by the above-mentioned ATP- development 88. These results demonstrate the broad
chromosome fusion. dependent chromatin-remodelling enzymes, for exam- impact that H3.3 incorporation has for male chromatin
ple, through the disassembly of nucleosomes and/or in D. melanogaster. The possibility that a similar mecha-
Histone hyperacetylation the ‘unwinding’ of higher-order chromatin struc- nism, involving H3.3 incorporation, also maintains the
A state in which many Lys
residues are acetylated on
tures (BOX 3). Interestingly, the expression of many of global open chromatin state of ES cells warrants future
many of the histones present these chromatin-remodelling enzymes is significantly investigation, even though this variant is also present in
in a given region of chromatin. enriched in ES cells, including the esBAF complex and telomeric regions85.

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!
'WEJTQOCVKP *GVGTQEJTQOCVKP
$KXCNGPVFQOCKP *2 */6U
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2QN[EQOD 6+2 EQORNGZ ,/,&% 578*
- - - 578*
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-
- - - #E - - -
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&0/6.
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Figure 2 | The balance between euchromatin and heterochromatin in eS cells. Several epigenetic regulators
orchestrate the open chromatin state of embryonic stem (ES) cells and set the stage for the transcriptional network.
Relevant epigenetic marks include histone modifications and incorporation of different core histone variants (yellow
and orange cylinders) that alter access and efficiency of the transcriptional machinery. The main histone marks, the
active H3 tri-methylated on Lys 4 (H3K4me3) and the repressive H3K9me3 and H3K27me3 (represented by the circles
K4, K9 and K27), are positively regulated by specific histone methyltransferases (HMTs; including G9a (also known as
EHMT2), SUV39H1, SUV39H2 and SETDB1) and negatively regulated by the respective histone demethylases (HDMs;
including jumonji domain-containing 2C (JMJD2C; also known as KDM4C) and JMJD1A (also known as KDM3A)).
Active (K4) and repressive (K27) marks can be present in the promoter regions of developmental genes to prevent
their expression while allowing rapid activation by transcription factors such as the polycomb proteins Enhancer of
zeste homologue 1 (EZH1) and EZH2 (termed bivalent domains). Histone acetylation also marks active chromatin, and
the acetyl group (the orange triangle, Ac) can be added through complexes such as TAT-interacting protein of 60 kDa
(TIP60; also known as KAT5)–p400 and removed by histone deacetylases (HDACs), which can be part of repressive
complexes such as the nucleosome-remodelling (NuRD) complex. DNA (dark blue line) methylation is typically present
on CpG islands in promoter regions and heterochromatin (marked by H3K9me3 and heterochromatin protein 1 (HP1)).
DNA can be hypermethylated, as a result of the action of DNMTs, such as DNMT3a–DNMT3b or DNMT3L, but in
euchromatic regions DNA is generally unmethylated. Chromatin-remodelling proteins such as chromodomain
helicase DNA-binding 1 (CHD1) and BRG1 in the ES cell-specific BRG- or BRM-associated factor (esBAF) complex
may regulate the open chromatin state, possibly by contributing to boundary determination between euchromatin
and heterochromatin. There is growing evidence that the formation of euchromatin can repress the establishment
of heterochromatin nearby (as it has not been confirmed in ES cells, this is denoted by a question mark).
Alternatively, or in addition, other mechanisms may Lessons from reprogramming somatic cells
directly protect H3K4me3 from demethylation. Binding The process of generating iPS cells reverts somatic cells
of chromatin remodellers such as CHD1 directly to back to a pluripotent stem cell state that is very similar
H3K4me3 via its chromodomains89 may protect against to that of ES cells and may provide an alternative to the
the action of demethylases and selectively cooperate use of ES cells for dissecting the relationship between
with HMTs to maintain the H3K4me3 mark. For exam- open chromatin and pluripotency 94. Although molecular
ple, CHD1 interacts, through its chromodomain, with landmarks that arise during the course of reprogram-
the HMT ASH2, which methylates H3K4 (ref. 90). This ming have been identified, the process remains largely
histone mark prevents the binding of repressive com- unsolved at the mechanistic level. Upon expression of
plexes such as the NuRD deacetylation complex 91,92 and the reprogramming factors (generally OCT4, SOX2,
the DNMT subunit DNMT3L (ref. 93). The opening of MYC and KLF4), alkaline phosphatase (AP) activity and
chromatin can also be complemented by histone hyper- expression of the cell surface marker SSEA1 (also known
acetylation, as shown for telomeres in yeast 80. In fact, as FUT4) are early markers of the undifferentiated state.
the HAT and remodelling complex TIP60–p400 recog- AP and SSEA1 can be detected as early as 3 and 9 days,
nizes H3K4me3 and depends on this mark to bind its respectively, after the onset of reprogramming in mouse
targets62. cells. Endogenous expression of OCT4 and NANOG can
All of these mechanisms may orchestrate a complex, be detected only after about 10 days post-induction, and
dynamic regulation of open versus compact chroma- the four exogenous factors, generally delivered by viral
tin in ES cells (fIG. 2). It will therefore be important to constructs, need to be expressed during all of that period.
determine, in a genome-wide manner using ChIP–seq, However, cells only fully reprogramme upon silencing of
how epigenetic marks change when regulators of open the viral vectors95. The main question that arises is: what
chromatin such as CHD1 are lost. Further genetic and are the immediate downstream effects of the reprogram-
biochemical studies, in particular epistatic analyses ming factors that trigger induction of pluripotency?
and dissection of protein–protein interactions, should OCT4 and SOX2 are part of an autoregulatory loop that
also help define the relative contribution of these maintains pluripotency in ES cells96, and MYC binds to
mechanisms to the chromatin state and pluripotency a separate class of genes not bound by OCT4, SOX2 or
of ES cells. KLF4 (ref. 97), in concert with self-renewal regulators

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Box 4 | Open chromatin and the undifferentiated state in cancer cells

The acquired ability of cancer cells to divide perpetually and at the same time to support tumour growth, metastasis and
invasiveness, bears resemblance to stem cell biology117. It is thought that this acquired immortality is obtained through
the activation of stem cell-specific pathways that are essential for self-renewal, such as Wnt, sonic hedgehog (SHH) or
Notch pathways118,119. There is also a correlation between the transcriptomes of stem cells and highly undifferentiated
cancer cells from tumours with higher proliferation rates and poorer prognosis120–124. For example, myelocytomatosis
oncogene (MYC) can reactivate an embryonic stem (ES) cell-like programme in normal and cancer cells124. However,
MYC has several functions, and the mechanism by which MYC activates this ES cell-like programme could be
independent of its canonical transcription factor activity125. In particular, MYC regulates large domains of euchromatin,
possibly by inducing histone hyperacetylation126,127. It is therefore possible that there are commonalities between
undifferentiated cancer cells and ES cells that include a shared transcriptional programme linked with reorganization
of the chromatin to include euchromatic histone marks128.
Some aspects of higher order chromatin conformation may have similarities between ES cells and certain
undifferentiated types of cancer. For example, loss of heterochromatin markers such as heterochromatin protein 1α
(HP1α)129,130 and H3 di-methylated on Lys 9 (H3K9me2)30 have been observed in metastatic breast cancer and lymphoid
cancer cell lines, respectively. In addition, many genes marked with bivalent domains in ES cells, including those
encoding tumour suppressors and pro-differentiation factors, further acquire H3K9 methylation in embryonic carcinoma
cells and DNA methylation in adult cancer cells121. These additional repressive marks may contribute to a higher-order
chromatin organization and permanent silencing of tumour suppressors and pro-differentiation factor genes in cancer
cells131. Furthermore, the process of inducing pluripotency has similarities to cellular transformation and is facilitated
by the activation of oncogenes such as MYC and the inhibition of tumour suppressors such as p53 (for reviews, see
refs 94,132). It will therefore be of interest to explore potential parallels between the regulation of the chromatin
state in pluripotent stem cells and cancer cells.
such as E2F1 and zinc-finger X-chromosomal (ZFX). mechanisms remain unknown. Interestingly, some of the
MYC is not essential for reprogramming 17,98,99 but it same epigenetic barriers may also be overcome in cancer
facilitates early stages of the process, possibly through progression (BOX 4).
its direct action on chromatin100 or indirect action via Recent insights have been gained from treating repro-
repression of differentiation genes101. The ability to dis- gramming cells with agents that affect the chromatin
sect how individual factors contribute to the generation state. In particular, treatment with agents that promote
of iPS cells would greatly benefit from methods that chromatin decondensation, such as the DNMT inhibitor
allow high-efficiency synchronized reprogramming, 5-aza-cytidine, the HDAC inhibitor valproic acid or a G9a
ideally, coupled with analysis at the single cell level, nei- methyltransferase chemical inhibitor, leads to increased
ther of which is as yet possible. Nevertheless, studies so efficiency of iPS cell generation and sometimes can sub-
far have already provided insights into chromatin-level stitute for a particular transcription factor 103,105–107. It is
regulation of reprogramming. likely that a key step in the generation of iPS cells is the
re-opening of the somatic cell chromatin. Consistent with
Chromatin reconfiguration during reprogramming. A this, in a recent unbiased screen for components of ES cell
large reconfiguration of the chromatin structure, from extracts that facilitate reprogramming, the BAF family
DNA methylation to histone modifications and nucleo- components BRG1 and BAF155 (ref. 108) could sub-
some spacing, occurs during reprogramming. Such layers stitute for MYC. Moreover, they promoted the opening of
of epigenetic regulation are often used as repressive chromatin during the reprogramming process, through
mechanisms in somatic cells to prevent unwanted gene DNA demethylation, and increased H3K4me3 in the
expression from other lineages. How these epigenetic promoter regions of important transcription factors108.
barriers to reprogramming are overcome is a key ques- Suppression of CHD1 also inhibits the generation of iPS
tion. Several lines of evidence support the notion that cells69. Additional evidence comes from other reprogram-
the process of reprogramming involves rare stochastic ming assays, such as somatic cell nuclear transfer 109. Here
epigenetic events. The reprogramming process is slow again, BRG1 is an essential nuclear factor for nuclear
and gradual, with several intermediate states 101–103. reprogramming 110. Furthermore, treatment with HDAC
Reactivation of endogenous ES cell genes such as OCT4 inhibitors enhances efficiency of development after
can occur at very different time points in different iPS nuclear transfer 111. These results suggest that the chro-
cell lines derived from the same clone102. Eventually, matin remodellers that maintain the ES cell state, includ-
almost all cells are reprogrammed to pluripotency, ing BRG1, BAF155 and CHD1, may re-open chromatin
albeit with different and often long latency periods104. during reprogramming and set the stage for activating
Inhibition of the p53/p21 pathway and overexpression the transcriptional network for pluripotency.
of LIN28 accelerate the kinetics of reprogramming by
increasing the cell division rate, which may facilitate Transcriptional memory. A final insight into the epi-
the acquisition of DNA and/or histone modifications. genetic regulation of cell states comes from the recent
This reinforces the idea that reprogramming is a com- observation that, although iPS cells are remarkably
plex process that may use stochastic events to overcome similar to ES cells, they may have transcriptional dif-
epigenetic barriers; however, the underlying molecular ferences112,113. Mouse iPS cells appear to retain a residual

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Genetic epistasis DNA methylation signature from their original somatic in our understanding of pluripotency is how the dif-
The relationship or order in cells114,115, and a similar phenomenon is observed in ferent layers of epigenetic regulation of the chromatin
which two genes act in a human iPS cells (M.R.-S. laboratory, unpublished obser- state impact one another and the transcriptional net-
pathway (that is, upstream or vations). The transcriptional profile of human iPS cells work. Clearly, much effort should now focus on inte-
downstream, synergistic or
antagonistic), which can be
becomes more similar to that of human ES cells after sev- grating the various levels of epigenetic regulation in
studied by analysing single eral passages112, suggesting that some form of reprogram- pluripotent stem cells — for example, using analyses
and double mutants. ming happens with continued culturing. The functional of genetic epistasis and protein–protein interactions
significance of these transcriptional differences remains — and understanding how such information may be
DamID
to be fully understood. Interestingly, in frog embryos parsed out during differentiation. New approaches for
A method that is used to
analyse binding of proteins to
generated by nuclear transfer of muscle cells, which defining the chromatin landscape are being established,
DNA. Genetically modified express the muscle-specific gene myogenic differentia- which will allow for a better understanding of the chro-
Drosophila melanogaster tion 1 (MYOD1), expression of this gene is maintained matin structure and its significance for the identity of
culture cell lines express a in non-muscle lineages even after several divisions87. This a particular cell type. For example, the use of DamID
protein of interest fused with
a bacterial DNA adenine
transcriptional memory may be mediated through depo- in D. melanogaster has identifed five different types of
methyltransferase. Local DNA sition of the histone variant H3.3 (ref. 87). This chromatin chromatin (instead of the classic three: euchromatin,
methyltransferase activity mark could establish, through an unknown mecha- heterochromatin and facultative heterochromatin),
indicates protein binding. nism, a memory of the genes that had been previously according to the chromatin proteins that are bound to
transcribed in the somatic cell. these domains116. They include three types of silenc-
Such epigenetic memory, potentially mediated by ing or repressive chromatin — one bound by HP1,
DNA methylation or histone variant incorporation, another bound by Polycomb and a third with no appar-
may contribute to differences between iPS cells and ES ent known repressive or active marks — which encom-
cells and suggests that competing epigenetic influences pass more than 50% of the genome. The euchromatic
may affect chromatin re-opening during reprogram- regions are divided into two domains, one enriched
ming. A mechanistic understanding of these epigenetic with H3K36me3 and the other mostly bound by regu-
influences, which is at present lacking, should shed latory factors, and include most developmental genes.
light not only on how iPS cells are generated but also, Studies such as this in mammalian cells will hopefully
more broadly, on cellular transitions that occur during provide a more comprehensive picture of ‘open’ and
differentiation or transformation. ‘closed’ chromatin.
In addition, much remains to be learned about the
Conclusions mechanisms that regulate epigenetic reprogramming
Significant new insights have been gained into the during the generation of iPS cells. We must remember
regulation of pluripotency and reprogramming at the that ES cells and iPS cells are cultured in vitro, and that
chromatin level. The emerging picture is that a globally the molecular mechanisms that underlie their biology
open chromatin state that is accessible for transcrip- evolved for processes in the context of the whole embryo
tional activation is actively maintained in pluripotent that remain poorly understood and deserve further inves-
stem cells. In this context that is permissive for trans- tigation. Finally, it will be important to assess the signifi-
cription, there are additional epigenetic mechanisms cance of the intriguing epigenetic similarities observed
that promote silencing of lineage-specific genes while between pluripotent stem cells and undifferentiated
leaving them poised for rapid activation. A major gap cancer cells (BOX 4).
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embryonic stem cells are distinguished by gene (2010).

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Open chromatin in pluripotency

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Table 1 | Chromatin remodellers in ES cells

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Box 4 | Open chromatin and the undifferentiated state in cancer cells

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