Mutation Research 637 (2008) 1–15

Available online at www.sciencedirect.com

Mini review

Histone acetylation and chromatin signaturein stem cell identity and cancer

Vivek Shukla a,b, Thomas Vaissiere a, Zdenko Herceg a,∗a International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, 69008 Lyon, France

b Genetics of Development and Disease Branch, National Institute of Diabetes 10/9N105,Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

Received 3 April 2007; received in revised form 30 June 2007; accepted 17 July 2007Available online 1 August 2007

Abstract

Cancers are traditionally viewed as a primarily genetic disorder, however this view has recently been modified by compellingevidence arguing that epigenetic events play important roles in most human cancers. Deregulation of epigenetic information(encoded in DNA methylation and histone modification patterns) in cells with pluripotent potential may alter defining proper-ties of stem cells, self-renewal and differentiation potential, leading to cancer initiation and progression. The level of compactionof chromatin dictates accessibility to genomic DNA and therefore has a key role in establishing and maintaining distinct geneexpression patterns and consequently pluripotent state and differentiation fates of stem cells. Unique properties of stem cellsdefined as “stemness” may be determined by acetylation and methylation of histones near gene promoters that regulate gene

transcription, however these histone modifications elsewhere in the genome may also be important. In this review, we discussnew insights into possible mechanisms by which histone acetyltransferases (HATs) and histone acetylation in concert with otherchromatin modifications may regulate pluripotency, and speculate how deregulation of histone marking may lead to tumourigene-sis.© 2007 Elsevier B.V. All rights reserved.

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Keywords: Epigenetic mechanisms; Chromatin modifications; Histon

1. Introduction

The traditional view of cancer implies that it devel-ops through sequential acquisition of genetic changes(mutations) that inactivate tumour suppressor genes,

activates oncogenes and alters functions of other cancer-associated genes leading to a clonal expansion ofmutated cells with growth advantage over normal cells.

∗ Corresponding author at: Epigenetics Group, International Agencyfor Research on Cancer (IARC), 150 Cours Albert Thomas, F-69008Lyon, France. Tel.: +33 4 72 73 83 98; fax: +33 4 72 73 83 29.

E-mail address: herceg@iarc.fr (Z. Herceg).

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0027-5107/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2007.07.012

ation; Histone acetyltransferases; Stem and progenitor cells; Cancer

owever, recent studies indicate that aberrant epige-etic information plays an important role in virtuallyvery type of human neoplasia. Interestingly, accumu-ating evidence argues that the patterns of epigeneticnformation established during early development mayredispose to cancer promoting events and that mostuman cancer may have a common basis associated withltered epigenetic patterns in tissue stem/progenitor cells1]. Consistent with this idea, aberrant epigenetic regu-ation of a set of genes (“tumour progenitor genes”) may

e an origin of tumour development and malignant phe-otype, although the precise contribution of epigeneticvents to tumour development and tumour phenotypeemains to be established.

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It is increasingly obvious that many cancers ariserom deregulated control of cells with pluripotent poten-ial, i.e. stem and progenitor cells. Stem cells weresolated a quarter century ago [2], however it was notntil isolation of the first human ES cells [3] that theechanisms of pluripotency begun to receive more atten-

ion. Importance of epigenetic states in the maintenancef stem/progenitor cell pluripotency is illustrated byhe fact that virtually all cell type can be convertednto more immature cell stage with wider differentia-ion potential [4–6]. Therefore, unique characteristic oftem/progenitor cells, self-renewal and pluripotent dif-erentiation potential, appear to be largely controlled byNA methylation and chromatin modifications.Chromatin modifications occur at the level of his-

ones that are the targets for several post-translationalovalent modifications, including acetylation, phospho-ylation, methylation, ubiquitination, and poly(ADP)-ibosylation. Emerging evidence suggests that deregu-ated patterns of chromatin modifications are implicatedn many steps of tumour development and progression.he aim of this review is to discuss the role of chromatinodifications, with emphasis on histone acetylation, in

he maintenance of stem/progenitor cell identity andow deregulation of chromatin modification patternsn these pluripotent cells may lead to tumour develop-

ent.

. Stem and progenitor cells

Stem cells constitute a minority of cell populationn adult tissues, yet they play the key role in the devel-pment and tissue homeostasis. The main properties oftem cells are the self-renewal, essential for maintenancef the stem cells pool, and the ability to differentiate inifferent lineage required for the integrity and functionf tissues. Given their special properties, stem cells areightly regulated by multiple genes and gene networks.his control prevents the shift in the balance betweenelf-renewal and differentiation. There are two differentypes of stem cells: the embryonic stem (ES) cells andhe adult stem cells.

.1. Embryonic and adult tissue stem cells

ES cells are able to give rise to any cell typespluripotent cells) and normally differentiate into thehree germ layers (ectoderm, mesoderm and endoderm)

uring embryonic development. ES cell can be main-ained in cell culture in non-differentiated state for a longeriod of time. Adult stem cells are a tiny population ofndifferentiated cells present in specific adult tissues and

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hey are lineage or tissue specific. For example, all bloodineages and mature blood elements are derived from amall pool of hematopoietic stem cells (HSC) in bonearrow. The adult stem cells are present in many other

issues such as retina, liver, brain, epithelium, epidermnd follicule, however, it is likely that stem cells areresent in any tissue that undergoes renewal.

Since stem cells in any given organisms share andentical genome with other cell types, it is obvi-us that their ES cell plasticity is due to non-geneticvents. Gene expression patterns controlled by epi-enetic mechanisms (histone modifications and DNAethylation) largely confer unique properties defined as

stemness”. Therefore, it is quite plausible that misreg-lation of chromatin modification patterns may lead ton altered potential of stem cell self-renewal and expan-ion of epigenetically modified stem cell pools (Fig. 1).tem cells modified in this manner exhibit no genetichanges, yet they may represent a precursor pool suscep-ible to acquisition of mutations and further epigeneticlterations. In this scenario disruption of chromatin mod-fication states is the first step of tumourigenesis ands a contributing factor to polyclonal tumour phenotype1].

.2. Cancer stem cells

Cancer stem cell (CSC) is an operational term to func-ionally define a distinct subpopulation of tumour cellsith unlimited renewal potential [7]. These cells are able

o form tumour upon transplantation and to recapitulateumour heterogeneity. CSC and ES cells share many keyeatures including the infinitive proliferation potentialnd the capacity to invade tissues and organs, to grow innd promote vascularisation of “foreign” tissues. Therere different views for the origin of cancer stem cellsuch as cell–cell fusion and horizontal gene transfer [8].lthough numerous studies provided strong support for

he existence of cancer stem cells in both hematopoi-tic and epithelial malignancies, it should be noted thathey are primarily defined by functional features andot by a distinct molecular (e.g. cell surface antigens)r mutation profiles. However, better characterizationf cancer stem cells in terms of molecular signaturesill be required for the design of efficient strategies

n cancer therapy. While genetic alterations (mutations,ranslocations, deletion and duplications) have long beenmplicated in the development and progression of can-

er likely through establishment and maintenance ofancer stem cells, recent evidence indicates that chro-atin modifications may also play major roles in this

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Fig. 1. Epigenetic disruption in stem/progenitor cells may be an earlcontribute to tumour heterogeneity. Epigenetic changes induced by emutational events and give rise to cancer stem cells. Abnormal expressto reprogramming into a pluripotent state and also cancer stem cells.

3. Chromatin modifications

In eukaryotes, the several meters long genomic DNAneeds to be packed into chromosomes with a diameterof only several micrometers. This is achieved throughan elaborated scheme of packaging that starts with theformation of nucleosome from DNA winding around

an octomer of two subunits of each of the core his-tones H2A, H2B, H3, and H4 as well as the linkerhistone H1. Therefore, eukaryotic DNA is packaged intoa highly compacted DNA–protein complex, chromatin,

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in the development of cancer and give rise to cancer stem cells andental and endogenous factors in stem/progenitor cells predispose tofunction of a set of genes in more differentiated cells may contribute

hich fulfils essential functions not only as a struc-ural element but also in preserving genetic information.hromatin is presented in two main forms: less con-ensed and transcriptionally permissive euchromatin,nd highly condensed and typically silent heterochro-atin [9]. Once thought of as static, non-participating

tructural elements, it is now clear that histones are

ntegral and dynamic components of the machineryesponsible for regulating gene transcription and otherNA-based cellular processes. The core histones con-

ain a central histone fold domain, which is flanked

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F cations. Post-translational modifications of histones occur primarily on N-t etylation (A), methylation (M), phosphorylation (P), and ubiquitination (U).N lated.

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Fig. 3. Histone acetylation occurs on histone tails and is mediatedby histone acetyltrasferases (HATs). (A) A representation of chemicalreaction of histone acetylation. This reaction is mediated by HATs and

ig. 2. Schematic representation of a nucleosome and histone modifierminal tails of core histones (H2A, H2B, H3 and H4) and include acote that several lysines (e.g. Lys 9) can be either acetylated or methy

y N- and C-terminal tails. These tails, which pro-rude from the surface of the chromatin polymer andre protease sensitive, comprise 25–30% of the massf individual histones, thus providing an exposed sur-ace for potential interactions with other proteins. Onearticular feature of histone tails is the fact that theyre the targets of several forms of covalent modificationncluding acetylation, methylation, ubiquitination, andhosphorylation (Fig. 2). These modifications may serves signals for binding of chromatin remodelling com-lexes, which modify the chromatin into repressive orermissive configurations [10], although different viewsave been expressed [11]. These modifications are alsoroposed to form “histone code” that may have con-istent and combinatorial character and therefore mayictate cellular outcomes [10,12]. Factors that mediatehromatin modifying and remodelling activities include:istone acetyltransferases (HATs); histone deacety-ases (HDACs), histone methyltransferases (HMTs) anddenosine triphosphate (ATP)-dependent nucleasome-emodelling complexes [10,13–15].

. Histone acetylation and histonecetyltransferases

Acetylation of the lysine residues at the N-terminusf histone proteins (Fig. 3) is believed to remove pos-tive charges, thereby reducing the affinity betweenistones and DNA. It is believed that this facilitatesccess to the promoter region for RNA polymerase

nd transcription factors. However, there is evidencehat histone acetylation may mediate transcription andther chromatin-based processes by providing bind-ng surfaces for regulatory protein or acting in concert

involves a covalent addition of acetyl group at the � amino groupsof evolutionary conserved lysine residues. (B) Acetylation of histonesis leads to an open, transcriptionally permissive chromatin. Histoneacetylation is reversible modification and acetyl groups are removedby several histone deacetylates (HDACs).

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with other modification to form a histone code (dis-cussed below in detail). Therefore, in most cases,histone acetylation enhances transcription while his-tone deacetylation represses transcription. In agreementwith this idea, the acetylation of lysine residues in his-tone tails is associated with active gene transcription[16].

4.1. Histone acetyltransferases

Although the phenomenon of histone acetylation hasbeen known for over 30 years [17], the biology ofhistone acetylation received little attention until sev-eral years ago, when several known transcriptionalco-activators proved to exhibit an intrinsic histoneacetyltransferase (HAT) activity [18–22], sparked agreat interest. Subsequent studies identified a num-ber of other proteins with HAT activity [14,23]. MostHATs can be divided into several families on thebasis of conserved structural motifs. These include: theGNAT (Gcn5-related N-acetyl transferase) family, theMYST (MOZ (monocytic leukaemia zinc finger pro-tein, Ybf2 (Sas3), Sas2 (something about silencing-2),TIP60 (HIV Tat-interacting 60 kDa protein) family, andthe p300/CBP family. Different HATs seem to displaydifferent mechanisms of histone substrate binding andcatalysis.

The GNAT family of HAT proteins function as co-activators for a subset of transcriptional activators. Theycontain a HAT domain of around 160 residues anda conserved bromodomain at C-terminus, which hasbeen shown to recognise and bind acetyl-lysine residues.The wide distribution of the bromodomain among HATenzymes highlight the importance of lysine acetyla-tion in self-maintenance of a transcriptional active state,but also in recruitment of other chromatin modify-ing/remodelling enzymes [24].

The MYST family is a major HAT family whose nameis derived from four founding members: MOZ, Ybf2,Sas2, and TIP60 [14,23]. The MYST family membersare grouped together on the basis of their close sequencesimilarities, including a particular highly conserved 370residue MYST domain, which uses an acetyl-cysteineintermediate in the acetylation reaction, so the catalyticmechanism involved is different from that shared by theother families of HATs [23]. The members of the MYSTfamily are involved in a wide range of regulatory func-tions including transcriptional activation, transcriptional

silencing, dosage compensation and cell cycle progres-sion. In addition to the MYST domain, many memberscontain a cysteine-rich, zinc-binding domain within theHAT regions and N-terminal chromodomains. Since the

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ddition of an acetyl group to a lysine residue creates aew surface for protein association, and many transcrip-ion factors and chromatin regulators bind directly orndirectly acetylated histones, the maintenance of a spe-ific histone acetylation pattern is crucial to various cellrocesses such as cell cycle progression, DNA repair,ell proliferation and differentiation. Consequently, it isot surprising that mutations or chromosomal translo-ations involving HAT genes result in development ofalignancies [15,23].The p300/CBP family is another major group of

uclear HATs that has been extensively characterized.he members of this family are more global regulatorsf transcription [25]; contain a considerably larger HATomain of about 500 residues, and other protein domains,ncluding a bromodomain and three cysteine–histidineich domains (TAZ, PHD and ZZ) that are believed toediate protein–protein interactions.The list of proteins that harbour HAT activity is grow-

ng and a number of proteins that could not be classifiedn these three major HAT families were shown to acety-ate histone or non-histone proteins in different contexts23]. HATs are typically found in large multiprotein com-lexes that share certain degree of similarity in subunitomposition. For example, Gcn5 is the catalytic sub-nit shared by the SAGA [26], SALSA [27], and SLIKomplexes [28]. Different non-catalytic components ofAT complexes are believed to be involved in the locus

argeting, thus providing chromosomal domain speci-city, whereas the substrate specificity may be conferredy individual acetyltransferases. In agreement with thisdea, biochemical studies have shown that these com-lexes exhibit a higher activity in comparison to theirespective catalytic subunits.

. How does histone acetylation mediatehromatin-based processes?

A large body of evidence indicate that acetylationf histones is not a random process, but a highly spe-ific post-translational modification at selected lysineesidues in histone tails. The addition of acetyl groups believed to neutralize the positive charge of lysine,hich affects the interaction of the histone tails withNA, but also with RNA and proteins. Several recent

tudies indicated that the acetyl group attached to histoneails may also provide a specific binding site for certainroteins via their bromodomain. Broadly, acetylation of

istones is linked to transcriptional activation, therefore,t is not surprising that many of the enzymes respon-ible for acetylation of histones at different residuesere first known as transcriptional co-activators and

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ater as enzymes. In general, there are three possi-le mechanisms, which are not mutually exclusive, byhich histone acetylation regulates transcription. In therst model, covalent addition of acetylation group toistone tails weakens histone–DNA contacts by neutral-zing the positive charge of the histone tails, leading topen, permissive chromatin structure (Fig. 3). In thisegard, histone acetylation may facilitate chromatin-ased processes by unwinding chromatin structure andnsuring DNA accessibility [29–31]. The second modelredicts that acetylated histone tails serve as platformsor recruitment of different regulator proteins involvedn gene transcription. Several recent studies supportedhis model by providing evidence that proteins con-aining bromodomain interact with acetylated lysinesf histone tails [32,33]. Furthermore, some transcrip-ion factors, such as c-Myc, associate preferentially withre-acetylated histones in the chromatin [34]. The thirdodel, known as “histone code” [10,12] implies that

istone tails undergo different modifications (acetyla-ion, methylation, phosphorylation and ubiquitination) incombinatorial and consistent manner forming a code

hat is read by cellular machineries to dictate cellularutcomes.

. Bromodomains

Bromodomains are small (approximately 110-amino-cid) protein domains that form an extensive familyf evolutionarily conserved protein modules [24,35].he Drosophila protein Brahma was the first identified

o contain bromodomain [36], and subsequent studiesdentified bromodomains in many chromatin-associatedroteins. Interestingly, most HAT enzymes and nearly allnown HAT-associated transcriptional activators wereound to contain bromodomains [24,37].

Although bromodomains were originally found inroteins associated with chromatin, it is relativelyecently that they have been discovered to function ascetyl-lysine binding domains. Several in vitro stud-es demonstrated that bromodomains exhibit preferentialinding to acetylated peptides, suggesting that acety-ated histones could serve as binding surfaces for theinding of bromodomain-containing factors [24,38–41].his suggestion was confirmed by the work fromorkman’s laboratory showing that acetylation of his-

ones is required for retention of bromodomains onhe promoters [33]. A number of structural studies

f bromodomain/peptide ligand complexes have pro-ided better insights into the ligand selectivity ofromodomains [24,32,39–41]. These studies show thatromodomain/acetyl-lysine recognition can serve as a

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ivotal mechanism for regulating protein–protein inter-ctions in numerous cellular processes including histoneodifications and chromatin remodelling as well as in

rocesses such as transcriptional activation and DNAepair.

. Genome-wide patterns of histone acetylation

It is well established that DNA-binding activatorsecruit HATs to promoters, thereby generating local-zed domains with high levels of histone acetylationhyperacetylation) that influence transcriptional activity.cetylation of histones, notably histone H4, is con-

entrated at CpG island regions of active loci ratherhan just being targeted to promoters [42]. At thend of transcriptional activity, transcriptional repres-ors recruit HDACs and acetylation states are reversed.owever, this targeted promoter-specific histone acety-

ation occurs against the background of global histonecetylation patterns across the genome (Fig. 4). Interest-ngly, hyperacetylated regions of genome are not alwaysssociated with active genes [43]. Therefore, the cur-ent view that specific HAT complexes are targetedo select promoter elements by DNA-binding elementsnd that subsequent recruitment of HDACs reverseshis reaction needs to be revisited. While some pro-

oters are strongly acetylated and deacetylated in aromoter-specific manner, surrounding regions are alsocetylated and deacetylated [44]. Therefore, acetylationnd deacetylation seem to be rather global modifi-ations, whereas localized hyperacetylation occurs inromoter-dependent context. The same is true for his-one deacetylation. Global histone modifications bycetylation is constantly kept in balance with histoneeacetylation for the two main reasons: (i) it maintainsasal state of underacetylated histones by preventingull acetylation, and (ii) it allows chromatin to returno its default, underacetylated state (after the signalor transcriptional activation is switched off) by rapidurnover of acetyl groups at majority of nucleosomal tails44].

In addition, heterochromatic regions of the yeastenome exhibit histone hypoacetylation over signifi-antly larger regions such as the silent mating-type locind telomeric regions [45,46]. Similar patterns of his-one modifications are also present in higher eukaryotes.or example, at some loci in vertebrate genome, his-

one hyperacetylation spreads over a region as large

s 10–100 kb. The best example of such a region ishe chicken �-globin locus in erythroid cells, whichxhibits histone hyperacetylation over 30 kb of DNA47,48]. Other studies have revealed that several other

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Fig. 4. Balance between histone acetylation and deacetylation as wellas with other histone modification (methylation, ubuiquitination andphosphorylation) contribute to the establishment of different chromatinstates such as heterochromatin (A) and euchromatin (B). Lysine acety-lation contributes to the establishment of transcriptionally permissive

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chromatin, whereas histone hyperacetylation occurs at transcription-ally active loci (C).

loci exhibit global patterns of histone hyperacetylation.These include �- and �-globin locus in chicken, mouse,and humans [49–52], HOX loci in lung fibroblasts [53],the immunoglobulin heavy chain (IgH) locus and themajor histocompatibility complex class II gene HLA-DRA in B-lymphocytes [54,55], and the interferon-�locus in T-cells [56]. These regions are dubbed “hyper-acetylated domains” [43], however, it should be notedthat hyperacetylation of these regions is accompaniedby other histone marks including methylation of H3K4[48], among others. These studies provide evidence thatthere are a growing number of foci that are hyperacety-lated over broad regions and that they occur in the cell

type specific manner. This suggests that varied acetyla-tion levels over broad chromatin regions may be a part ofchromatin signature for cell identity and differentiationfate.

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. Propagation of chromatin compaction statesnd histone acetylation

Chromatin-mediated regulation of gene expressionnd function is characterized by a remarkable degreef stability and consistency, which is believed to ensuren appropriate state of cell differentiation, however,he underlying mechanism is poorly understood. Whilepigenetic inheritance contained in DNA methylationatterns is relatively well understood, molecular eventsy which histone modifications are propagated over cellenerations is largely unknown.

The nuclear DNA can exist within at least twoasic chromatin conformations; euchromatin (generallyctive) and heterochromatin (generally inert). This dis-inction dates back to 1928 and is based on the worky Emil Heitz who identified cytologically detectableongitudinal differentiation of chromosomes [57]. Thisasic conformation has permitted a greater understand-ng of gene controls within individual cells in higherrganisms, although with recent discoveries of specifichromatin modifications and chromatin domains, it maye possible to subdivide chromatin in many more sub-ypes [58]. Heterochromatin is usually a tightly packedorm of DNA and its major characteristic is that it is notranscribed. Heterochromatin replicates later in S phasef the cell cycle and is found only in eukaryotes. It iselieved that it serves several functions, from gene regu-ation to the protection of the integrity of chromosomes.ll these roles can be attributed to the dense packingf DNA, which makes it less accessible to protein fac-ors that bind DNA or its associated factors. In contrast,uchromatin is a lightly packed form of chromatin thats rich in genes, and is often, but not always, under activeranscription. Unlike heterochromatin, euchromatin isound both in eukaryotes and prokaryotes. Euchromatinarticipates in the active transcription of DNA to mRNAroducts. The unfolded structure allows gene regulatoryroteins to bind the DNA sequences, which then can ini-iate the transcription process. The boundary betweenuchromatin and heterochromatin is determined by thentagonistic function of euchromatic regulators and byhe SU(VAR)39 HMT that mediates the histone H3ysine-9 (H3K9) di- and tri-methylation [59]. Histoneysine methylation plays an important role in mark-ng differentiation between chromosomal subdomainsf heterochromatin and euchromatin [59]. Studies in thession yeast Schizosaccharomyces pombe have begun to

eveal the genetic pathways critical for the assembly andpigenetic maintenance of heterochromatin, includingey roles played by the RNAi machinery, H3K9 methyla-ion and heterochromatin protein 1 (HP1). Recent studies

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ave also identified a novel E3 ubiquitin ligase, a proteinniversally required for H3K9 methylation and hete-ochromatin formation [60].

. Histone acetylation and stem cell identity

Recent studies provide strong evidence that the pat-ern of chromatin modifications play a major role innique stem cell properties. The main properties of stemells are the self-renewal, essential for maintenance ofhe stem cells pool, and the ability to differentiate inifferent lineage required for the integrity and functionf tissues. Given their special properties, stem cells areightly regulated by multiple genes and gene networks.merging evidence suggests that mechanisms utilizingistone modifications play a fundamental role in theaintenance of stem cell identity and differentiation

ates. Self-renewal and pluripotency, represent oppos-ng demands on genome of stem cells. Self-renewalotential requires a long-term memory system for sta-le maintenance of transcriptional patterns. In contrast,he potential for multi-lineage differentiation requireslasticity of the genome allowing multiple differentia-ion decision. This apparent dichotomy of stem cells iseflected by the presence of specific chromatin config-ration that has begun to emerge from recent studies.apping of histone methylation patterns in mouse ES

ells revealed distinct chromatin domains termed ‘biva-ent domains’ that may silence developmental geneshile keeping them poised for transcriptional activa-

ion [61]. These bivalent chromatin domains consist ofarge regions of chromatin with histone H3K27 methy-ation harbouring smaller regions with histone H3K4ethylation. These domains appear to dissolve duringS cell differentiation and are proposed to be a novelhromatin-based mechanism for the maintenance ofluripotency [61]. While it appears that DNA sequencetself largely define this initial “chromatin landscape”n ES cells, different endogenous and environmentaltimuli are likely to trigger chromatin re-configurationnd lineage-specific gene expression program. Consis-ent with these findings, it was found that certain tumouruppressor genes and pro-differentiation genes are heldn a “transcription-ready” state mediated by “bivalent”romoter chromatin patterns [62]. These patterns con-ain H3K27 methylation, a repressive mark mediated byolycomb complex. Given their importance in stem celldentity, deregulation of bivalent domains may make the

umour suppressor genes and pro-differentiation genesulnerable to abnormal DNA methylation events lead-ng to cancer initiation and progression, although furthertudies are needed to fully understand this process.

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Recent studies revealed the existence of correlationetween histone acetylation, transcriptional competencend DNA replication timing in mammalian cells [63–67].zuara et al. [63] investigated the patterns of DNA

eplication timing in the function of transcriptionalotential in cells with different pluripotency includingS cells, HSCs, and their differentiated progeny. Inter-stingly, silent, lineage-specific genes replicated earliern pluripotent cells than in tissue specific stem cellsHSCs) and differentiated progeny and exhibited highevels of acetylated H3K9 and methylated H3K4, bothf which are histone marks associated with active chro-atin. Early replication thus correlates with histone

cetylation, where the genes showing high levels ofcetylated H3K9 replicated early in S phase regard-ess of their expression status, whereas low levels of3K9 acetylation were associated with late replication.urprisingly, the presence of these markers of openhromatin was combined with the presence of H3K27rimethylation (histone mark usually associated withepressive chromatin) at non-expressed genes. Thus,ineage-specific genes may be primed for expressionn ES cells but their expression is prevented by repres-ive H3K27 methylation mark. These results argue thatluripotency of stem cells may be defined by specificatterns of histone acetylation and methylation whereineage-specific loci are accessible to transcriptional

achinery but they are not expressed due to the pres-nce of opposing histone mark. Therefore, replicationiming defined by histone modifications may generate ahromatin signature for ES cells and may be the basesor distinguishing ES cell from progenitors with moreestricted lineage potential [63]. It would be of interesto investigate whether abnormal patterns of replicationiming contribute to the development of cancer.

The genome of stem cells undergoes global changesn gene expression during the transition from a pluripo-ent to a committed state. Recent studies identified aumber of genes that are essential in the maintenancef stem cell properties. These include transcription fac-ors, Oct4, Nanog and Sox2 [68]. In addition, severalignalling pathways such as Wnt and Notch have beenhown to play important role in stem cell pluripotency69]. However, despite intensive efforts, no pathwayroved to be specific for stem cells. Although, such path-ay may exist, it appears that global changes in gene

xpression patterns during stem cell differentiation maye mediated by chromatin structure and modifications.

herefore, gene expression is primarily determined byhromatin state and interaction of chromatin bindingactors, although underlying mechanisms remain to belucidated.

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Several recent studies suggested that pluripotentcells may have a distinct chromatin configuration thatallows additional layers of interaction with chromatin-associated factors. In general, the building blocks ofchromatin in eukaryotes are nucleosomes that consistof DNA wrapped around core histones. An interest-ing feature of chromatin is the presence of severalhistone variants such as histone H2AX and histoneH3.3 which play important roles in cellular processessuch as DNA damage response and DNA replicationcontrol [70–72]. Nucleosomes are further structuredto form a higher-order chromatin organisation thatdefines the accessibility to and function of chromatinDNA. Recent evidence indicates that the interactionbetween chromatin and different chromatin-associatedproteins is highly dynamic. A study using fluorescentrecovery after photobleaching (FRAP) demonstratedthat chromatin-associated proteins appear to be mobilewithin the nucleus and interact with chromatin onlytransiently [73]. Interestingly, with the exception ofthe core histones, even structural chromatin compo-nents such as HP1 protein and linker histones, bindto chromatin in a transient manner [74,75]. Therefore,establishment and maintenance of structural and func-tional chromatin domains may involve a highly dynamicinteraction of different proteins with chromatin. By anal-ogy, chromatin-associated proteins may play importantroles in gene expression patterns associated with mainte-nance of ES cell pluripotency and during differentiation.

Major architectural chromatin proteins appear to bemore dynamic than previously thought. Using FRAPapproach, Meshorer et al. [76] monitored affinity andexchange rate of different chromatin-associated pro-teins in pluripotent mouse ES cells and differentiatedcell types, and found that different histone variants arerapidly exchanged ranging from several seconds to fewminutes. Moreover, a significantly larger fraction (25%)of total cellular pools of certain histone variants areloosely bound to chromatin in ES cell compared todifferentiated cells (3%). This led to the concept ofhyperdynamic (“breathing”) chromatin [76]. The hyper-dynamic chromatin seems to be a special feature ofpluripotent cells, as committed progenitor cells (lineage-restricted cells lacking pluripotency) do not exhibit theproperty of hyperdynamic chromatin fibre. Therefore, itappears that hyperdynamic chromatin characterized byloosely bound structural chromatin components is notonly a hallmark of pluripotent state but may play an

important role in the maintenance of the pluripotency ofES cells. In addition, hyperdynamic chromatin may beimportant for early stages of ES differentiation throughmaintaining chromatin in relatively open, plastic state

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nd reshaping the global architecture of the genome.y maintaining loosely bound histone variants and otherhromatin associated proteins, pluripotent ES cells mayaintain the potential to rapidly differentiate to multiple

ell lineages according to changing need of a tissue.The molecular mechanism controlling the hyperdy-

amic state of chromatin in pluripotent cells remainsnknown. Interestingly, the changes in global organisa-ion of the genome during differentiation of pluripotentS cells were associated with changes in histone mod-

fications. It was found that differentiation of ES cellsnduced an increase in H3K9 trimethylation (associatedith silent chromatin) [76,77]. As cells differentiate,

he number of H3K9 trimethylation foci per nucleusncreased while their size decreased. In contrast, thecetylation levels of both histone H3 and H4, whichre associated with transcriptionally active chromatin,ere reduced in differentiated cells compared to ES cells

76]. These findings provide evidence that chromatin islobally reorganized as pluripotent ES cells differentiatend that chromatin in differentiated or partially differ-ntiated cells exhibits a more closed (non-permissive)onformation compared to that in ES cells. Therefore,olecular events provoking changes in the pattern of

istone acetylation and methylation appear to governransition from pluripotent to committed state. Although

olecular mechanism underlying the maintenance ofyperdynamic chromatin in ES cells remains to be elu-idated, these observations have important implicationsor stem cell research and development of therapeutictrategies. It will thus be interesting to identify moleculesnvolved in controlling the breathing state of ES chro-

atin and the complexes involved in this process.Recent studies indicated that in stem cells during early

mbryonic development, the promoter DNA methyla-ion is accompanied by specific histone modificationshat are typical of heterochromatin [78,79]. Specific epi-enetic modifications mark the degree of activity ofhe transcribing genes. Genes that are being activelyranscribed generally contain nucleosomes with acety-ated histone proteins, histone H3 and H4, as well asethylated H3; in contrast, DNA segments with inac-

ive expression normally have deacetylated histones.he study by Heintzman et al. [80] used a combina-

ion of chromatin immunoprecipitation and microarraynalysis to map histone modifications, transcription fac-or binding, and nucleosome density within 30 Mb ofhe human genome and found that active human pro-

oters were nucleosome-depleted. In addition, theseromoters also showed enrichment of H3K4 trimethy-ation, whereas the enhancers showed enrichment ofonomethylated H3K4. Interestingly, these distinct pro-

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oter and enhancer chromatin signatures appeared tollow the prediction of the identities of many promot-rs and enhancers of other genes in the region undernvestigation [80].

An important unsolved issue regarding the devel-pment of cancer stem cells concerns a possibilityhat differentiated cells may undergo malignant trans-ormation by reacquiring stem cell features throughedifferentiation. A recent study revealed that reversibleene repression may be replaced by permanent silenc-ng, locking the cells into a perpetual state of self-renewaleading to tumour-prone state [79]. It has already beeneported that stem cells rely on polycomb group (PcG)roteins to reversibly repress genes encoding transcrip-ion factors required for differentiation [81]. The geneshich are targeted for transcriptional repression inuman ES cells by the PcG proteins, suppressor ofeste 12 (SUZ 12) and embryonic ectoderm develop-ent (EED) proteins, form the polycomb repressive

omplex 2 (PRC2) and are associated with nucleosomeshat are trimethylated at H3K27 [82,83]. Global DNA

ethylation patterns established at the time of implanta-ion is thought to be maintained at each cell divisiony the action of DNA (cytosine-5)-methyltransferase(DNMT1) during DNA replication [84]. As EZH2

tself can recruit DNMTs [85], it is plausible that underppropriate conditions EZH2 can serve as a platformor attracting methyltransferases that bring about deovo methylation. Epigenetic inactivation of tumour sup-ressor genes have been identified in cancer and thisnactivation is accomplished by active de novo DNAethylation in cells in which one allele is alreadyutated or deleted, serving as a mechanism to repress

he second allele [86]. More recently, it has been demon-trated that both gene targeting and adaptive mechanismre involved in the de novo methylation that occurs inancer and that the polycomb-directed de novo methy-ation may play important part in carcinogenesis [78].herefore, abnormal chromatin pre-marking involvingistone methylation and acetylation in pluripotent cellsuring early embryonic development may predisposeertain genes to cancer promoting DNA methylationvents and tumourigenesis.

Acetylation of specific lysine residues within themino-terminal tails of core histones is mediated byeveral HATs whose activity is dependent on the mul-iprotein HAT complexes. Recently, we have studiedhe role of HATs and histone acetylation in cellular

unctions using loss-of-function approach in cells andice. We found that TRRAP, a common component ofAT complexes, is essential for embryonic development

nd stem cell clonogenic potential [87] (and Z. Herceg,

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arch 637 (2008) 1–15

npublished results). Our genetic and cellular studiesave shown that TRRAP/HAT is involved in a con-erted and context-coupled recruitment of HAT activityo chromatin, providing a functional link between spe-ific chromatin modifications and cellular functions31,88]. Furthermore, we have found that TRRAP andRRAP-mediated histone acetylation may participate in

he maintenance of stem cell identity through chromatinignature (Loizou, Shukla and Herceg, manuscript inreparation). These results suggest that stem/progenitorell identity is defined by histone modifications includ-ng histone acetylation that generate an epigeneticignature for pluripotent cells. Although the mechanismy which TRRAP/HAT participate in the regulation ofluripotency is unclear, the role of TRRAP in the main-enance of pluripotency could be through its interactionith other key regulators such as c-Myc, a transcrip-

ion factor which directly interacts with TRRAP [89] andlays a role in differentiation and apoptosis whose dereg-lation alters stem cell pools [90]. Interestingly, c-Mycas identified as a part of a minimal set of four genesecessary to derive pluripotent stem cells from differ-ntiated cell [5,91]. TRRAP may also participate in theegulation of stem cell pluripotency through its physicalr functional interaction with polycomb group (PcG) ofroteins. PcG proteins are involved in the maintenance ofmbryonic and adult stem cells through their regulationf epigenetically mediated long-term repression, whoseeregulation has been implicated in cancer development92]. Interestingly, TRRAP is found in the same chro-atin modifying complex with enhancer of polycomb

E(Pc)] protein [93–97], and genetic studies in C. elegansevealed that both proteins seem to act in a linear geneticathway [98]. The role of polycomb proteins is to main-ain stable and heritable repression of specific stemnessenes; thus it is possible that TRRAP/[E(Pc)] interactionay participate in self-renewal and differentiation fate

ecisions.

0. Genetic versus epigenetic changes in cancernd implications for cancer therapy

Cancer can be defined (histo-pathologically) as anbnormal mass of tissue with uncoordinated and exces-ive growth even after the cessation of the stimuli thatvoked the change. Tumours can be benign (localizednd non invasive) or malignant (able of invading sur-ounding and distant tissues due to metastasis) composed

f transformed cells and supporting cells and structures.he clonal genetic model is widely held view of cancerevelopment which implies that tumour mass arises fromclonal expansion of a single progenitor cell that has

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incurred genetic damage. This view thus implicates thattumours are monoclonal and that different categories ofgenes are involved in the process such as oncogenes andtumour suppressor genes. In agreement with this notion,accumulation of mutations during tumour progressionhas been found in many cancers. However, despite itsstrengths, the clonal genetic model has several importantweaknesses that may prevent its universal acceptance. Toreconcile these issues, Feinberg, Ohlsson and Henikoffproposed the epigenetic progenitor model of cancer [1].According to this model, tumours develop in three steps:(1) epigenetic disruption in stem/progenitor cells due toaberrant regulation of so-called tumour progenitor genes,(2) genetic changes in gatekeeper genes and oncogeneswithin the subpopulation of epigenetically disruptedprogenitors, and (3) genetic and epigenetic plasticity(enhanced ability to stably evolve tumour phenotype)[1]. Although further studies are needed to fully validatethe epigenetic progenitor model, this model may havefar-reaching implications for cancer risk assessment andcancer therapy.

Several recent studies suggested an interesting linkbetween epigenetic and genetic alterations in cancer.Genetic changes including mutations, rearrangementand microsatellite instability, hallmarks of most malig-nancies, may be secondary to and a consequence ofepigenetic alterations. The transcriptional inactivationby CpG hypermethylation of promoters of specificgenes involved in DNA repair (MLH1, MGMT andBRCA1) and carcinogen detoxification (GSTP1) cancause genetic changes triggering neoplastic transforma-tion and contributing to cancer phenotype [86]. Furtherstudies on the hypermethylation of these genes and themutational pathways that their inactivation triggers mayprove very useful for the development for diagnosticmarkers and anti cancer drugs.

Misregulation of histone modifications and DNAmethylation patterns appears to be a widespread phe-nomenon and may lead to an altered potential of stemcell self-renewal and expansion of epigenetically mod-ified stem cell pools. Universal presence of epigeneticchanges in stem/progenitor cells may not only explainphenotypic heterogeneity associated with cancer growth,invasiveness, and metastatic potential, but also bearsimportant implications for cancer therapy. Since epige-netic changes are reversible, identification and targetingepigenetic alterations in cancer stem/progenitor cellsmay provide an unprecedented potential for therapeutic

intervention. Development of specific drugs that targetabnormal patterns of histone modifications and DNAmethylation (for example, histone deacetylase inhibitorsor DNA demethylating agents) in early lesions may be

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arch 637 (2008) 1–15 11

n attractive strategy to modulate gene expression pat-erns that confer unique properties in stem and cancerells defined as “stemness”.

Therapeutic strategies targeting aberrant histonecetylation and DNA methylation proved to be promis-ng in treating different types of human neoplasia.or example, strategies applying HDAC inhibitors, aew class of potential therapeutic agents for cancerreatment, showed promising results as potential ther-peutic agents [99,100]. HDAC inhibitors enable thee-expression of epigenetically modified genes havingmportant roles in processes such as differentiation,ell cycle regulation, apoptosis, angiogenesis, invasionnd metastasis. Clinical trials have been carried outor HDAC inhibitors such as SAHA (suberoylanilideydroxamic acid) and depsipeptide which were admin-stered by continuous intravenous infusion [101,102].n this study, acetylated histone H3 levels in periph-ral blood mononuclear cells (PBMCs) were used as aiomarker of efficiency. The phase I trials with depsipep-ide has also shown responses with T cell lymphomas103]. HDAC inhibitors undergoing clinical trials forreatment of hematopoietic malignancies include theatural HDAC inhibitor trichostatin, hydroxamic aciderivatives (such as suberoylanilide hydroxamic), theyclic tetrapeptide depsipeptide, benzamide derivativesike MS-275 and CI-994, and aliphatic acid such as val-roic acid and phenylbutyrate.

In addition to histone acetylation, DNA methylationhanges may also be an excellent target for epigeneticherapy. The promoter region of many tumour suppressorenes are hypermethylated in various cancers, includingematopoietic malignancies such as MLH1 mismatch-epair gene implicated in colorectal and other cancers,he VHL (von Hippel-Lindau) gene in renal cancer,RCA1 in early breast cancer [104]. Methylation of p15ccurs in patients with malignancies like myelodysplas-ic syndrome (MSD) and acute myelogenous leukaemiaAML), and increased methylation frequency have beenorrelated with the advancement of the disease [105].ince p15 can be reactivated after treatment with methy-

ation inhibitors [106], therapeutic strategies have beeneveloped to effectively inhibit methylation in thesealignancies.Since methylation of CpG islands occurs infre-

uently in normal cells, methylation provides a selectiveumour-specific therapeutic target. In contrast to mutatedene, the DNA sequence product and protein product

f methylated genes remain unaltered. Pharmacologi-al inhibition of methylation-mediated suppression canesult in restoration of normal gene function of inappro-riately silenced genes. A very potent specific inhibitor

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f DNA methylation, 5-aza-2′-deoxycytidine, has beenidely used as a demethylating agent in vitro, and

s used clinically in the treatment of acute leukaemiand myelodysplasia [107]. It should be noted that,t least in this context, methylation is dominant toistone deacetylation, so transcription can only occurhen demethylating agent is applied first, followedy HDAC inhibitors. In a recent study, significantntitumour effects has been observed in HCC (hepato-ellular carcinoma) model systems after treatment withDAC inhibitor SAHA/demethylation inhibitor 5-aza-′-deoxycytidine [108]. Interestingly, this combinationreatment did not impair cellular integrity of untrans-ormed hepatocytes.

Studies on the effects of HDAC inhibitors andemethylating agents in stem cells harbouring deregu-ated epigenetic states would be highly useful to addressotential utility of these epigenetic drugs in targetingancer stem cells.

1. Concluding remarks

Stem/progenitor cells in any given organism share andentical genome with other cell types, therefore, it isbvious that their plasticity is due to epigenetic events.mportantly, cancer may originate from the transforma-ion of normal stem/progenitor cells, therefore similarpigenetic mechanisms may regulate self-renewal androwth potential of stem cells and cancer cells. Genexpression patterns controlled by histone modificationsnd DNA methylation confer unique properties defineds “stemness” that include the capacity to self-renewnd differentiate into many cell lineages (pluripotency).herefore, it is plausible that misregulation of epige-etic states may lead to an altered potential of stemell self-renewal. Stem cells modified in this mannerxhibit no genetic changes, yet they may represent arecursor pool susceptible to acquisition of mutationsnd further epigenetic alterations. In this scenario, epi-enetic disruption is the first steps of tumourigenesisnd is a contributing factor to polyclonal tumour pheno-ype.

Recent studies provided the first insights into theossible mechanisms by which chromatin modificationsay control the maintenance of the stem cell pluripo-

ency and differentiation fates. There is accumulatingvidence that there are several distinct mechanismsy which chromatin modifications including histone

cetylation and methylation may contribute to uniqueroperties of stem/progenitor cells. These include, butay not be limited to (i) bivalent chromatin domains, (ii)

yperdynamic (“breathing”) chromatin, and (iii) DNA

arch 637 (2008) 1–15

eplication timing. Despite the existence of multiple con-rolling mechanisms, there is a striking instability of thetem cell state (pluripotency) compared to the state ofore differentiated cells. Although the causes of this

nstability are unclear, this feature of stem cells con-rolled by mechanisms involving histone modifications

ay be more sensitive to disruption. Further studies areeeded for a better understanding of changes in chro-atin structure and function and possible changes in

omposition of key complexes (such as polycomb andAT complexes) as well as identity of the interactingroteins. Given that all cell types and many cancers areerived from pluripotent cells, a better understandingf epigenetic mechanisms controlling pluripotency andeprogramming will greatly impact on many areas ofodern biology and will help to tailor efficient thera-

eutic strategies.

cknowledgement

We apologize to colleagues whose relevant publi-ations were not cited due to space limitation. Ourork is supported by grants from Association for

nternational Cancer Research (AICR), United King-om; the National Institutes of Health/National Cancernstitute (NIH/NCI), United States; Institut Nationalu Cancer (Epigenetic profiling Network, EpiPro),rance; L’Association pour la Recherche sur le CancerARC), France; la Ligue Nationale (Francaise) Contree Cancer, France; the European Network of Excellencenvironmental Cancer Risk, Nutrition and Individualusceptibility (ECNIS), and the Swiss Bridge Award (to.H.).

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