Review
Epigenetic Regulation of Pluripotency and Differentiation
Michael J. Boland, Kristopher L. Nazor, Jeanne F. Loring
Abstract: The precise, temporal order of gene expression during development is critical to ensure proper lineage
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commitment, cell fate determination, and ultimately, organogenesis. Epigenetic regulation of chromatin structure
is fundamental to the activation or repression of genes during embryonic development. In recent years, there
has been an explosion of research relating to various modes of epigenetic regulation, such as DNA methylation,
post-translational histone tail modifications, noncoding RNA control of chromatin structure, and nucleosome
remodeling. Technological advances in genome-wide epigenetic profiling and pluripotent stem cell differentiation
have been primary drivers for elucidating the epigenetic control of cellular identity during development and
nuclear reprogramming. Not only do epigenetic mechanisms regulate transcriptional states in a cell-type–specific
manner but also they establish higher order genomic topology and nuclear architecture. Here, we review the
epigenetic control of pluripotency and changes associated with pluripotent stem cell differentiation. We focus
on DNA methylation, DNA demethylation, and common histone tail modifications. Finally, we briefly discuss
epigenetic heterogeneity among pluripotent stem cell lines and the influence of epigenetic patterns on genome
topology. (Circ Res. 2014;115:311-324.)
Key Words: epigenomics
■
methylation
T
■
stem cell, pluripotent
human PSCs (hPSCs; ie, hESCs and hiPSCs) has allowed
laboratories across the world to integrate these cells into their
research programs.
Because this widespread adoption of hPSC technologies
has placed hPSCs in the spotlight, early successes or failures
with these cells will have profound effects on hESC-based disease modeling and regenerative medicine. Irrespective of the
specific disease or developmental process of interest in any
given study, it is of critical importance to ensure that hPSCderived cell types are well defined and biologically relevant.
In this regard, determining epigenetic and transcriptional profiles of the population of cells under study may be the best
readout of biological relevance and cellular identity.
Epigenetics is classically defined as a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.9 Bird10 has revised this definition to
the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states. The different
modes of epigenetic regulation include DNA modifications,
such as 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), post-translational histone tail modifications, energy-dependent nucleosomal remodeling, and long noncoding
RNA regulation of local chromatin structure and chromosomal
organization. It has been known for ≥3 decades that epigenetic modification of DNA by methylation of deoxycytidine
residues has a powerful effect on gene expression.11–13 More
recently, with the growing understanding of histone-mediated
he growing use of human pluripotent stem cells (hPSCs)
in biomedical research, coupled with advances in techniques to probe the epigenome and genome, has shown that
a complex interplay of DNA elements and dynamic epigenetic processes underlies the coordinately regulated patterns of
gene expression during human development. Here, we provide a global overview of the current knowledge of epigenetic
regulation in pluripotent cells and during mammalian development. We focus on 2 of the major epigenetic controls, DNA
methylation and histone modifications, assessing both studies
performed in the mouse and with human cells.
Human embryonic stem cells (hESCs) are self-renewing
and pluripotent, with the capacity to differentiate into any
somatic cell type. These qualities are valuable because they
can provide an inexhaustible source of cells for drug screening and potentially for a wide range of cell replacement therapies. Recent work has shown that hESC differentiation can
recapitulate endogenous developmental processes, such as
neocortical development,1–3 and can even generate structures
resembling immature organs.4–6 Therefore, hESCs provide the
possibility of unprecedented insight into human development.
With the discovery that somatic cells could be reprogrammed
into induced PSCs (iPSCs), which harbor a developmental potential indistinguishable from ESCs,7,8 it became possible to
generate disease-specific hiPSC lines by reprogramming cells
from individuals having the disease of interest. Furthermore,
the refinement of methods for reprogramming and culturing
Original received March 18, 2014; revision received June 2, 2014; accepted June 4, 2014. In May 2014, the average time from submission to first decision
for all original research papers submitted to Circulation Research was 14.87 days.
From the Department of Chemical Physiology, Center for Regenerative Medicine, The Scripps Research Institute, La Jolla, CA 92037.
Correspondence to Jeanne F. Loring, PhD, Department of Chemical Physiology, Center for Regenerative Medicine, The Scripps Research Institute, La
Jolla, CA 92037. E-mail [email protected]
© 2014 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.115.301517
311
312 Circulation Research July 7, 2014
Nonstandard Abbreviations and Acronyms
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5caC
5fC
5mC
5hmC
BER
CTCF
DMRs
DNMT
ESC
hESC
hPSC
iPSC
LAD
PRC
PSC
RNAPII
TDG
5-carboxylcytosine
5-formylcytosine
5-methylcytosine
5-hydroxymethylcytosine
base excision repair
CCCTC-binding factor
differentially methylated regions
DNA methyltransferase
embryonic stem cells
human embryonic stem cells
human pluripotent stem cells
induced pluripotent stem cells
lamina-associated domains
polycomb repressive complex
pluripotent stem cells
RNA polymerase II
thymine-DNA glycosylase
chromatin remodeling (the histone code),14 the complex interplay of different epigenetic modes is beginning to be revealed.
Epigenetic modes that involve DNA or histone modifications
include the enzymes that catalyze the particular modification (writers), proteins that recognize and bind the modification (readers), and the enzymes that remove the modification
(erasers). For example, histone 3 lysine 27 trimethylation
(H3K27me3) is catalyzed by the histone methyltransferase
(HMTase) enhancer of zeste homolog 2 (EZH2), read by chromobox homolog 7 (CBX7), and erased by the lysine-specific
demethylase UTX. Readers possess characteristic recognition
motifs, such as the methyl-CpG binding domain for 5mC and
the bromodomain for lysine acetylation. Several different domains recognize methylated lysines or arginines in a residue/
modification-specific manner. They include chromodomains
and tudor, WD40 repeat, and plant homeodomain (PHD) finger
domains.15 Figure 1 shows a list of common readers, writers,
and erasers pertaining to the epigenetic marks reviewed here.
In this review, we provide an overview of DNA methylation
and histone modifications and their crosstalk on how they affect specific regulatory elements, namely, promoters and enhancers. We will then review available knowledge pertaining
to epigenetic dynamics during the developmental processes of
differentiation and X-chromosome inactivation (XCI), including the role that epigenetics plays on nuclear organization and
genome topology. Where appropriate, we provide examples
pertaining to cardiovascular research and direct the reader to
more in-depth reviews of the topics we discuss.
DNA Methylation
DNA methylation is a heritable, yet reversible, epigenetic
modification that plays a central role in transcriptional repression, suppression of retroelement transposition, genomic imprinting, XCI, and higher order chromatin organization. DNA
methyltransferases (DNMTs) catalyze the transfer of a methyl
group from cofactor S-adenosylmethionine to carbon 5 of the
cytosine ring to generate 5mC.16 In mammals, the de novo
DNMTs (DNMT3A, DNMT3B, and cofactor DNMT3L) are
responsible for establishing postfertilization genomic methylation patterns, whereas DNMT1, together with its cofactor
ubiquitin-like with PHD and ring finger domains 1 (Figure 1),
localizes to the replication fork to maintain methylation patterns on the newly synthesized DNA strand during replication. Although the minimal sequence requirement for DNA
methylation is that the target cytosine typically resides within
a CpG dinucleotide, sequence preferences have been identified for the de novo DNMTs that seem to be conserved from
mice to humans.17–19
In mouse development, after fertilization and before pronuclear fusion, zygotic genome activation and the first-cell
division, the paternal genome is demethylated by enzymatic
modification of 5mC (discussed later), whereas the maternal
genome is passively demethylated during subsequent rounds
of replication via nuclear exclusion of an oocyte-specific
Dnmt1 isoform.20 Thus, the totipotent zygote is essentially
devoid of DNA methylation except at imprinted regions. The
genome is gradually remethylated during subsequent cleavage
divisions that generate the morula and early blastocyst. The
genomes of PSCs derived from the inner cell mass/epiblast are
highly methylated.20 Erasure of gametic methylation patterns
and genomic remethylation in the developing zygote is necessary for specification of initial lineage commitment. However,
DNA methylation is not required for pluripotency because
the genomes of Dnmt3a, Dnmt3b, and Dnmt1 triple-knockout
mouse ESCs are hypomethylated, yet the cells retain self-renewal and pluripotency but exhibit a host of differentiation
defects.21 This suggests that establishment of zygotic methylation patterns is crucial for germ layer specification and lineage
commitment. Underscoring the importance of DNA methylation for cellular differentiation and embryonic development,
Dnmt1 and Dnmt3b knockout mice exhibit embryonic lethality, whereas Dnmt3a knockouts display stunted growth and
neonatal mortality.22,23
The DNA methylation machinery works in conjunction
with other modes of epigenetic regulation to regulate gene
expression via local chromatin structure and higher order
genomic topology. Methyl-DNA binding proteins (MBD1,
MBD2, and MBD3; methyl-CpG binding protein 2 [MeCP2];
and ZBTB33 [Kaiso]) bind 5mC and recruit histone deacetylases, repressive histone methyltransferases, and other chromatin remodeling proteins, such as ATP-dependent chromatin
remodeling complexes, to repress transcription actively.24
The genomic location of DNA methylation affects its
role on transcription and higher order chromatin structure.
The highly and constitutively methylated centromeric and
pericentromeric DNA satellite repeats play a large role in
heterochromatin organization at these regions. Indeed, the
heterochromatic centromeres from many chromosomes aggregate to form structures called chromocenters that play roles in
overall nuclear architecture. Many studies have demonstrated
that promoter CpG methylation is inversely correlated with
gene expression. Genes regulated by methylation usually
contain a low density of promoter CpG sites. Most low CpG
density promoters are methylated in ESCs and subsequently
demethylated and expressed in a lineage or cell-type–specific
manner during differentiation.25–27 Areas termed CpG islands,
Boland et al Epigenetics of Pluripotency and Differentiation 313
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Figure 1. Proteins involved in the regulation of common epigenetic modifications. Writer refers to the enzyme(s) that catalyze
and establish a particular modification. Readers are proteins or multiprotein complexes that recognize and bind the modification, and
erasers are a group of enzymes that catalyze removal of the mark. The coordinated action of these 3 groups not only ensures the faithful
maintenance of epigenetic heritability but also determines the dynamic nature of epigenetic regulation during development. The list
of histone modification writers, readers, and erasers is exceedingly long. Therefore, we have listed only a few well-studied examples
for each modification. New nomenclature guidelines have been proposed for many of these proteins9; however, we have used the
old nomenclature because it is more widely used in the literature. Proteins denoted in blue text exhibit heart defects when mutated.
See also reference 10. DPF3B–Tetralogy of Fallot, MLL2–Kabuki Syndrome, CHD7–CHARGE Syndrome, WHSC1–Wolf-Hirschhorn
Syndrome. 5mC indicates 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; ASH1L, (absent, small or homeotic)-like; ATF, activating
transcription factor 4; BPTF, bromodomain PHD finger transcription factor; CBX, chromobox; CDYL, chromodomain Y-like; CHD,
chromodomain; CTBP, C-terminal binding protein; DNMTs, DNA methyltransferases; DPF3B, D4 Zinc and double PHD Fingers, family
3; EAF (Eleven nineteen lysine-rich leukemia gene)-associated factor; EZH, enhancer of zeste; GCN5, general control of amino-acid
synthesis 5; H3K27me3, histone 3 lysine 27 trimethylation; HAT, histone acetyltransferase; HDAC, histone deacetylase; ING2, inhibitor
of growth family member 2; JHDM1D, jumonji C domain-containing histone demethylase 1; KDM, lysine demethylase; MeCP2, methylCpG binding protein 2; MLL, mixed-lineage leukemia; MPP8, M-phase phosphoprotein 8; MYST1, MOZ YBF2 SAS2 TIP60 family
member 1; NO66, nucleolar oxygenase 66; NSD1, nuclear receptor binding SET domain protein 1; PCL, polycystin-like; PHF2, PHD
finger protein 2; RSC4, remodel the structure of chromatin complex subunit 4; SETD, su(var)3-9 and enhancer of zeste domain; SNF5,
switch/sucrose nonfermentable (SWI/SNF) homolog 5; PRDM, PR domain-containing; TAF3, TATA box binding protein associating factor
3; TET, ten eleven translocation; UHRF1, ubiquitin-like PHD and Ring finger domain-containing protein 1; and WHSC1, Wolf-Hirschhorn
syndrome candidate 1.
which are regions of high CpG density found within or near
proximal promoters or transcription start sites, are typically
devoid of DNA methylation.28
Recent studies suggest that promoter hypomethylation
coupled with methylation within gene bodies strongly correlates with expression.29,30 Differentially methylated regions
(DMRs), generally found at regulatory elements such as enhancers and promoters, display lineage- or cell-type–specific
methylation patterns. DMRs can be used to predict whether a
cell belongs to a certain lineage or tissue based on the genes
they regulate.27 As a general rule, the extent of genomic methylation exhibits an inverse relationship to developmental potential such that pluripotent genomes are highly methylated,
whereas differentiated cells from a variety of lineages display
reduced levels of global DNA methylation.29,31 These data indicate that there is a progressive loss of methylation as differentiation proceeds.27,29 Inhibition of DNA methylation has
been shown to result in strong induction of cardiomyocyte
differentiation.32 In addition, a subset of genes involved in
cardiac structure undergoes cardiomyocyte-specific demethylation during development.33 However, there are specific
examples of methylation gains at DMRs during differentiation. For example, promoter methylation and silencing of the
pluripotency regulators, POU5F1 and NANOG, occurs during
lineage specification, and the HOX gene cluster becomes progressively methylated as development proceeds.
Non-CpG Methylation
Non-CpG methylation (CpH, where H=A, T, or C) is enriched in PSCs and lost on differentiation in all cell types/tissues except the brain, where it accumulates in neurons during
postnatal and adolescent development, coinciding with periods of synaptogenesis and synaptic pruning.34,35 It is far less
abundant than CpG methylation in somatic cells, constituting
only 0.02% of total 5mC in somatic cells, but in hESCs, 25%
of 5mC are in CpH.29,35 The most prevalent non-CpG methylation is found at CpA dinucleotides with mCpT and mCpC
constituting a small fraction of CpH methylation.29,34,36,37 NonCpG methylation seems to be catalyzed by DNMT3A34,36,37
and DNMT3B.29 The functions of non-CpG methylation remain elusive, but high-resolution global-profiling techniques
suggest that it may have a myriad of functions. For example,
non-CpG and CpG methylation have been implicated in the
regulation of RNA splicing in ESCs and neurons, respectively,29,38 and enrichment of CpH methylation on the template
strand within gene bodies in ESCs correlates with high expression.35 However, in the brain, CpH methylation is associated with transcriptional repression.18,34 Interestingly, CpH
methylation, but not CpG methylation, is depleted at pluripotent-specific enhancers in ESCs,35 whereas CpG methylation
is lost at cell-type–specific enhancers during differentiation.27
Thus, it seems that PSCs use the DNA methylation machinery
differently than somatic cells for gene regulation.
314 Circulation Research July 7, 2014
Oxidation of 5mC and DNA Demethylation
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Recently, it was discovered that the ten eleven translocation (TET) family of 2OG-Fe(II) dioxygenases catalyze the
hydroxylation of 5mC to generate 5hmC.39 Genomic 5hmC
was first characterized in cerebellar Purkinje neurons, where
it is ≈40% as abundant as 5mC.40 The genomes of PSCs also
possess high levels of 5hmC.39,41,42 At low resolution, 5hmC
and 5mC exhibit mutually exclusive localization patterns:
centromeric and pericentromeric regions tend to have higher
concentrations of 5mC, whereas 5hmC is more enriched on
chromosome arms.42 High-resolution global mapping demonstrates an anticorrelation between 5mC and 5hmC levels at
regulatory elements. Moreover, 5hmC signatures are enriched
at sites of DNaseI hypersensitivity, which are indicative of
genomic regions bound by regulatory proteins, whereas 5mC
is generally depleted at sites of DNA–protein interaction.43
5hmC is particularly enriched at bivalent domains (discussed
later), near transcription start sites, at CpG-rich proximal promoters, and at active and poised enhancers.41,43,44 Interestingly,
both 5mC and 5hmC are enriched within the gene bodies of
actively transcribed genes.30,41–43,45
To date, there are 3 identified TET isoforms that play different roles during development. TET3 primarily functions early
in embryogenesis, where it rapidly converts 5mC to 5hmC in
the male pronucleus. The other 2 isoforms, TET1 and TET2,
not only share some targets but also have mutually exclusive
functions during development. Depletion of Tet1 or Tet2 in
ESCs results in globally reduced levels of 5hmC but has no
discernible effect on pluripotency. Tet1 or Tet2 single knockouts display different differentiation defects—Tet1 knockout
differentiation is skewed toward trophectoderm (in agreement
with in vitro differentiation data using Tet1 knockdown mouse
ESCs [mESCs]), and Tet2 knockout animals possess dysregulated hematopoietic stem cells.46–49 The majority of double
Tet1/Tet2 knockout animals die at birth; however, a small percentage of double knockout animals are phenotypically normal and fertile but possess imprinting abnormalities,50 thereby
implicating TET proteins and 5hmC in the proper maintenance of genomic imprints. These results suggest that TET1
and TET2 have nonoverlapping albeit mild functions during
development: TET1 helps maintain pluripotency by suppression of trophectoderm differentiation, and TET2 maintains
normal homeostasis in the hematopoietic lineage.
Originally it was thought that 5hmC was merely an intermediate in the demethylation pathway, but its putative role in
gene regulation has grown, along with increased controversy
about the details of its function. Recent studies indicate that it
might be a stable mark complete with its own readers51 some
of which seem to overlap with MBDs. Since the discovery of
5hmC, a significant question is whether MBDs also recognize
5hmC. The literature is contradictory on whether proteins that
recognize 5mC also function as 5hmC readers. For example,
several studies have shown that many of the proteins that directly bind methylated DNA (eg, MBD1, MBD2b, MBD3,
MBD4, MeCP2, and ubiquitin-like with PHD and ring finger domains 1 [UHRF1]) either do not recognize and cannot
bind 5hmC or exhibit low and variable affinity for 5hmC.51–54
However, another report indicates that MBD3 does bind 5hmC
and influences the expression of genes marked with 5hmC.55
Furthermore, there are conflicting reports as to whether
MeCP2 is able to recognize and bind 5hmC. Much emphasis
has been placed on MeCP2’s putative role in reading 5hmC
because of the high levels of both MeCP2 and 5hmC in the
brain, and MeCP2’s role in the neurodevelopmental autistic
disorder, Rett Syndrome. Again, there are conflicting reports
as to whether MeCP2 is able to recognize and bind 5hmC.
Mellén et al56 identify 5hmC as a high-affinity substrate of
MeCP2 and observe MeCP2 and 5hmC colocalization at
genes expressed in the brain. However, Szulwach et al44 demonstrate an inverse relationship between MeCP2 and 5hmC
levels in the brain. Moreover, other reports suggest that 5hmC
is a poor physical substrate for MeCP2 in vitro.54 Although
recombinant MeCP2 may possess low affinity for 5hmC in
vitro, its affinity in vivo may be enhanced by either accessory
proteins or by post-translational modifications. It will be important to rectify these differences and to identify molecules
that read 5hmC and its oxidized derivatives further.
DNA demethylation has both fascinated and frustrated
scientists for decades. The most dramatic example of DNA
demethylation occurs early in development when the paternal genome is rapidly demethylated before pronuclear fusion
and the first-cell division occurs.57,58 Other examples of demethylation are more localized, such as the derepression of
genes during germ layer formation, lineage specification and
terminal differentiation, and the activity-dependent epigenetic
remodeling events related to synaptic plasticity and memory
formation.27,38,59–61
The process of active, or enzymatic, DNA demethylation
has been the subject of speculation for years.62 Recent studies
indicate that active demethylation involves a DNA repair event,
such as base excision repair (BER).63 However, none of the glycosylases that initiate BER (thymine-DNA glycosylase [TDG],
MBD4, or SMUG1) possess high affinity for 5mC as a substrate. Therefore, for demethylation to proceed through a BERbased mechanism, 5mC must be modified into a form that is
efficiently recognized by the BER glycosylases. Spontaneous
deamination of 5mC to thymidine yields a T:G mismatch, and
deamination of 5hmC results in a 5-hydroxyuracil (5hmU):G
mismatch, both of which are recognized by either of 2 mammalian BER glycosylases; TDG or MBD4. TDG and MBD4
have both been implicated in DNA demethylation,64 and recent
studies have defined a clear role for TDG in BER-mediated
DNA demethylation.65,66 Deamination of 5hmC by activationinduced deaminase (AID) results in demethylation of reporter
constructs containing 5hmC in mammalian cells,38 and AID has
been shown to be important for the demethylation events that
occur during somatic cell reprogramming to pluripotency.67
The precise role of AID in active DNA demethylation remains
unclear because recently it has been shown that recombinant
AID displays little activity toward 5mC and no activity toward
5hmC as substrates68 although unidentified cofactors may enhance AID substrate affinity in vivo.
Currently, experimental evidence suggests a model of active DNA demethylation that can be summarized as follows
(Figure 2): TET-mediated hydroxylation of 5mC generates
5hmC, which is recognized by a complex containing TDG and
AID (and possibly GADD45).69 AID then deaminates 5hmC to
Boland et al Epigenetics of Pluripotency and Differentiation 315
enzymatic cascade catalyzed by multiple proteins. In the original studies documenting rapid demethylation, it seemed that
the paternal genome was actively demethylated because the
antibody-based methods used to visualize 5mC did not recognize 5hmC. It has more recently been demonstrated that
5mC in the paternal genome is rapidly converted to 5hmC by
TET3, which is then passively lost during subsequent rounds
of replication.75–77
Histone Code
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Figure 2. DNA methylation dynamics. Cytosine is methylated
at carbon 5 of the pyrimidine ring by DNA methyltransferases
(DNMT) to generate 5-methylcytosine (5mC), which can be
hydroxylated to 5-hydroxymethylcytosine (5hmC) by ten eleven
translocation (TET) dioxygenases. 5hmC can be deaminated by
the cytidine deaminase, activation-induced deaminase (AID),
to generate 5-hydroxymethyluracil (5hmU), or further oxidized
by TETs to 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC).
5hmU, 5fC, and 5caC are all substrates for thymine-DNA
glycosylase (TDG); the primary glycosylase that initiates base
excision repair-mediated DNA demethylation.
5hmU thereby generating a suitable substrate for TDG, which
initiates the BER cascade ultimately ending with restoration
of a CpG dinucleotide. Two other DNA glycosylases (Mpg
and Neil3) have been recently identified as 5hmC readers, but
their role in DNA demethylation remains to be determined.51
It has also been shown that TET enzymes possess the ability to oxidize 5hmC in a step-wise manner further to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).70
5caC is a high-affinity substrate for purified TDG but not for
MBD4,70,71 and TDG has been shown to excise both 5fC and
5caC rapidly.72 Depletion or knockdown of Tdg in ESCs results in drastic reduction or ablation of glycosylase activity toward 5caC, and increased levels of 5fC and 5caC at promoters
also marked with 5hmC.71,73,74 Interestingly, 5fC also seems
to be preferentially enriched at a specific class of regulatory
elements, called poised enhancers (discussed later), concomitant with increased levels of 5hmC and decreased 5mC levels.
Furthermore, increased levels of 5fC are observed at poised
enhancers in Tdg null ESCs, suggesting Tdg-mediated demethylation occurs at these regulatory elements.74
To return to the most striking example of rapid demethylation, the genome-wide demethylation of DNA in the male
pronucleus of a zygote,57,58 evidence indicates that this occurs
by TET3-mediated conversion of 5mC to 5hmC rather than a
BER-mediated mechanism, mainly for 2 reasons: (1) 5mC is
symmetrical at CpG sites, where the cytosine on each strand
is methylated; therefore, BER of a symmetrically methylated
CpG site would result in a double-strand break. Genome-wide
demethylation within a short temporal window would generate hundreds of thousands of double-strand breaks and likely
trigger apoptosis. (2) BER is a laborious and time-consuming
Post-translational modification of histone tails results
in a combinatorial readout that affects gene expression
through the regulation of local chromatin structure.14 A
comprehensive review of all histone tail modifications,
which include acetylation, methylation, citrullination
(also known as deimination), phosphorylation, ubiquitylation, sumoylation, and biotinylation, is beyond the scope
of this article. The precise role of histone modifications,
such as phosphorylation, sumoylation, and biotinylation,
and their effects on chromatin structure and transcriptional
state remain unclear and warrant further investigation.78
Interestingly, citrullination, the enzymatic conversion of
arginine to citrulline, has recently been implicated in the
establishment and maintenance of pluripotency.79 Here,
we will focus on the 5 most commonly studied modifications of histone 3 (H3), specifically H3 lysine 4 methylation (H3K4me), H3 lysine 9 methylation (H3K9me), H3
lysine 27 acetylation (H3K27ac), H3 lysine 27 methylation
(H3K27me), and H3 lysine 36 methylation (H3K36me3)
because they have been shown to correlate with and predict
chromatin/transcriptional states of regulatory elements in
many different cell types accurately.
High-resolution, genome-wide studies have elucidated
common themes of gene regulation by modified histones.
Genomic regulatory regions that respond to developmental
and environmental stimuli (ie, enhancers and promoters) are
marked by histone modifications that confer transcriptionally permissive (euchromatin) or repressive (heterochromatin)
chromatin states, which are mediated by the Trithorax group
proteins and polycomb group proteins, respectively. Common
euchromatin modifications are H3K4me3, H3K9ac, H3K27ac,
and H3K36me3. They are found primarily at active enhancers
(H3K9ac and H3K27ac), promoters (H3K4me3), and within the bodies of actively transcribed genes (H3K36me3).80
H3K4me3 displays a punctate localization pattern within
1 to 2 kb of active promoters that contain CpG islands.28 It
promotes transcription by recruiting nucleosome remodeling
complexes and histone acetylases.
The 2 most studied modifications found at repressed chromatin are H3K9me3 and H3K27me3, generally localized to
constitutive and facultative heterochromatin, respectively. The
histone methyltransferase that catalyzes H327me3 is enhancer
of zeste homolog 2 (EZH2), which is a member of the multisubunit polycomb repressive complex 2 (PRC2) that also contains embryonic ectoderm development, suppressor of zeste
12, and retinoblastoma binding proteins 4 and 7. PRC2 is targeted to genomic regions in response to developmental cues
where it catalyzes H3K27me3. This modification recruits another multiprotein complex, PRC1, which contains members
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of the chromobox family that recognize H3K27me3. Also included in PRC1 is the ring finger protein, RING1B, an ubiquitin ligase that catalyzes monoubiquitination of histone H2A,
a modification that impedes RNA polymerase II (RNAPII)
elongation, resulting in transcriptional repression. Originally
identified in Drosophila, the suppressor of variegation 3 to 9
family of histone methyltransferases, which do not associate
with PRC complexes, catalyzes trimethylation of H3K9. This
modification is heavily enriched at centromeric and pericentromeric DNA where, together with the DNA methylation
machinery, it mediates the constitutive higher order heterochromatin structure necessary for mitotic spindle assembly.
PRCs play many roles during the determination of cell
identity.81 Abolishment of either PRC1 or PRC2 activity in ESCs results in differentiation, albeit skewed toward
ectoderm and endoderm, respectively. However, double
knockouts (Eed−/−, Ring1B−/−) completely lose the ability to initiate differentiation.82,83 These results indicate that
the 2 complexes have both redundant and nonoverlapping
functions during development and the maintenance of PSC
identity. Furthermore, in an excellent example of epigenetic crosstalk, PRC2 has been physically coupled to long
noncoding RNA regulation of chromatin structure during
cardiomyocyte differentiation.84
In general, the genome of PSCs is enriched for open or
transcriptionally permissive/poised chromatin and contains
less heterochromatin relative to somatic cells (see below).
PSCs contain more acetylated chromatin and smaller regions
of H3K9me3 and H3K27me3 relative to differentiated cells.
Knockout of the histone acetyltransferase MYST1 (MOZ
YBF2 SAS2 TIP60 family member 1), a member of the core
pluripotency network, results in enhanced heterochromatin
formation and loss of differentiation potential in ESCs.85
Bivalent Domains
Traditionally, it was thought that genes required for downstream developmental process were actively repressed until
needed. The discovery of bivalent domains in ESCs changed
the way we view gene regulation in pluripotency and differentiation.28 Bivalent domains were first identified as broad
H3K27me regions that also contain smaller, narrowly defined
regions of H3K4me localized at transcriptional start sites.28
Bivalent domains can be further categorized based on histone
modifications and polycomb group (PcG) protein occupancy.
Domains that contain PRC2 only are weakly conserved and
generally correspond to membrane proteins or genes of unknown function.86 However, domains containing both PRC1
and PRC2 complexes are highly conserved evolutionarily and
show strong enrichment for many developmentally regulated
transcription factors and other factors involved in morphogenesis and in cell signaling. Polycomb targets are commonly
associated with CpG-rich DNA, and approximately half of
PRC2 binding sites correspond to CpG islands.86 Interestingly,
PRC2 is present at nearly all bivalent regions, whereas PRC1
occupies less than half (39%) of bivalent promoters.86 The
majority of bivalent domains in ESCs directly overlap transcription start sites of genes that encode transcription factors
and approximately half of the bivalent domains contain binding sites for ≥1 of the pluripotency transcription factors.28
Promoters
Promoters surround transcriptional start sites and serve as
the platform on which the basal transcription machinery and
RNAPII assemble during transcription initiation. They are
generally categorized based on CpG content/DNA methylation status and histone modifications as active, repressed, or
poised (Figure 3). Active genes possess promoters enriched for
H3K4me3, H3K9ac, H3K27ac, and 5hmC and are typically
devoid of 5mC. Conversely, the promoters of repressed genes
are marked by H3K27me3 and H3K9me3, and 5mC. Poised
genes, which are either activated or repressed during development, have bivalent promoters, marked by the presence of both
H3K4me3 and H3K27me3. Poised genes are generally associated with more complex expression patterns and include key
developmental transcription factors, morphogens, and cell surface molecules. In addition, several bivalent promoters seem to
regulate transcript for lineage-specific microRNAs.26
Highly expressed genes, such as metabolic and housekeeping genes, usually contain hypomethylated CpG islands
within their promoters. It is estimated that 95% of transcriptional start sites marked by H3K4me3 contain CpG islands.28
Promoters with low CpG content are more sensitive to regulation by DNA methylation and are typically found at genes
expressed in a highly tissue-specific manner.87 In ESCs, only
a small percentage of low CpG promoters contain significant levels of H3K4me3 and essentially none are marked by
H3K27me3.26
Ren et al87 have proposed that the epigenetic mechanisms
regulating a given promoter may be influenced by the promoter’s sequence. In support of this, they find that genes
preferentially expressed in ESCs and early cell types during
differentiation (neural progenitor cells and mesendoderm) are
CpG rich and contain CpG islands, whereas a much smaller
percentage of genes containing CpG islands are expressed in
tissue-specific manner. Furthermore, they find that developmentally regulated genes are regulated by promoters with high
CpG content and are typically marked by H3K27me3, in contrast to tissue-specific promoters that possess low CpG density
and are preferentially enriched for DNA methylation.88
Enhancers
About 400 000 putative enhancers have been defined in the
human genome based on analysis of histone modifications
and proximity to transcription start sites.89 Enhancers are
typically 200 to 300 bp in length and exhibit nucleosomal
depletion when compared with flanking chromatin.90 They
are enriched for DNaseI hypersensitivity sites and the histone
acetyltransferase, E1A binding protein p300, suggesting an
overall open chromatin structure.90 Indeed, most enhancers
are bound by multiple transcription factors. The Mediator
complex, a transcriptional coactivator, is also commonly
found at enhancers. The Mediator complex is a large multiprotein complex that is necessary for RNAPII recruitment to
the promoters of enhancer-regulated target genes.91 In ESCs,
co-occupancy by Mediator proteins, Oct4, Sox2, and Nanog
is predictive of an enhancer.92,93
Enhancers can be distinguished from promoters by their
unique histone modification signatures and, similar to promoters, these histone modifications can be used to classify
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Figure 3. Epigenetic control and remodeling of regulatory elements during development. A, Poised enhancers/promoters are
typically found in pluripotent stem cells. They possess epigenetic modifications indicative of both transcriptionally active (green marks)
and repressed (red mark) chromatin. For instance, the Trithorax Group (TrxG) proteins and polycomb group (PcG) proteins, which catalyze
H3K4me3 and histone 3 lysine 27 trimethylation (H3K27me3), respectively, localize to poised promoters. Poised regulatory elements
are also generally devoid of 5-methylcytosine (5mC) and enriched for 5-hydroxymethylcytosine (5hmC). Poised enhancers can be
distinguished from poised promoters by the presence of the enhancer-specific mark, H3K4me1, and bound Mediator complex. Chromatin
loops organized by CCCTC-binding factor (CTCF) binding localizes distal poised (or active) enhancers and promoters within close
physical proximity to facilitate transcription. B, The transition from poised to active regulatory elements involves loss of PcG localization,
and demethylation and concomitant acetylation of H3K27. Gains of H3K36me3 within gene bodies are also observed on gene activation.
C, Heterochromatin formation is associated with gene repression. This is mediated, in part, by erasure of activating histone modifications
(H3K4me1, H3K27ac, and 5hmC), establishment of repressive marks, such as H3K27me3 and 5mC, and nucleosomal compaction.
DNMT, DNA methyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; MBD, methyl-DNA binding domain protein;
MED, mediator complex; PcG, polycomb group complex; POL II, RNA polymerase II; TET, ten eleven translocation dioxygenase; TF,
transcription factor; TrxG, trithorax group complex.
them as active, poised, or repressed (Figure 3). For example, although both promoters and enhancers typically possess H3K27ac, enhancers are characteristically enriched
for H3K4me1, in contrast to the H3K4me3 seen at promoters.80,94,95 Heintzman et al90 and Rada-Iglesias et al95 have categorized bivalent enhancers based on histone modification
signatures; class I elements represent active enhancers and
are enriched for H3K4me1 and H3K27ac and the Mediator
complex. Class II elements exhibit high levels of H3K27me3
in addition to H3K4me1 and are considered to be poised enhancers. Repressed enhancers are devoid of both H3K4me1
and H3K27ac and instead are marked by H3K27me3. Class
I elements are expressed at a higher level than class II elements, which agrees with the RNAPII occupancy observed at
class I elements but not at class II enhancers.95 Active enhancers are also enriched for 5hmC and depleted of 5mC; whereas
inactive and developmentally poised enhancers are marked
by 5mC and 5hmC, respectively.41,42,80
Enhancers are highly variable between lineages and cell
types and confer cell-type–specific or lineage-restricted gene
expression.88,94,96,97 Interestingly, only a small percentage of
either class I or II enhancers overlap with CpG islands, and it
is estimated that 94% of lineage-restricted enhancers are CpG
poor and depleted for 5mC.88,95 Furthermore, most lineagerestricted enhancers have low CpG density and are marked by
H3K27ac in ESCs and cells from the early germ layers. These
same enhancers are enriched for H3K27me3 and DNA methylation in terminally differentiated cells, which negatively
correlates with expression of their target genes.88
Super enhancers are a recently identified class of enhancers
that play a major role in determining cellular identity.97,98 They
are characterized as large genomic regions (≥50 kb) possessing
enhancer-like features: enrichment for H3K4me1, H3K27ac,
Mediator complex, and transcription factors. However, unlike typical enhancers, they contain unusually high levels of
Mediator complex proteins and transcription factors and display enrichment for cohesin and Nipbl (Nipped-B homolog), a
protein involved in enhancer–promoter communication.97 The
≈200 super enhancers are often observed as clusters of smaller
enhancers enriched for transcription factor motifs involved in
cell identity.97,98 Therefore, genes regulated by super enhancers are highly cell type specific and are usually expressed at
higher levels than genes regulated by typical enhancers. For
example, nearly all genes associated with ESC identity (eg,
POU5F1 [OCT4], SOX2, NANOG, and KLF4) are regulated
by super enhancers.98
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Epigenetic Dynamics During Differentiation
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The epigenome of PSCs is dramatically different from that of
somatic cells. When comparing histone modification and DNA
methylation patterns between differentiated cells and ESCs,
Hawkins et al99 estimate that approximately one third of the
genome differs in chromatin structure. Many of these differences can be attributed to differential transcription factor binding and chromatin state remodeling during differentiation.96,99
For example, differentiated cells have larger regions marked by
H3K9me3 and H3K27me3 when compared with ESCs.99 Most
of these H3K9me3 and H3K27me3 domains are significantly
larger (2–3-fold) in fibroblasts and T lymphocytes when compared with ESCs, whereas H3K36me3 domains are similar between ESCs and differentiated cells.99,100 These large expanded
H3K27me3 domains are remodeled to an ESC-like state during reprogramming.100 This suggests that the expansion of
H3K27me3 domains accompanies cellular differentiation.
Bivalent domains in ESCs often transition to H3K27me3
domains that expand in lineage-committed cells such that
H3K27me3 affects ≈40% of the genome in differentiated cells compared with only ≈8% of the ESC genome.100
Differentiation results in a progressive, global H3K27me3
enrichment that occurs in a cell-type–specific manner. These
results identified Polycomb-mediated repression as an important mechanism for cell fate determination and lend support
to the long-standing idea that differentiation results in an increasingly restrictive chromatin landscape.88,99,100 The majority of bivalent domains observed in undifferentiated ESCs
resolve to a monovalent status in cells committed to a given
lineage.26,96 Bivalent domains found in promoters encoding
transcription factors typically resolve to either H3K4me3 only
or H3K27me3 only in a lineage-specific manner.96 A clear
example of this can be found during cardiomyocyte differentiation from ESCs. Many key transcription factors, such as
GATA6, GATA4, NKX2.5, HAND2, TBX5, and signaling molecules (BMP2 and WNT5A) involved in cardiac development
gradually lose H3K27me3 and gain H3K4me3 concomitant
with transcription in a stage-specific manner as differentiation proceeds.101,102 Conversely, later stages of cardiac differentiation require EZH2–mediated epigenetic repression of
progenitor-specific transcription factors for cardiomyocyte
maturation.102,103 Loss of H3K4me1/me3 is usually accompanied by the gain of DNA methylation.96 Transcription factor
motifs are particularly enriched for gains of DNA methylation during germ layer specification. For example, the number
of transcription factor motifs that gain DNA methylation are
≈2-fold greater in the definitive endoderm than in either the
early ectoderm or the mesoderm.96 Loss of transcription factor
occupancy during differentiation may trigger DNA methylation; for example, putative regulatory elements downstream of
the proneural gene DBX1 (developing brain homeobox 1) gain
DNA methylation in the endoderm and mesoderm but not in
the ectoderm where this gene is expressed.96
In contrast, many poised enhancers become active (assessed
by the loss of H3K27me3, gain of H3K27ac, and acquisition
of RNAPII) during the transition from pluripotency to germ
layer specification.95 Bruneau and colleagues have found that
the majority of enhancers are poised at any given stage during
cardiomyocyte differentiation. However, a small group of enhancers become active in a cell-type–specific manner. For example, the enhancers that become active in cardiac progenitors
regulate genes essential for heart morphogenesis and development, whereas later during differentiation there is enrichment
for genes encoding cardiac structural and functional proteins.104
Loss of DNA methylation at regulatory elements is also common during germ layer formation and lineage commitment and
is especially prevalent in ectoderm formation relative to the
other germ layers.27,96 Similar to the idea of poised regulatory
elements, there are examples of lineage-specific epigenetic
priming during development. Specifically, many regions that
are associated with fetal and adult brain development exhibit
high levels of DNA methylation in ESCs. These regions are
demethylated and resolve to an H3K4me1 state early during
ectoderm differentiation yet remain methylated in the other lineages.96 Many of the genes that acquire H3K4me1 do not show
immediate expression changes and, therefore, can be said to be
primed for transcription at later stages. There are similar examples of epigenetic priming in cardiogenesis and lymphogenesis, suggesting that epigenetic priming is a common feature
of development.104,105 Other examples of priming include distal
CpG-poor regions that exhibit high DNA methylation levels in
ESCs are demethylated and transition to H3K27me3 only in a
lineage-specific manner. These regions are generally required
at later stages of development in that particular lineage,96 further emphasizing the dynamic nature of histone-mediated epigenetic regulation when compared with DNA methylation.
Genes that temporally regulate cellular identity during differentiation can also be the subject to cyclic methylation events.
For instance, a subset of genes necessary for neuronal progenitor fates are demethylated in neural precursors, expressed in
progenitors, and then remethylated and repressed in later cell
types, such as neurons.
Epigenetic Effects on Nuclear Organization
and Genome Topology
At the most basic level, chromatin can be viewed as beads
on a string where DNA (string) is coiled around nucleosomes (beads) with ≈165 bp periodicity to form the 11-nm
fiber. Subsequent compaction generates the 30- and 300-nm
fibers, ≈700-nm chromosomal domains, and ≈1400-nm mitotic chromosomes.106 During interphase, each chromosome
occupies its own defined space within the nucleus, referred
to as a chromosome territory. This allows nonrandom juxtapositions or physical interactions between genomic regions
both in cis (intrachromosomal) and trans (interchromosomal). These interactions are characterized using chromosome
conformation capture (3C) techniques and their derivatives,
such as 4C, which permits interrogation of multiple genomic
contacts at a single locus, and Hi-C or Chromatin Interaction
Analysis by Paired-End Tag Sequencing (CHIA-PET),
which have the power to identify many associations, thereby
providing information on genome topology.107 Chromosomal
regions of high gene density and high transcriptional activity tend to loop out of their respective chromosome territories and indiscriminately associate in trans with each other.
Unlike associations of active regions, subnuclear movement
of inactive regions is more locally constrained and hence
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these interactions tend to be intrachromosomal within a
chromosome territory core.108
Somatic nuclei possess a nuclear organization different
from pluripotent cells, and these differences are largely reflected by chromatin state.109 Pluripotent cells contain less
heterochromatin in general and less chromatin associated
with the nuclear lamina and nucleoli than differentiated cells.
Lamina-associated domains (LADs) are a class of stable
chromatin domains found at the nuclear periphery that typically possesses low levels of gene expression.110 There are
≈1300 LADs in mammalian cells ranging in size from 0.1
to 10 Mb. Coincident with their transcriptional status, LADs
are enriched for the heterochromatic marks, H3K9me2/3 and
H3K27me3, and their borders usually contain CpG islands
and binding sites for the chromatin insulator, CCCTC-binding
factor (CTCF).110 The interaction pattern and number of LADs
seem to be cell type specific; however, some LADs are common across cell types.111 During differentiation, specific LADs
containing genes destined for activation in the differentiated
progeny translocate from the nuclear periphery to the interior. Conversely, genes to be repressed in the next cell type
condense and are often relocated to the periphery where they
interact with the nuclear lamina.111 Interestingly, genome reorganization occurs sequentially during differentiation. For
example, specific genomic regions are relocated from the
nuclear lamina during differentiation of ESCs to neural precursor cells.111 These regions remain spatially constant during
the differentiation of neural progenitor cells to astrocytes, but
a new, distinct group of LADs relocates away from the periphery during this subsequent differentiation step. Repressed
genes that relocate away from the nuclear lamina become unlocked and are primed/poised for activation later during differentiation, whereas active genes that shuttle to the nuclear
lamina become locked and stably repressed.
In addition to LADs, mammalian genomes contain ≈2000
large domains called topologically associating domains,
which are megabase-sized local interaction domains that inhibit spreading of heterochromatin in cis.112 They are generally bounded by invariant CTCF-binding sites, tRNAs or small
interspersed elements, and contain coordinately regulated
enhancers and promoters that cluster together. The CTCFbinding sites that demarcate the boundaries of a large number
of topologically associating domains are highly conserved between human and mouse and between cell types. Thus, they
are unlikely to play a role in the establishment of cell-type–
specific genomic topology during differentiation. Subregions
within each topologically associating domains, however, seem
to be dynamic.112 These dynamic regions may confer cell-type–
specific genome organization via transcription factor complexes bound at the regulatory elements of active genes (Figure 4).
The genome of PSCs is divided into a large complex pattern
of chromatin loops bounded by CTCF-binding sites.113 These
loops can be classified according to epigenetic modifications
found within the loop and at the loop boundary.113 Loops
are generally enriched for either active or repressive histone
modifications inside the loop in a mutually exclusive manner
although some loops do not seem to be enriched for any particular histone modification. Interestingly, ≈20% of loops are
enriched for enhancer marks (H3K4me1/2) within the loop,
Figure 4. Schematic of cell-type–specific genome topology.
Genetic loci (depicted as colored circles) cluster together making
both intra (Chr A:Chr A) and inter (Chr A:Chr B) chromosomal
interactions in a cell-type–specific manner. This establishes
coregulated transcription of multigene networks that confer cellular
identity. Differing topological interaction patterns are expected to
be associated with different cell types, such as embryonic stem
cells (cell type 1) and progenitor cells (cell type 2).
promoter marks (H3K4me3) at the loop boundary, and marks
for active transcription (H3K36me3), and repressed chromatin
(H3K27me3) outside the loops on opposite sides. CTCF and
cohesin function together to form chromatin loops that segregate heterochromatin from euchromatin.113,114 An example of
chromatin dynamics that occur during differentiation can be
found at the HOXA locus. In ESCs, the HOXA locus consists
of bivalent chromatin.114 On differentiation, chromatin reorganization results in demarcation of a euchromatic and a heterochromatic domain at a highly conserved CTCF-binding site
bound by both CTCF and cohesin.114
It has become increasing clear that interactions between transcription factors and Mediator complex bound to regulatory elements influence genome topology through their interactions
with CTCF and cohesin. For example, Oct4 has been shown
to interact with CTCF during the establishment of XCI and the
maintenance of bivalent chromatin.114,115 Conversely, loss of
Oct4 expression during differentiation permits the CTCF/cohesin-mediated heterochromatin formation at the HOXA locus114
mentioned earlier. Interactions between Mediator and cohesin
at enhancers have been shown to regulate gene expression in a
cell-type–specific manner by regulating the formation of chromatin loops that bring distal enhancers into close proximity
with promoters of active genes.93 Interestingly, both active and
poised enhancers participate in looping interactions in differentiated cells, suggesting that chromatin looping may precede
and influence epigenetic priming116 during differentiation.
Promoter clusters play a central role in chromosome topology
but their contribution seems to not be tissue specific because
promoters have an equally probable chance of interaction in
ESCs and in somatic cells. Surprisingly, high-resolution analyses have determined that promoter states are largely invariant
and highly stable between diverse cell types.80,90 Furthermore,
the majority of promoter clusters exhibit activity in multiple
cell types.80 However, enhancers contribute to genome topology in a more tissue-specific manner.93,117,118 For example, in
ESCs, the SOX2 promoter interacts with a cluster of enhancers that are specifically active in ESCs, whereas a different set
of enhancers is associated with the SOX2 promoter in neural
progenitor cells.118,119 In general, cell type-specific enhancers
are highly methylated in cells of other lineages and a direct
correlation exists between DNA demethylation and enhancer
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activation.118 Inactive enhancers are highly methylated and
become demethylated in a tissue-specific manner during development. Interestingly, in addition to the cell-type–specific
transcription programs regulated by enhancers, differential
enhancer usage may also regulate common sets of promoters.
Indeed, cell-type–specific enhancers have been shown to interact with promoter clusters common to multiple cell types,
suggesting that different cell types use specific enhancers to
regulate broadly expressed housekeeping genes.118,119
During reprogramming to iPSCs, cells acquire a spatial
genomic organization specific to pluripotent cells. In ESCs,
genes involved in the maintenance of pluripotency (ie, Dppa5,
Zfp42, Zfp281, Lefty, and Lin28) make interchromosomal contacts with the Nanog locus, suggesting that pluripotency genes
aggregate together.117 Reprogramming-induced reactivation of
the Nanog locus results in the establishment of pluripotencyassociated gene contacts that are not observed at the Nanog
locus in somatic cells.117 Furthermore, in ESCs, it has been
shown that the majority of genes associated with reprogramming are spatially connected to the SOX2 locus within 1 large
gene cluster, implying they may be coordinately regulated
within an active structure termed a transcription factory.119
Imprinting and XCI
Two specialized modes of epigenetic regulation are imprinting
and XCI. Both rely on combinatorial epigenetic silencing (ie,
a complex interplay of repressive histone modifications, CpG
methylation, and noncoding RNA modulation of chromatin
structure). Genomic imprinting is parent-of-origin allele-specific expression thought to have arisen to allocate extraembryonic nutrient resources to the developing fetus.120 The number
of imprinted genes is a matter of debate. Estimates in mice
range from ≈150 to >1000 imprinted genes,121 but recent work
indicates that there are probably not many more than ≈200.18,122
The majority of imprinted genes contain DMRs that possess
parent-of-origin–specific DNA methylation patterns (CpG and
non-CpG) at enhancers and promoters that confer allele-specific expression. Many imprinted genes are expressed in the brain
in a temporal manner to regulate neurodevelopment. Loss of
imprinting, either through gain or loss of DNA methylation
(or genetic deletion of regulatory elements/DMRs), results in
biallelic gene silencing or expression that has been implicated
in cancer and many neurodevelopmental disorders, including
Beckwith–Wiederman Syndrome, Prader–Willi Syndrome,
and Angelman Syndrome.123 Genomic imprints are known to
be unstable in mESCs. Recent studies examining a large number of hPSC lines come to the same conclusion. Tissue samples
and androgenetic and parthenogenetic hPSC lines have been
used to establish the normal DNA methylation status of several
imprinted genes. Many hPSC (ESC and iPSC) lines exhibit
both and gains and losses of methylation at imprinted loci leading to loss of imprinting and either gene silencing or biallelic
expression. These same loci seemed to be epigenetically stable
in primary cell lines and tissues. Moreover, certain imprinted
genes (PEG3, MEG3, and H19) exhibit loss of imprinting via
hypermethylation and gene silencing in several hPSC lines.
More importantly, aberrations in genomic imprints and XCI in
undifferentiated hPSCs are faithfully maintained during differentiation and cannot be reset with further reprogramming.27,124
XCI is an excellent example of many epigenetic modes acting synergistically to control gene expression via chromatin
structure and chromosome topology. XCI is a means of dosage
compensation between XY males and XX females. Shortly
before embryo implantation, one of the X-chromosomes
is randomly chosen for transcriptional repression. The earliest event in XCI is expression of XIST, a long noncoding RNA expressed from the X-inactivation center on the
X-chromosome marked for inactivation. XIST then spreads
along the X-chromosome beginning with regions either interacting with or in close proximity to the XIST locus followed by spreading to more distant regions. Specific domains
of XIST recruit PRC2 to establish transcriptional repression.
Together, PRC1 and DNA methylation maintain heritable XCI
in somatic cells. In the naïve state of undifferentiated mESCs,
both X-chromosomes are active (Xa/Xa). However, hPSCs
generally exhibit XCI (Xa/Xi) on derivation. Recently, it has
been shown that the inactive X in hPSCs often becomes reactivated over time in culture.27,124 Once reactivated, XCI is
not re-established by differentiation.124 Erosion of XCI during
time in culture can have profound effects on the use of female
hPSCs for cell therapy, disease modeling, or drug screening.125
Careful analysis using SNP (single nucleotide polymorphism)
genotyping combined with allele-specific reverse transcription quantitative polymerase chain reaction demonstrates that
erosion or loss of XCI is common among late passage female
hPSCs.27 Loss of XCI in undifferentiated hPSCs is associated
with XIST repression, loss of H3K27me3, and derepression of
genes silenced on the Xi. Once lost, the active state is stable
and XCI cannot be re-established by differentiation.124
Epigenetic Heterogeneity Among PSCs
Reprogramming of a somatic cell fate to a pluripotent state
is largely an epigenetic process.126 Indeed, removal of certain
epigenetic roadblocks either by chemical inhibition of DNA
methylation or knockdown of MBDs during reprogramming
enhances iPSC generation.127,128 In addition, inclusion of histone deacetylase inhibitors during exogenous transcription
factor–based reprogramming and somatic cell nuclear transfer
also increases the proficiency of PSC derivation.129,130 The 3
different types of PSCs, ESCs, iPSCs, and SCNT ESCs (derived by somatic cell nuclear transfer), display global methylation patterns that are similar to each other and different
from somatic cells. The first in-depth study to examine the
epigenomic differences among the 3 classes of mouse PSCs
found that SCNT ESC global DNA methylation patterns were
more similar to ESCs than iPSC patterns were to ESCs.131 This
suggests that the ooplasm is more robust at reprogramming
somatic genomes than transcription factor–based reprogramming. Now that human SCNT ESC lines are available132–134;
it will be interesting to compare the 3 hPSC epigenomes and
contrast them with data generated from mouse-derived PSC
types to determine whether conclusions drawn from this study
are applicable to humans.
It is generally accepted that transcription factor–based reprogramming does not completely remodel the epigenome,
at least initially. Multiple studies have shown that a certain
degree of epigenetic memory is retained in early passage iPSCs, and this can lead to biased differentiation propensities
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depending on the donor cell source.131,135–137 This somatic
memory is attributable to incomplete silencing of donor
(source) cell-specific expression patterns. DNA methylation
patterns account for the majority of somatic memory131,138
because histone modification patterns between iPSCs and
ESCs exhibit minimal differences.139,140 During reprogramming, large regions of the genome fail to reset non-CpG
methylation patterns, and many DMRs are specific to and
common across iPSC lines when compared with ESCs.17
Recently, however, it has been demonstrated that these epigenetic memories account for only a small percentage of
the variability among iPSCs and ESCs.141 In addition, it has
been shown that expansion of iPSCs and time in culture diminishes the epigenetic differences between ESCs and iPSCs.135,142 This suggests that iPSC global epigenetic patterns
stabilize over time although certain hotspots of variation
remain, particularly at imprinted regions.27,142,143 A closer
examination of global methylation patterns and gene expression profiles reveals that the variability among ESCs and
iPSCs is largely because of variations among individual cell
lines rather than differences between classes of PSCs.27,141
Genes with the highest variability in expression are genes
induced on differentiation, and genes exhibiting the most
epigenetic variability in PSCs are imprinted genes, such as
H19, PEG3, and MEG3.27,141 Furthermore, when DNA methylation patterns and gene expression profiles are considered
together, the majority of iPSC lines cannot be distinguished
from the majority of ESC lines.27,140,141 Together these studies suggest that the epigenetic heterogeneity observed in
iPSC lines is not substantially different from the heterogeneity seen in ESC lines. Thus, when compared with ESC
epigenetic patterns established during preimplantation development, it seems that iPSCs display a small amount of
epigenetic variation, which can be attributed to reprogramming; however, hiPSCs and hESCs are essentially identical
in their epigenetic profiles and their differentiation abilities.
Conclusions
We have discussed several of the best-understood epigenetic
mechanisms and linked them to current knowledge of PSCs
and cardiac lineage development wherever possible. We did
not touch on nucleosome remodeling, histone variants, histone replacement, or noncoding RNA regulation of chromatin.
This is a fast moving field, so our intention in this review is to
provide a general background for appreciating future studies.
hPSCs are an ideal model for studying the dynamic epigenetic changes that accompany the development of cellular
diversity. High-resolution global analyses of epigenetic dynamics during hPSC differentiation have greatly advanced our understanding of the mechanisms that regulate aspects of human
embryonic development. In the past few years, gene expression
signatures have become a routine means to define specific cell
types that in the past were identified by histology or expression
of a few antigens. Epigenetic technologies hold the promise
to characterize cells by their epigenetic landscape, which can
give tremendous insight into the functional aspects of cell type
specificity. Global epigenetic profiling of heterogeneous cell
populations, such as those found in tissues, has to date been useful to establish the general themes of epigenetic transcriptional
regulation. However, such analyses are ultimately limited, and
epigenetic events important for rare cell types will undoubtedly
be missed. Further refinements in cellular differentiation and
characterization have permitted the study of nearly homogenous populations of cells. As genome-wide, single cell profiling
methods and technologies become more sensitive and inexpensive, there will be far more fine-grained information related to
human development and stem cell differentiation.
New discoveries (eg, 5hmC and its oxidized derivatives)
have begun to shed light on some of developmental biology’s
long-standing questions, such as the epigenomic remodeling events that occur in the earliest stages of development.
Although epigenetic research has advanced greatly in the past
2 decades, much remains to be discovered. For example, evidence suggests the existence of epigenetic readers able to recognize multiple histone marks simultaneously,144 but we have
a limited grasp of the combinatorial nature of histone modifications on the same histone and between histone tails.
The potentially, most powerful epigenomic processes involve changes in genome topology: precise rearrangements of
inter- and intrachromosomal associations that occur in response
to developmental or environmental stimuli. Genomic association studies have shown that growth factor induction results in
hierarchical gene expression networks mediated by the spatial
relocalization of genes.145 It will be exciting to examine the role
of epigenetic changes in chromosomal interactions that result
in gene network activation or repression during differentiation.
Understanding epigenetic regulation of differentiation and
cellular identify is critically important for the use of hPSCs
and their derivatives in clinical applications. Evolving in vitro differentiation protocols increasingly incorporate knowledge about the normal processes of embryogenesis. Because
protocols are fine-tuned to mimic morphogen gradients and
subtle signaling events in the developing embryo, researchers
are learning to develop hPSC-derived cell types that are more
physiologically similar to their in vivo counterparts. Knowledge
of cellular epigenetic states will allow researchers to develop
cell types or combinations of cell types that faithfully represent
human cells and tissues better. Ultimately, this knowledge will
lead to tremendous improvements in predictive in vitro assays
for drug toxicity and efficacy, which will greatly enhance the
effectiveness and safety of clinical trials. Because regenerative
medicine, in the form of cell replacement therapies, begins to
be a viable medical treatment, epigenetic profiling will allow
exquisite quality control of cells used for transplantation.
Acknowledgments
Because of the broad nature of this review and to length restrictions,
we apologize to those investigators whose work was not cited. We
thank the article reviewers for insightful suggestions. We thank members of the Loring lab for helpful comments and suggestions.
Sources of Funding
This work was supported by National Institutes of Health grant
5R33MH087925 and California Institute for Regenerative Medicine
grants RM1-01717, CL1-00502, RT1-01108, and TR1-01250 to J.F.
Loring.
Disclosures
None.
322 Circulation Research July 7, 2014
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Epigenetic Regulation of Pluripotency and Differentiation
Michael J. Boland, Kristopher L. Nazor and Jeanne F. Loring
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Circ Res. 2014;115:311-324
doi: 10.1161/CIRCRESAHA.115.301517
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