Epigenetic Dynamics of Stem Cells

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The life cycle of an organism during developmentfrom a zygote to an adult is characterized by phasesof reprogrammingand differentiation. The changes ingene expression that guide these processes are causedby transcription factors and epigenetic modifications(BOX 1), which impose heritable cellular memories thatdefine both specific cell lineages and cell types1. Thus,on the whole, somatic cells progressively acquire anincreasing array of epigenetic marks (by a processknown as epigenetic programming) that are importantfor cell specification because they establish a heritablememory of cellular states and differentiation pathways.By contrast, germ cells and early embryos can reset(or reprogramme) epigenetic marks24. This epigeneticreprogramming of germ cells is an essential characteris-tic of their immortality and occurs in preparation of theeventual acquisition of totipotency: the epigenetic marks

(termed imprints) that distinguish the parental originof some genes, for example, are erased in primordialgerm cells (PGCs) and then reset in a parent-of-origin-dependent manner, such that they are in place at thetime of fertilization.

The combination of oocyte and sperm in the zygoterepresents the climax of cellular potency: the zygote isthe only unequivocally totipotent cell in the life cycle.During this temporally unique period immediatelyafter fertilization, the sperm nucleus is extensivelyremodelled, replacing protamineswith histones, andundergoes paternal-specific active demethylation ofDNA57. Subsequent cleavage divisions in the early

embryo are characterized by a progressive reduc-tion in DNA methylation as a consequence of pas-sive demethylation caused by the exclusion of DNA(cytosine-5)-methyltransferase 1 (DNMT1) from thenucleus8. Genome-wide reprogramming of histonemodifications also occurs during this period. Thisprocess coincides with the gradual commitment ofcells towards the earliest lineages, which is accom-panied by lineage-specific epigenetic programmingevents.

The steps leading towards the earliest lineagedecisions have been best studied in mice, in whichseparation of the first cell lineages occurs in two suc-cessive, dichotomous differentiation events. First, atthe blastocyststage, the trophectoderm (TE) becomesfixed in its differentiation potential towards theextraembryonic trophoblast lineage and is set apart

from the inner cell mass (ICM). This is followed bythe separation of the ICM into epiblast (or primitiveectoderm) and primitive endoderm (or hypoblast) inthe late blastocyst (FIG. 1). The epiblast will form theembryo proper, whereas the primitive endoderm givesrise to the parietal and visceral endoderm layers that,together with the trophoblast, contribute to extra-embryonic tissues. It is only in the region overlyingthe epiblast at the egg cylinderstage that some cellsof the visceral endoderm will contribute to the defini-tive embryonic endoderm, such that embryonic endo-dermal structures are composed of cells originating inboth the epiblast and the primitive endoderm9.

Laboratory of Developmental

Genetics and Imprinting,

The Babraham Institute,Babraham Research Campus,

Cambridge CB22 3AT,

and Centre for Trophoblast

Research, University of

Cambridge, Downing Street,

Cambridge CB2 3EG, UK.

Correspondence to

M.H. and W.R.

e-mails: myriam.hemberger@

bbsrc.ac.uk;wolf.reik@bbsrc.

ac.uk

*These authors contributed

equally to this work.

doi:10.1038/nrm2727

Published online 15 July 2009

Reprogramming

The resetting of epigenetic

marks, usually to achieve wider

developmental potency.

Reprogramming occurs during

the life cycle in the early

embryo and in the germ line,

and also in experimental

systems in which differentiated

cells are converted into

induced pluripotent stem cells.

Epigenetic dynamics of stem cellsand cell lineage commitment:digging Waddingtons canalMyriam Hemberger*, Wendy Dean* and Wolf Reik

Abstract | Cells of the early mammalian embryo, including pluripotent embryonic stem (ES)

cells and primordial germ cells (PGCs), are epigenetically dynamic and heterogeneous.

During early development, this heterogeneity of epigenetic states is associated withstochastic expression of lineage-determining transcription factors that establish an

intimate crosstalk with epigenetic modifiers. Lineage-specific epigenetic modification of

crucial transcription factor loci (for example, methylation of the Elf5promoter) leads to the

restriction of transcriptional circuits and the fixation of lineage fate. The intersection of

major epigenetic reprogramming and programming events in the early embryo creates

plasticity followed by commitment to the principal cell lineages of the early conceptus.

CHROMATI N DYNAMI CS

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Epiblast

|

PGCs

Developmentalstage

Stem cells

Morula

EG cells

Midlate blastocyst

Somatic cell lineagesEndodermMesodermEctoderm

Dedifferentiationand reprogamming

ES cellsXEN cells TS cells iPS cellsEpiSCs

Fertilization

TEPrimitive

endoderm

Totipotency

The differentiative capacity

of a cell to form all cell types of

the conceptus and the adult

organism, including

extraembryonic membranes,

the placenta and germ cells.

Protamine

A highly basic non-histone

protein that replaces histones

in the sperm to achieve dense

packaging of DNA.

Blastocyst

The embryo before

implantation that contains at

least two distinct cell types:

an outer epithelial cell layer

(trophectoderm) and an inner

group of cells (inner cell mass).

Egg cylinder

The embryonic stage after

implantation that comprises a

double-layered structure of the

outer endoderm and the inner

embryonic or extraembryonic

(trophoblast) ectoderm,

enclosing a narrow lumen.

Primitive streak

A structure that is formed at

the posterior end of amniote

embryos at gastrulation stages

and is the area of mesoderm

formation.

After these initial decisions have been made, cells

of all three lineages (epiblast, primitive endoderm andtrophoblast) undergo successive steps of differentiationto form all cell types of the organism, as well as the pla-centa. A few cells, however, (the germ cells) are set asideearly on in the egg cylinder to close the life cycle. Thegerm cells represent the end point of a differentiationcascade and can be considered highly specialized. Atthe same time, however, they have the capacity to returnto totipotency (as a consequence of germ cell-specificepigenetic reprogramming) when the gametes cometogether in the zygote.

In this Review, we focus on the epigenetic dynamicsthat underlie differentiation and reprogramming eventsduring two key time points in the life cycle of the mousethat might be particularly important for the regulation oflineage decisions and define the potency of a cell. In thiscontext, we concentrate on recent insights into the rolesof DNA methylation and modifications of histones inthe epigenetic control of developmental pathways.

Epigenetic dynamics during the life cycle

The progression of PGC specification, migration anddifferentiation gives rise to mature gametes, spermand oocytes, which come together in the zygote to forma new organism.

Epigenetic reprogramming in PGCs.PGCs are uniquecells that are designated to regain the capacity to forma new organism. They arise from a few cells expressingB lymphocyte-induced maturation protein 1 (Blimp1; alsoknown as Prdm1) that can first be identified at embryonicday 6.25 (E6.25) of mouse development. PGCs are speci-

fied at E7.25 as a group of approximately 40 cells in theextraembryonic region posterior to the primitive streak,which are demarcated by expression of Blimp1and Stella(also known as Dppa3)1012. PGCs undergo extensive epi-genetic reprogramming during and after their migrationthrough the hindgut to the developing gonads, culmin-ating in the erasure of parental imprints and many otherepigenetic marks throughout the genome2,13,14.

Although the mechanisms of erasure and the exacttiming of these reprogramming events are still unclear,recent studies have revealed important details of the

Box 1 | Epigenetic modifications

Epigenetic modifications are instructions that are imposed on the DNA sequence

to provide an additional level of gene regulation and to establish heritable

transcriptional states. In mammals, DNA methylation and covalent modifications of the

amino-terminal tails and the core of nucleosomal histones constitute most epigenetic

modifications. DNA methylation occurs symmetrically on the cytosine base in CpG

dinucleotides and is generally associated with gene repression and formation of

heterochromatin. Histone modifications encompass a range of modifications, includingacetylation, methylation, phosphorylation, ADP ribosylation and ubiquitylation, that

extend the information content of the underlying DNA sequence and confer unique

transcriptional potential. Histone modifications can have both repressive and

activating functions. The best-understood modifications are the trimethylation of the

Lys9 and Lys27 residues of histone H3 (H3K9me3 and H3K27me3), which have

repressive functions, and H3K4me3 and H3K9 acetylation (H3K9ac), which are

associated with active genes. Many other modifications on histones have been

identified that regulate gene activity, and many enzymes are now known that modify

histones or methylate DNA. More recently, activities have also been discovered that

can reverse some of these epigenetic marks (see also the epigenetic modifiers in FIG. 4).

Figure 1 |Key differentiation stages in development and derived stem cell types. Alife cycle diagram of the first

principal cell lineages of the early embryo and the stem cell populations that can be derived from them. The firstdefinitive differentiative decisions are made at the blastocyst stage, in which the trophectoderm (TE) layer is set

aside from the inner cell mass (ICM; not shown). The ICM subsequently delaminates a layer of primitive endoderm.

The remaining cells form the epiblast. All three cell lineages that is, TE, primitive endoderm and epiblast give rise

to distinct stem cell types: trophoblast stem (TS) cells117, extraembryonic endoderm stem (XEN) cells118and embryonic

stem (ES) cells, respectively. These three types of stem cell can also be established from earlier morula stage embryos,

and their lineage allocation programme is presumably recapitulated during the in vitroculturing process47. Immediately

after implantation, the epiblast can give rise to a different stem cell population, termed epiblast stem cells (epiSCs)48,49.

The three embryonic germ layers, endoderm, mesoderm and ectoderm, are formed during gastrulation. Primordial germ

cells (PGCs) originate from a small mesodermal cell population at the posterior tip of the epiblast2,11,12. Embryonic

germ (EG) cells can be derived from early stage PGCs between embryonic day 8.5 (E8.5) and E11.544,45,50. The three

embryonic germ layers go on to differentiate into all cell types of the adult organism. Recent breakthroughs in

dedifferentiation techniques allow the reprogramming of fully differentiated somatic cells into induced pluripotent

stem (iPS) cells, which closely resemble ES cells in their developmental potency100103.

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=12142&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=73708&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=73708&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=12142&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum


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Facultative heterochromatin

The fraction of chromatin that

is condensed and inactive in agiven cell lineage, which can be

decondensed and active in

another.

Constitutive

heterochromatin

The fraction of

heterochromatin that stays

compact throughout the cell

cycle. It is mainly composed of

repetitive sequences (satellite

DNA) and is concentrated in

characteristic regions, such as

centromeres.

Pericentromeric

heterochromatin

The heterochromatin structure

composed mostly of satellite

repeats that are located

adjacent to the centric

satellites. Together these

comprise the centromeres and

form an important structural

element for chromosome

stability.

Centromere

The region of a chromosome

that is attached to the spindle

during nuclear division.

DNA remethylation takes place several days aftermany of the histone marks have been reset and occursfrom E15E16 onwards in the male germ line but onlypostnatally during oocyte growth in the female germline2. This process results in a DNA methylation state thatis globally similar to that of somatic cells but with impor-tant pattern differences, including at differentially meth-ylated regions that serve as imprinting control regions(ICRs). ICRs are marked either by maternal or paternalmethylation in the germ line and regulate parent-of-origin-specific expression of imprinted genes in thenext generation. It is worth noting that several aspects ofreprogramming in PGCs and later stage germ cells (forexample, the erasure and re-establishment of imprints)result in specialized epigenetic programmes in the maturegametes, some of which are carried forward and are likelyto influence epigenetic reorganization in the early embryo,

potentially with lasting effects on pluripotency andpossibly also lineage commitment events.

Epigenetic reprogramming in early embryos. The organ-ization of the genome in the two types of gamete differsdramatically. Sperm chromatin is packaged extremelydensely using predominantly protamines in addition toa modest amount of sperm-specific histone variants25.Although the mechanism of protamine removal is stillnot known, it follows that the differences between maleand female chromatin organization should be resolvedshortly after fertilization to ensure correct chromosomesegregation in the first cleavage division of the zygote.

Remarkably, however, the chromatin organization ofthe two pronuclei remains strikingly different. Thus, thepaternal genome lacks the repressive heterochromaticH3K9me3 and H3K20me3 modifications that normallymark pericentromeric heterochromatinand are essential forcentromeric function during mitosis in somatic cells26,27.The paternal pronucleus retains H3K9me1 in pericen-tromeric regions along with residual DNA methyl-ation. These marks, together with a different repressivemechanism that is mediated by Polycomb group (PcG)repressive complex 1 (PRC1), which is provided by theegg cytoplasm, serve as a specialized repressive mecha-nism in the male pronucleus to mediate pericentromericheterochromatin formation2831.

This organization ensures that, despite their differentchromatin features, male and female pronuclei display acomparable centromereorganization that allows propermetaphase plate arrangement and chromosome segrega-tion during nuclear division31. Thus, although the genomesare functionally equivalent, the distinction of their paren-tal origin is preserved during the return to totipotency in

the zygote. It remains to be seen whether preservation ofthese epigenetic parent-of-origin marks (which are dis-tinct from those that mark imprinted genes) is importantfor early development and for the first cell determinationevents, or whether these marks are merely a remnant ofthe paternal or maternal origin of the genomes.

Fertilization initiates a second wave of epigeneticreprogramming that is characterized by rapid activeDNA demethylation before the onset of DNA replica-tion57and is followed by passive DNA demethylation upto the morulastage3234. In contrast to the reprogrammingof DNA methylation in PGCs, parent-specific methylationat ICRs is retained during this reprogramming phase35,36.

De novoDNA methylation is initiated before theblastocyst stage and thus coincides with the earliest dif-ferentiation event that separates the embryonic andtrophoblast lineages (represented by the ICM and TE,respectively)7. Intriguingly, global DNA methylation levelsare markedly different between the embryonic and extra-embryonic lineages; the TE is relatively hypomethylatedcompared with the ICM, as revealed by 5-methylcytosinestaining 7 (FIGS 2,3). These global differences persistthroughout development and are retained in the embryoand placenta37,38. However, more recent whole genomeapproaches have revealed that promoters might repre-sent an exception to this pattern, which might account forthe similarity in overall transcriptional activity between the

lineages39,40. Hence, methylation differences are mostlylocalized to intergenic regions and non-promoter genicsequences. Similar to DNA methylation, several his-tone modifications, including H3K27me1, H3K27me2,H3K27me3 and H3K9ac, also exhibit an asymmetrybetween the ICM and TE and might therefore have arole in lineage allocation and/or lineage commitment, asdiscussed in more detail below4143.

Epigenetic interface with pluripotency

Three principal phases in murine development allowthe derivation of pluripotent cell lines in vitro(FIG. 1).These developmental stages include early PGCs between

Box 2 | A geneticepigenetic network

A simple binary view of the presence or absence of epigenetic modifiers and

lineage-specifying transcription factors is insufficient to explain their functions. Their

activities are additionally modulated on various levels by different mechanisms. DNA

(cytosine-5)-methyltransferase 1 (DNMT1), for example, can be methylated, and this

modification reduces its stability. Lys-specific demethylase 1 (LSD1; also known as

KDM1) is required for demethylation and stabilization of DNMT1 (REF. 112).

Phosphorylation of heterochromatin protein 1(HP1) by casein kinase II (CK2) causesits release from methylated histone H3 at Lys9 (H3K9) 113. Also, automethylation of the

euchromatic H3K9 methyltransferase G9A (also known as EHMT2) creates binding sites

for HP1and HP1, thereby redistributing these chromatin proteins114. Similarly, thedistinction between activating and repressive functions of the pluripotency

transcription factors might be imposed by post-translational modifications (for

example, sumoylation of OCT4, which is encoded by Pou5f1) that in turn might alter

stability, DNA binding, dimerization and protein complex association capacities115.

Another crucial regulatory mechanism relies on the subcellular shuttling of epigenetic

modifiers between the nucleus and cytoplasm. This is particularly important in the zygote

with respect to the activity of DNMT1: nuclear exclusion from the first S phase results in

the sequential reduction of DNA methylation of much of the genome during the crucial

period that leads up to lineage determination116. In the germ line, the highly defined

window of nuclear exclusion of chromatin proteins around embryonic day 11.5 (E11.5), at

a time when erasure of epigenetic marks takes place, suggests a causal relationship with

this process. Interestingly, this change of localization might be permanent (for example,histone H2A.Z), temporary (for example, histone macroH2A) or reciprocal (for example,

the histone chaperone chromatin assembly factor 1 (CAF1), which translocates from the

nucleus to the cytoplasm, and nucleosome assembly protein 1 (NAP1), which translocates

from the cytoplasm to the nucleus)15. The exclusion of DNMT3B from the nucleus at E12.5

might prevent pre-emptive remethylation of the germline genome18.

These examples establish a range of secondary epigenetic modifiers that are not

necessarily epigenetic in nature but impose an additional level of regulation and are of

equal importance to the primary modifiers.

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OCT4

NANOG

SOX2

SALL4

STELLA

CDX2

EOMES

ELF5

DNAmethylation

TS cells

ES cells

Lineage fixation

Trophoblast lineage

Embryonic lineage

Absent StrongElf5 Stella

Egg cylinder

Zygote 2 cell 4 cell 8 cell 16 cell

Early blastocyst

Late blastocystPrimitiveendoderm

TE

ICM

Expression in PGCs

Levels assumed

Epiblast

Epiblast

Trophoblast

E8.5 and E12.5 that can give rise to EG cells, morula-to-blastocyst stage embryos from which embryonic stem (ES)cells can be derived, and E5.5E6.5 epiblast tissue that cangive rise to epiblast stem cells (epiSCs) 4449. Interestingly,

human ES cells might be more similar to mouse epiSCsthan to mouse ES cells, which is an important considera-tion for the interpretation of studies that compare ES celldata from different species48,49.

Figure 3 |Stochasticity of gene expression, developmental potency and lineage commitment. Thestochasticity

of gene expression and mutual interactions between lineage-determining transcription factors with antagonizing

functions gradually lead to the first cell lineage decisions. These transcription factors include OCT4 (which is encoded

by Pou5f1)74,119, the homeobox protein NANOG74, SRY box-containing factor 2 (SOX2)120, Sal-like protein 4 (SALL4)121,

STELLA (also known as DPPA3)122, caudal-type homeobox 2 (CDX2)74,123, eomesodermin (EOMES)75,124and ELF5 (REFS 90,91).

This process is accompanied by epigenetic programming events that impose a permissive (or non-permissive) environment

for cell fate or might predispose a cell towards a particular lineage. It is feasible that the expression of each transcription

factor oscillates between a minimum and maximum level, and positive or negative interference of such transcriptional

noise introduces a developmental bias in individual blastomeres (indicated by the cell shading). Expression of keytranscription factors remains mosaic in individual blastomeres until the blastocyst stage, when the embryonic (the inner

cell mass (ICM), epiblast and derived embryonic stem (ES) cells) and trophoblast (the trophectoderm (TE) and derived

trophoblast stem (TS) cells) lineages are established. De novoDNA methylation is initiated between the morula (16-cell

stage) and blastocyst stage7. Lineage fixation coincides with methylation of Elf5in the ICM, which establishes a tight

epigenetic boundary between the embryonic and trophoblast lineages91. Methylation of Stellaoccurs in the same overall

time window (although probably slightly later than Elf5) and might have a similar gatekeeper function to establish the

post-implantation epiblast77.

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OCT4

NANOG

SOX2

SALL4

SUZ12, EED and EZH2

PHC1 and PHC2

JMJD1A*

JMJD2C

EHMT1 and G9A

DNMT3A, DNMT3B,DNMT3L and HELLS

ARID3BARID3AARID5BCDYLCDYL2CHD9JARID1BJARID2JMJD1CJMJD2A

MYST4PRDM14*RESTSETSETD1BSMARCA2SMARCA3SMARCAD1*SMARCD1

H3K9me2 andH3K9me3

H3K27me3

De novo

DNA methylation

Epigenetic modifiers regulatingpluripotency factors

Pluripotency factors regulatingepigenetic modifiers

* In human ES cells

Morula

A ball of cells that is created

by the first cleavage divisions,

before compaction, that

initiates the formation of the

blastocyst.

It is worth pointing out that the stem cell aspect ofthese different types of pluripotent mouse cell lines, inparticular their self-renewing capacity, is a feature thatis only acquired during culture in vitro, presumably bydisrupting the normal context of cellcell interactionsand signalling, and by the preservation or acquisitionof epigenetic states that allow continued proliferation.

Crucial factors necessary to maintain pluripotency. Akey feature that reflects the developmental capacity ofthese EG, ES and epiSCs cell lines is that, as in the earlyembryo, they express markers of pluripotency suchas OCT4, NANOG and SOX2 (REFS 4751)(FIGS 3,4).

The genetic basis of pluripotency has been elegantlyelaborated in ES cells, in which a transcriptional net-work driven by the core transcription factors OCT4,NANOG and SOX2, is essential to maintain the undif-ferentiated state. This network activates genes that arerequired for ES cell survival and proliferation whilerepressing target genes that are activated only duringdifferentiation52,53. With growing insights into the fac-tors required for pluripotency, this core triumvirate isexpanding; for example, with the notable addition ofthe zinc finger transcription factor sal-like protein 4(SALL4)5457. It seems that the co-expression and co-regulation of these factors is one of the key featuresof pluripotency. Intriguingly, OCT4, NANOG, SOX2and SALL4 regulate their own expression and controla set of target genes through mutual heterodimeriza-tion and/or shared promoter occupancy, thus forminga self-reinforcing circuit of pluripotency52,5760.

In addition to these transcription factors, chroma-tin organization and epigenetic modifications are alsokey elements for controlling gene expression during

ES cell self-renewal and differentiation. Gene repres-sion mediated by PcG proteins and the conferredH3K27me3 mark is required for ES cell pluripotencyand plasticity during embryonic development 61,62.Chromatin immunoprecipitation studies have shownan unexpected but potentially key concept in the biol-ogy of ES cell pluripotency: genes that are repressedin ES cells but are required for later differentiation aremarked by bivalent H3K4me3 and H3K27me3 domainsthat render them poised for activation6365. In fact, theH3K4me3 and H3K27me3 marks can effectively dis-criminate genes that are expressed (H3K4me3), poisedfor expression (H3K4me3 and H3K27me3) or stablyrepressed (H3K27me3), and therefore reflect the cellstate and lineage potential65. Approximately one-third ofgenes, however, are not marked by either H3K4me3 orH3K27me3, yet are mostly repressed in ES cells. Thesegenes tend to be marked by DNA methylation, whichis therefore a complementary mechanism to histonemodifications to ensure appropriate gene expressionand heritable gene repression simultaneously39,66,67.

Epigenetic regulation of pluripotency networks. Thecombination of global detection methods for transcrip-tion factor target genes and epigenetic modificationshas revealed the existence of an intriguing interplaybetween pluripotency factors and epigenetic modifi-

ers (FIG. 4). In fact, the dynamic balance between thesetwo regulatory systems probably forms the basis forthe pluripotent state. Thus, several epigenetic modi-fiers that confer gene silencing are bound by OCT4,NANOG, SOX2 and/or SALL4. This includes the genesthat encode the PRC components embryonic ectodermdevelopment (Eed), suppressor of zeste 12 homologue(Suz12) and enhancer of zeste homologue 2 (Ezh2),which confers H3K27me3. Other genes bound byOCT4, NANOG, SOX2 and/or SALL4 include poly-homeotic-like 1 (Phc1), the H3K9 methyltransferaseG9a (also known as euchromatic histone methyl-transferase 2(Ehmt2)) and its cofactor Ehmt1(also

Figure 4 |Crosstalk between pluripotency factors and epigenetic modifiers.Epigenetic modifiers that are bound and, presumably, transcriptionally regulated by one

or several of the pluripotency factors OCT4 (encoded by Pou5f1), the homeobox protein

NANOG, SRY box-containing factor 2 (SOX2) and sal-like protein 4 (SALL4)53,56,69. In turn,

some of these epigenetic modifiers are known to regulate the activity of pluripotency

factors. Jumonji domain-containing 1A (JMJD1A; also known as KDM3A) and, in

particular, JMJD2C (also known as KDM4C) are required to reverse the trimethylation

at Lys9 of histone H3 (H3K9me3) at the Nanoglocus, which keeps the gene active69.

On differentiation, Oct4and Nanogare rapidly silenced by H3K9me2 and H3K9me3;

this is mediated by euchromatic histone methyltransferase 1 (EHMT1; also known as GLP)

and G9A (also known as EHMT2)7072, leading to de novoDNA methylation by the DNA

methyltransferases DNMT3A and DNMT3B73, potentially in conjunction with their

co-regulators DNMT3L and lymphoid-specific helicase (HELLS; also known as LSH).

Nanogis also silenced by H3K27me3 mediated by the Polycomb repressive complex (PRC)

component enhancer of zeste homologue 2 (EZH2)61,72. Other components of the PRCs

are embryonic ectoderm development (EED), suppressor of zeste 12 homologue (SUZ12)and polyhomeotic-like 1 (PHC1) and PHC2. The abundance of other epigenetic modifiers

as targets of the pluripotency network suggests their importance for maintaining

pluripotency; these include several AT-rich interactive domain (ARID) and JARID factors,

the chromodomain CDYL proteins, chromodomain helicase DNA-binding protein 9

(CHD9), the histone acetyltransferase MYST4, PR domain containing 14 (PRDM14),

RE1-silencing transcription factor (REST), SET and SET domain-containing 1B (SETD1B)

and the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin

(SMARC) proteins. The co-activation of, and co-regulation by, epigenetic modifiers might

be a fundamental feature of the metastability of the pluripotent state.

The genes encoding the proteins in bold are bound by all four pluripotency factors, and

the asterisk indicates factors identified as targets in human embryonic stem (ES) cells. It is

likely that similar networks also operate in the trophoblast lineage.

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http://www.uniprot.org/uniprot/Q8BX22http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13626&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=52615&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=14056&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13619&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=110147&ordinalpos=5&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=77683&ordinalpos=3&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=77683&ordinalpos=3&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=110147&ordinalpos=5&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13619&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=14056&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=52615&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13626&ordinalpos=1&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.uniprot.org/uniprot/Q8BX22


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known as Glp), the lymphoid-specific helicase andregulator of DNA methylationHells(also known asLsh) and the de novoDNA methyltransferases Dnmt3aand Dnmt3b52,53,56,57,61,68. At the same time, several epi-genetic modifiers with activating functions, includingthe H3K9 demethylases jumonji domain-containing 1a(Jmjd1a ; also known as Kdm3a)and Jmjd2c (alsoknown as Kdm4c),are also regulated by these pluripo-tency factors and their expression is crucial to preventES cell differentiation69.

SALL4 seems to have a unique role in this cross-talk between pluripotency factors and epigeneticmodifiers as it binds cooperatively with PRC1 andPRC2 to H3K27me3 at some loci but does not requirethese repressive complexes for binding at other sites57.Although the importance of this observation is notclear, it shows a direct interaction between pluripotencyfactors and epigenetic modifiers in the maintenance ofpluripotency. Notably, a dominant function of the targetgenes of OCT4, NANOG, SOX2 and SALL4 that encodeepigenetic modifiers is to regulate the expression of

the pluripotency factors themselves, thereby creating aself-reinforcing loop of transcriptional control (FIG. 4).For example, JMJD2C is required to reverse H3K9me3marks at the Nanoglocus and thereby keeps the geneactive. By contrast, on the induction of differentiation,Oct4is rapidly silenced by H3K9me2 and H3K9me3;this is mediated by the G9AEHMT1 complex and sub-sequent DNMT3A- and DNMT3B-dependent de novoDNA methylation. Concomitantly, reduced JMJD2Clevels owing to lower amounts of OCT4 lead to Nanogrepression by H3K9me2, H3K9me3, H3K27me3 andDNA methylation6973. Therefore, it seems that thebalance between epigenetic modifiers with opposingfunctions can maintain pluripotency by reinforcingOct4and Nanogexpression. At the same time, it isobvious that a slight disturbance of this equilibriumcauses repression of these genes and consequentlydifferentiation.

Such a fine-tuned system provides an explanationfor the lability, or unstable nature, of the pluripotentstate and the tendency of ES cells to differentiatein vitro. The positive regulation of several epigeneticrepressors by OCT4, NANOG, SOX2 and SALL4might also explain why pluripotent cells do not persistin vivo, providing a feed-forward control mechanismto prevent uncontrolled cell proliferation and terato-genesis. This view is supported by recent studies of

the endogenous protein complexes that OCT4 andNANOG are associated with, which identified pro-teins from the NuRD (nucleosome remodelling andhistone deacetylation), SIN3A (a transcriptional regu-lator) and PML (promyelocytic leukaemia) repressioncomplexes60. These findings highlight the repressiveroles of the key pluripotency factors that might under-lie the normal latency of the pluripotent state duringdevelopment. In addition to the probable integrationbetween genetic and epigenetic elements in the estab-lishment of the pluripotent state, it is also becomingclear that additional regulatory tiers exist that canfurther modify this crosstalk (BOX 2).

Stochasticity and metastable states of pluripotency.Animplicit characteristic of the crosstalk between transcrip-tion factors and epigenetic modifiers in the establish-ment of pluripotency and the differentiation of early celllineages is that it leaves considerable room for plastic-ity (FIGS 3,4). Such plasticity is greatly enhanced by theepigenetic reprogramming activities that are present inearly embryos. Perhaps as a consequence of this, thereis initially no clearly restricted expression of lineage-determining transcription factors in early development(FIG. 3). OCT4 in particular is observed in all cells until themid-blastocyst stage, gradually disappearing from the TEthereafter. NANOG levels in the morula are independentof cell position, whereas expression of the trophoblast-determining transcription factor caudal-type homeo-box 2 (CDX2) seems somewhat more restricted to outercells, which might correlate with the ability of this factorto contribute to asymmetric cell divisions at the eight-cellstage7476. These findings indicate that the earliest differ-entiation events are not decisions made by single or afew factors that are unique to one cell type, but that they

are brought about gradually by the relative abundance oftranscription factors with opposing functions.

Interestingly, this stochasticity of gene expression isnot only true for the early embryo but is also character-istic of ES cells. An intrinsic feature of ES cells is theirfluctuation between different epigenetic states that areassociated with the heterogeneous expression of vari-ous developmentally regulated genes77. Preservation ofthis metastability is presumably the crucial step in thederivation process of ES cells, whereas in the embryothe cells rapidly fall into stable states that might beenergetically favourable, such that they become morerestricted in their potency (FIG. 5).

The growing list of factors that are heterogeneouslyexpressed in ES cells include NANOG, platelet/endothe-lial cell adhesion molecule 1 (PECAM1), stage-specificembryonic antigen 1 (SSEA1), REX1(also known asZFP42) and STELLA7782. Expression levels of the genesthat encode these proteins in individual ES cells mightcorrelate with their potency, which can be tested experi-mentally by assessing the ability of ES cells to contributeto embryonic tissues (and ultimately the germ line) inchimeras. REX1-negative and OCT4-positive (REX1

OCT4+) ES cells, for example, exhibit a poor ability tocontribute to chimeras compared with REX1+OCT4+EScells, and their behaviour is in that regard more similar tothat of epiSCs82. This is also in line with the finding that

the gene expression profile of STELLAOCT4+ES cells ismore similar to that of epiSCs than the STELLA+OCT+population77. The fluctuation between the active andsilent state of Stellais accompanied by epigenetic differ-ences in these ES cell populations, with Stella-expressingcells showing an enrichment of the active chromatinmarks H3K9ac and H3K4me3 at this locus. Crucially,Stella-expressing and non-expressing ES cell subpop-ulations are interconvertible, such that the ES cell popu-lation as a whole seems to adopt similar proportions ofStella-positive and Stella-negative cells. Therefore, anongoing setting and resetting of epigenetic marks mightcontribute to the plasticity of the pluripotent state.

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http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=15201&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13435&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13436&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=104263&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=76804&ordinalpos=5&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.uniprot.org/uniprot/P43241http://www.uniprot.org/uniprot/Q96MM3http://www.uniprot.org/uniprot/Q96MM3http://www.uniprot.org/uniprot/P43241http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=76804&ordinalpos=5&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=104263&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13436&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=13435&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSumhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=15201&ordinalpos=2&itool=EntrezSystem2.PEntrez.Gene.Gene_ResultsPanel.Gene_RVDocSum


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|

Potency

Development pro ression

TrophoblastlineageEmbryonic

lineage

Zygote BlastocystLateblastocyst

Eggcylinder

Totipotency

ICM and ES cells iPS cells

epiSCs

TE and TS cells

Epigeneticreprogramming

Primitiveendoderm

Trophoblast

Epiblast

Blastomere

A cell that is generated during

the first embryonic cleavage

divisions after fertilization.

Epigenetics and lineage commitment

Developmental progression is fundamentally a linearprocess that involves a series of differentiation steps,proceeding from totipotency to pluripotency andmultipotency in committed cell lineages towards ter-minal differentiation (FIGS 3,5). Therefore, the embryoas a whole moves forward on the developmental scale,albeit individual cells exhibit some plasticity (the extentof which depends on the developmental stage). Theprogressive restriction of cellular plasticity is accompa-nied by epigenetic modifications that impose a cellularmemory and thereby stable cell fate.

The question of precisely when the earliest cell line-age decision event takes place that determines the fateof a blastomeretowards TE or ICM has been studied inconsiderable detail; the most widely accepted consensusis that a first bias might be introduced after the third cellcycle, and possibly even earlier between the four- andeight-cell stages83,84.

Transcription factors determining cell fate. At thegenetic level, key transcription factors are essentialto establish all three cell lineages of the early embryo.The precise regulation of Oct4seems to be particularlyimportant, and OCT4 levels can effectively distinguish

between trophoblast, epiblast and primitive endodermfate: Oct4is required for ICM development and embry-onic lineage determination, as in its absence these cellsadopt trophoblast cell fate85. Oct4overexpression, bycontrast, leads to primitive endoderm differentiation.As an Oct4regulating factor, SALL4 is equally impor-tant for epiblast and primitive endoderm developmentand, as with OCT4, its downregulation causes differ-entiation into trophoblast54. Nanog is also essentialfor ICM development, but Nanog deficiency causesdifferentiation towards primitive endoderm86.

As noted above, a feature of key importance for early

development and pluripotency is the co-regulation ofthese factors. OCT4 and SOX2 can heterodimerize, andOCT4, NANOG and SALL4 are found in shared proteincomplexes that co-occupy binding sites in promoters,including their own5860. Intriguingly, however, OCT4can also heterodimerize with CDX2, one of the earliestknown trophoblast determinants. The OCT4CDX2complex functions mostly as a transcriptional repressor,and it seems that the reciprocal inhibition of these tran-scription factors with antagonizing lineage-determiningcapacity might be a crucial characteristic of early devel-opment87. This mutual interaction reinforces the factthat it is not the absolute presence or absence, but the

Figure 5 |Cellular potency in development and reprogramming. The gradual loss of differentiative potency duringdevelopment can be likened to the downstream flow of a river, and epigenetic restrictions can be represented by

cascades. Distinct states of totipotency, pluripotency and multipotency can be likened to energetic plateaux that are

located just in front of a cascade (indicated by bars). The first major cascade is encountered at the blastocyst stage.

Trophectoderm (TE) cells reach this cascade first as they become committed to their lineage slightly earlier than cells of

the inner cell mass (ICM). Multipotent trophoblast stem (TS) cells are present in the conceptus until embryonic day 8.5

(E8.5), retaining their full differentiation potential for longer than cells of the embryonic lineage125. The epiblast of egg

cylinder-stage embryos gives rise to epiblast stem cells (epiSCs). EpiSCs are on a lower energetic plateau than embryonic

stem (ES) cells owing to their decreased potency to produce chimeras. Once such a plateau is reached, it can tolerate

complete (for example, NANOG in ES cells78) or temporary (for example, stochastically expressed genes) absence of some

factors that are essential to establish this state in the first instance. These findings indicate that a particular activation

energy is required to surmount the cascades in an upstream, counter-current direction to create states of increased

potency. This activation energy probably reflects the necessity to initiate epigenetic reprogramming events. Such an

interpretation has important implications for reprogramming strategies, indicating that short-term overexpression or

activation of additional factors is needed to reach an ES cell-like state in induced-pluripotent stem (iPS) cells.

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relative abundance of these transcription factors in pro-portion to each other that is likely the main determinantof cell fate.

In conjunction with CDX2, the T box factor eomeso-dermin (EOMES) is required for establishment of afunctional TE and for normal trophoblast prolifera-tion and differentiation in vivo, and has trophoblast-determining functions when overexpressed in ES cells87.Notably, despite their importance for the trophoblastlineage, genetic ablation of either of these genes does notprevent initial TE formation. Therefore, there must befactors upstream of Cdx2(and Eomes) that can functionas initial determinants of trophoblast fate. One such fac-tor, TEA domain family member 4 (TEAD4), has beenrecently identified88,89. After this initial determination,reinforcement and maintenance of the trophoblast lin-eage depends on the ETS-related family transcriptionfactor ELF5. Elf5ablation causes an absence of extra-embryonic ectoderm (normally made up from diploid,proliferating trophoblast cells) and an inability to deriveself-renewing trophoblast stem (TS) cell lines in vitro90.

It was recently found that ELF5 forms the crucial link ina self-reinforcing circuitry with CDX2 and EOMES thatis intriguingly similar to the SALL4OCT4NANOGSOX2 transcriptional network in ES cells91. Thus, CDX2,EOMES and ELF5 activate each other to establish theTS cell compartment.

Epigenetic environment for cell fate decisions. Inaddition to these crucial transcription factors, theepigenetic environment is similarly important todetermine the potential of a cell towards a particularlineage. The euchromatic H3K9 methyltransferase SETdomain-containing B1 (SETDB1) and the enzymati-cally active component of the PRC2 complex, EZH2,which confers H3K27me3, are important for the estab-lishment of the embryonic lineage92,93. The earliestrole to date of an epigenetic modifier in cell lineagedetermination has been described for co-activator-associated arginine methyltransferase 1 (CARM1),which confers H3R2, H3R17 and H3R26 methyl-ation. These marks are generally associated with geneactivity. Blastomeres with higher endogenous CARM1levels or, in particular, those that have been injectedwith Carm1mRNA to mediate overexpression, showa bias for preferentially localizing to the ICM94. Thus,not only can the epigenetic landscape impose a per-missive (or non-permissive) environment for cell fate,

but it might be able to predispose a cell towards aparticular lineage.

As is evident from the preceding examples and fromthe intricate interrelationship between pluripotencygenes and epigenetic modifiers, it is difficult to deter-mine whether key transcription factors with lineage-determining capacity set up a lineage-restrictingepigenetic environment, or whether pre-imposed epi-genetic restrictions determine the activity state of thesekey transcription factors and therefore determine line-age potential. It is probably the interplay between bothmechanisms combined with an element of stochastic-ity brought about by epigenetic reprogramming that,

in conjunction with cellcell interactions and cell-ular polarization processes95, eventually leads to cellcommitment. This model is further supported by therecent finding that cellcell contact and specific sig-nalling pathways regulate the nuclear accumulationof Yes-associated protein 1 (YAP1), a transcriptionalco-activator of TEAD4, thereby leading to the cellposition-dependent activation of lineage-specifyingtranscriptional networks in the early embryo96.

Fixation of cell lineage fate.The presence of a lineagepreference is followed by a loss of the ability to switchlineages, resulting in fixation of cell fate at the blasto-cyst stage. A crucial epigenetic lineage barrier betweenthe embryonic and trophoblast lineage compartmentsis established by DNA methylation91. HypomethylatedES cells owing to a deficiency of, for example, Dnmt1,Dnmt3a andDnmt3b, or ubiquitin-like PHD and RINGfinger domain-containing protein 1 (Uhrf1; also knownas Np95) lose their stable lineage restriction, can adopttrophoblast cell fate in vitroand contribute to the TE

in chimeras.The restriction of lineage potency is mediated

through epigenetic regulation of Elf5, which is meth-ylated and silenced in the embryonic lineage buthypomethylated and expressed in the trophoblastlineage. By forming a positive-feedback loop with theTS cell genes Cdx2 and Eomes, ELF5 reinforces com-mitment to the trophoblast lineage. By contrast, thisdifferentiation pathway is aborted in the embryoniclineage owing to epigenetic silencing of Elf5. Thus, Elf5functions as a gatekeeper between these earliest twodiverging cell lineages in development91.

An important feature of this circuitry is the positionof Elf5downstream of initial lineage determination thatcorrelates perfectly with the developmental fixation oflineage fate. It specifically allows the plasticity and regu-lation that is characteristic of early mammalian develop-ment in which expression of lineage markers remainsmosaic and individual blastomeres can still cross line-ages until the mid-blastocyst stage74. Only thereafter,activation of Elf5 in the TE of the late blastocyst and itshigh expression in the extraembryonic ectoderm rein-forces trophoblast cell fate, whereas the inability to acti-

vate Elf5 in the epiblast owing to promoter methylationrestricts its descendants to the embryonic cell lineage.Thus, a major role of DNA methylation is to impose aspecific cellular memory through the regulation of Elf5.

Methylation of Elf5coincides with increased Dnmt3bexpression in a time window when de novomethylationin the embryonic lineage becomes crucial for furtherdevelopment97,98. Recent findings also indicate that Stellais marked by DNA methylation in this developmentalperiod. Thus, whereas Stellais expressed in the ICM ofblastocysts, it becomes methylated and stably repressedin the epiblast of post-implantation embryos77. Thiscoincides with a clear restriction of potency in epiblastcells: ICM-derived ES cells readily populate embryonictissues when introduced into blastocysts, but epiblast-derived epiSCs cannot (or can only extremely rarely)contribute to chimeras48,49.

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http://www.uniprot.org/uniprot/O95936http://www.uniprot.org/uniprot/Q15561http://www.uniprot.org/uniprot/Q9UKW6http://www.uniprot.org/uniprot/Q9UKW6http://www.uniprot.org/uniprot/Q15561http://www.uniprot.org/uniprot/O95936


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The emerging picture from these observations is thatthe progressive reduction of developmental potency pro-ceeds in a stepwise manner in which, first, permissivechromatin states for lineage determination are establishedby histone modifications in a tight interplay with trans-criptional regulators, including OCT4, NANOG, SOX2,SALL4 as well as TEAD4, CDX2 and EOMES. Once thelineages have been specified, DNA methylation of a fewcrucial loci, notably Elf5and Stella,is then required torestrict their differentiation potential and to establishlineage-committed cell populations, the fate allocationof which is stably inherited by all descendants (FIGS 3,5).

Reversion of cellular restrictions

The stepwise acquisition of epigenetic marks culminatingin the fixation of lineage fate by differential DNA methyl-ation of gatekeeper genes, such as Elf5, forms the molec-ular basis for Waddingtons concept of the canalization ofdevelopmental pathways. Waddington compared the pathof a cell (lineage) towards terminal differentiation with aball travelling downwards along branching valleys; once

in its final valley, the ball cannot easily cross the mountaininto neighbouring valleys or return to the beginning99.This canalization explains how cellular differentiationpathways become stable and potentially irreversible.It also depicts the unidirectionality of differentiationthat dictates developmental progression and preventsteratogenesis during a time of exponential growth.

The ability to derive induced pluripotent stem (iPS)cells from fully differentiated cell types (FIG. 1)is thegroundbreaking reversion of this strict developmentalprogression, creating bidirectional developmental path-ways by experimental manipulation100103. Thus, tem-porally limited overexpression of the four transcriptionfactors OCT4, kruppel-like factor 4 (KLF4), SOX2 andMYCis sufficient to reactivate endogenous pluripotencygenes and to regain a developmental potency akin to thatof ES cells. Other combinations of factors that have beensuccessfully used for the derivation of iPS cells includethe oestrogen-related receptor- (ESRRB) in conjunctionwith OCT4 and NANOG in mouse fibroblasts104, andOCT4, SOX2, NANOG and the Caenorhabditis elegansLIN-28homologue in human fibroblasts105.

Reflecting the necessity for extensive epigeneticreprogramming in the iPS cell generation process, theefficiency of iPS cell derivation can be enhanced inthe presence of inhibitors of histone deacetylases, the his-tone methyltransferase G9A and DNA methylation106108,

and these components can substitute for some repro-gramming factors. In efforts to understand how suchdevelopmental reversion can be achieved in principle,recent computational modelling has produced importantinsights109,110. In these models, transcriptional feedbackloops create molecular switches that result in successivegene restrictions that correspond to a controlled differ-entiation cascade. Although cells are robustly resistantto reprogramming, the model also predicts that amplifi-cation of low-level fluctuations in expression (so-calledtranscriptional noise) might be sufficient to trigger thereactivation of the core pluripotency switch. The impor-tant insights from such studies are that reprogramming

towards pluripotency depends on the positive inter-ference of several factors, each with a certain noise ampli-tude of expression, thus explaining the stochasticity ofexperimental reprogramming as well as the certain extentto which reprogramming factors can be exchanged.

It is also becoming clear that the reversion of a cell intoa state of increased potency requires temporally higherlevels of gene expression, equivalent to an activationenergy, than those required once that state is reached. Itis even the case that cells on these plateaux can toleratecomplete (such as NANOG in ES cells78) or temporary(as in stochastically expressed genes) absence of somefactors that are essential to establish this state in the firstinstance without loss of developmental potency. We com-bine these data in a model in which development is like-ned to a stream and epigenetic restrictions to cascades;reprogramming is only possible by lifting a cell back upto the original level (FIG. 5). With better knowledge of theprecise molecular processes during reprogramming, itwill be possible to define the exact composition of thefactors that are required for each step.

Conclusions and future perspectives

Recent results highlight the characteristics and impor-tance of genome-wide epigenetic reprogramming thatoccurs both in the germ line and in the early embryo.The mechanistic aspects of reprogramming, whichinvolve histone marks, histone exchange and the use ofhistone variants along with demethylation of DNA, arecurrently under intense investigation. Reprogramming inPGCs is likely to be important for the erasure of genomicimprints and of epimutations, and could also be inte-gral for the control of transposon silencing or mobil-ity111. Epigenetic reprogramming in the early embryois crucial to erase further gametic gene expressionprogrammes and thus might be partly responsible forstochastic expression of lineage-determining transcrip-tion factors. The combinatorial stochastic expression oftranscription factors becomes subsequently restrictedto establish lineage-specific transcriptional circuits.This transcriptional focusing involves epigenetic mark-ing of gatekeeper genes, such as Elf5, that function toreinforce the trophoblast-specific transcriptional circuit.Perhaps there are similar gatekeepers that are involved inother lineage decisions, for example in maintaining theepiblast lineage. Thus, it is possible that methylation ofStella, which occurs during the transition from ES cells(and ICM) to epiSCs (and epiblast tissue), fulfils a similar

gatekeeper function.Future work will focus on finding out when and how

the first lineage-specific epigenetic marks arise. This willbe aided by the identification of major epigenetic modifi-ers and how they act in both a genome-wide and targetedmanner. The development of epigenomics technologiesthat can be applied to small numbers of cells will be ofcrucial importance to unravel key features of epi geneticplasticity and lineage commitment in mammaliandevelopment. Establishing the basic principles on whichcell lineages are defined and maintained will be crucialfor developing safe and succesful cell therapies to realizethe full potential of regenerative medicine.

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http://www.uniprot.org/uniprot/O43474http://www.uniprot.org/uniprot/P01106http://www.uniprot.org/uniprot/O95718http://www.uniprot.org/uniprot/P92186http://www.uniprot.org/uniprot/P92186http://www.uniprot.org/uniprot/O95718http://www.uniprot.org/uniprot/P01106http://www.uniprot.org/uniprot/O43474


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AcknowledgementsThe authors thank all their colleagues for advice and discus-

sion. Work in the authors laboratories is supported by the

Biotechnology and Biological Sciences Research Council,

the Medical Research Council, the European Union Epigenome

Network of Excellence, the Technology Strategy Board and

CellCentric.

DATABASESEntrez Gene:http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene

Blimp1| Dnmt3a|Dnmt3b| Eed| Ehmt1| Ezh2| G9a| Hells|Jmjd1a|Jmjd2c|Phc1| Stella| Suz12

UniProtKB:http://www.uniprot.org

CDX2| ELF5|EOMES|ESRRB| KLF4|LIN-28|MYC|NANOG

| OCT4| REX1| SALL4| SOX2| TEAD4

FURTHER INFORMATIONWolf Reiks homepage:

http://www.babraham.ac.uk/devgen/reik.html

Myriam Hembergers homepage: http://www.babraham.

ac.uk/devgen/hemberger.html

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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Epigenetic Dynamics of Stem Cells