MINIREVIEW / MINISYNTHESE

Epigenetic tete-a-tete: the bilateral relationshipbetween chromatin modifications and DNAmethylation1

Ana C. D’Alessio and Moshe Szyf

Abstract: The epigenome, which comprises chromatin, associated proteins, and the pattern of covalent modification ofDNA by methylation, sets up and maintains gene expression programs. It was originally believed that DNA methylationwas the dominant reaction in determining the chromatin structure. However, emerging data suggest that chromatin can af-fect DNA methylation in both directions, triggering either de novo DNA methylation or demethylation. These events areparticularly important for the understanding of cellular transformation, which requires a coordinated change in gene ex-pression profiles. While genetic alterations can explain some of the changes, the important role of epigenetic reprogram-ming is becoming more and more evident. Cancer cells exhibit a paradoxical coexistence of global loss of DNAmethylation with regional hypermethylation.

Key words: chromatin structure, DNA methylation, cancer.

Resume : L’epigenome, qui comprend la chromatine, les proteines associees et le patron de modifications covalentes del’ADN par methylation, determine et maintient les programmes d’expression genique. On a d’abord cru que la methylationd’ADN etait la reaction dominante de la determination de la structure de la chromatine. Cependant, des resultats recentssuggerent que la chromatine peut affecter la methylation d‘ADN dans les deux sens en provoquant la methylation ou la de-methylation d’ADN de novo. Ces evenements sont particulierement importants pour comprendre la transformation cellu-laire qui requiert un changement coordonne des profils d’expression genique. Alors que les alterations genetiques peuventexpliquer certains changements, le role important de la reprogrammation epigenetique devient de plus en plus evident. Lescellules cancereuses revelent une coexistence paradoxale d’une perte globale de methylation d’ADN et d’une hypermethy-lation regionale.

Mots cles : structure de la chromatine, methylation d’ADN, cancer.

[Traduit par la Redaction]

Introduction

The epigenome establishes gene expression profiles invertebrate cells. In contrast with the genome, which is iden-tical in different cell types, the epigenome is dynamic andvaries from cell to cell. The epigenome comprises 2 differ-ent components: the chromatin structure, which is associatedwith the DNA, and a pattern of DNA methylation, which ispart of the covalent structure of DNA. The basic unit ofchromatin is the nucleosome, which is a dimer of 2 identical

complexes, each consisting of 4 histone proteins: H2A,H2B, H3, and H4 (Pruss et al. 1995). The N-terminal tailsof histones H3 and H4 are highly charged and are tightly as-sociated with DNA. Chromatin had been viewed in the pastas a static entity, which packaged DNA in a condensed formand maintained its integrity. However, a vast body of litera-ture has established that histones undergo diverse covalentmodifications, which include acetylation, methylation, phos-phorylation, ubiquitination, and sumoylation (Strahl and Al-lis 2000; Zhang and Reinberg 2001). These modificationsare believed to form a histone code, which regulates chro-matin function by affecting the structural dynamics of thenucleosome and ultimately dictates gene expression patternsby defining the accessibility of the transcription machineryto genes, as well as gating the accessibility of the genometo other machineries, such as repair, DNA replication, andchromosomal segregation. Histone modifications regulatechromatin function either by altering the accessibility ofDNA to different trans-acting factors, or by recruiting spe-cific proteins that recognize a single or conformational setof modifications (Strahl and Allis 2000). Examples of pro-

Received 3 April 2006. Revision received 20 June 2006.Accepted 21 June 2006. Published on the NRC Research PressWeb site at http://bcb.nrc.ca on 8 August 2006.

A.C. D’Alessio and M. Szyf.2 Department of Pharmacology andTherapeutics, McGill University, 3655 Promenade Sir WilliamOsler, Montreal, QC H3G 1Y6, Canada.

1This paper is one of a selection of papers published in thisSpecial Issue, entitled 27th International West Coast Chromatinand Chromosome Conference, and has undergone the Journal’susual peer review process.

2Corresponding author (e-mail: moshe.szyf@mcgill.ca).

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Biochem. Cell Biol. 84: 463–476 (2006) doi:10.1139/O06-090 # 2006 NRC Canada

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teins whose interaction with DNA is defined by differenthistone modifications include bromodomain-containing pro-teins, which interact with acetylated histones (Filetici et al.2001), and chromodomain-containing proteins, which inter-act with methylated lysines (Lachner and Jenuwein 2002).A classic example is the heterochomatin-associated proteinHP-1, which recognizes histone H3 methylated at K9 and isbelieved to be involved in gene silencing in heterochromatin(Lachner et al. 2001). Different histone variants also playregulatory roles (Henikoff et al. 2004; Pusarla and Bhargava2005; Sarma and Reinberg 2005; Zhang et al. 2005). The re-modeling of chromatin is also critical for gene function, andinvolves ATPase-containing complexes, such as SWI/SNF,which propels energy-dependent nucleosomal movement re-quired for the opening up of chromatin around transcriptioninitiation regions (Gibbons 2005; Muchardt and Yaniv 1999;Wolffe and Hayes 1999).

Histone acetylation of H3 and H4 N-terminal tails is be-lieved to be a predominant signal for active chromatin byenhancing the accessibility of the transcription machinery.This signal is removed by the action of histone deacetylases(HDAC) (Kuo and Allis 1998). Histone methylation on K9,catalyzed by Suv39, recruits the heterochromatin proteinHP-1, which condenses the chromatin into an inactive struc-ture (Cao et al. 2002). Histone methylation at K4, on theother hand, is associated with gene activation (Santos-Rosaet al. 2002). Although it was long believed that histonemethylation is irreversible, histone demethylases that can re-move either repressive or activating histone methylationmarks have been identified (Metzger et al. 2005; Shi et al.2004).

The other component of the epigenome, DNA methyla-tion, occurs at position 5 of cytosines in the pyrimidine ringof CpG dinucleotides. This modification, which typically oc-cursat intergenic sites, has also been proposed to regulategene expression. An established consequence of CpG meth-ylation is transcriptional silencing (Razin and Riggs 1980).The process of DNA methylation takes place after DNA rep-lication (Araujo et al. 1998) and is catalyzed by DNA meth-yltransferases (DNMTs), which transfer the methyl groupfrom S-adenosylmethionine to the fifth position on the cyto-sine ring (Wu and Santi 1985). It is believed that DNAmethylation patterns are laid down during development byde novo DNMTs (DNMT3a and DNMT3b) (Okano et al.1999). These patterns are then copied in somatic cells by afaithful semiconservative maintenance DNA methyltransfer-ase (DNMT1), which replicates the methylation pattern froma methylated parental strand template to an unmethylateddaughter strand (Razin and Szyf 1984). Figure 1 summarizesthe main methylation reactions. This model has importantimplications that have dominated thinking in the field forthe last 2 decades. The first implication is that DNA methyl-ation patterns are fixed after birth once differentiation iscompleted. The second is that the parental DNA templateexclusively defines methylation patterns in somatic cells.These led to the idea that, while DNA methylation plays acritical role in development, it does not play a role in alter-ing and programming gene expression profiles after devel-opment.

However, what emerged in the last decade is that DNAmethylation is a reversible process, and therefore the distinc-

tion between maintenance and de novo methylation needs tobe revisited (reviewed and discussed in Szyf 2005). As atrue biological signal, DNA methylation is both targetedand reversible.

DNA methylation interferes with gene expression by di-rect and indirect mechanisms. First, methylation of a CpGsite in the recognition sequence of a transcription factor hin-ders its interaction with DNA recognition sequences (Comband Goodman 1990; Prendergast et al. 1991). Second, meth-ylation of a region around a transcription regulatory site at-tracts methylated DNA binding domain (MBD) proteins,such as MeCp2, which recruit corepressors and histone de-acetylases that inactivate the chromatin configuration aroundthe gene (Nan et al. 1998a, 1998b). This paradigm of genesilencing by DNA methylation is one of the finest examplesof the tight relationship between DNA methylation andchromatin structure.

The idea that these 2 mechanisms of epigenetic regula-tion, i.e., DNA and chromatin structure, are related goesback 3 decades to the pioneering work of Razin and Cedar,who showed that there is a tight correlation between inactivechromatin and DNA methylation (Razin and Cedar 1977). Itis as if the genome is compartmentalized into 2 distinctcompartments, a hypomethylated DNA compartment associ-ated with active acetylated chromatin, and a second compo-nent, which is hypermethylated and associated withhypoacetylated, inactive chromatin. Originally, attentionwas focused on the relationship between DNA methylationand inactive chromatin structure. Early studies have demon-

Fig. 1. DNA methylation reactions. DNA methyltranferases(DNMTs) catalyze the transfer of methyl groups onto DNA. Denovo methyltranferases introduce methyl groups (CH3) onto CpGsites that were not previously methylated. Once DNA methylationpatterns are set by de novo methyltranferases and demethylases,they are maintained during DNA replication by DNMT1, the main-tenance DNA methyltranferase. In the absence of DNMT1, cellsundergo passive DNA demethylation.

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strated that when methylated DNA is transfected into cells,it assumed an inactive chromatin structure (Eden et al.1998). This led to the notion of a unidirectional relationshipbetween DNA methylation and chromatin structure. There-fore, it was believed that although gene silencing involvesboth DNA methylation and chromatin inactivation, DNAmethylation leads to gene silencing, and therefore a methy-lated gene could only be activated if methylation is re-moved. It was generally assumed that HDAC inhibitorswould only activate methylated genes in cooperation with aDNA methylation inhibitor (Cameron et al. 1999). The no-tion that DNA methylation is a primary signal in gene inac-tivation received experimental support from the discovery ofmethylated DNA binding proteins and their ability to recruithistone-modifying proteins to methylated genes (Nan et al.1998a).

Recent data suggest, however, that this unidirectional pic-ture needs to be revisited and that chromatin structure couldalso dictate DNA methylation patterns. Proteins that are in-volved in the silencing of chromatin were also shown to re-cruit DNA methyltransferases to genes (Brenner et al. 2005;Fuks et al. 2000; Vire et al. 2006). Data from our laboratory,as well as those of others have suggested that DNA hypome-thylation might also be dictated by chromatin (reviewed inSzyf et al. 2004a). Thus, we propose that there is a bidirec-tional relationship between DNA methylation and chromatinstructure.

This has important biological and therapeutic implica-tions. In cancer, both hypomethylation and regional hyper-methylation are observed. First, what are the mechanismsresponsible for these processes and what are the effects ofblocking them? Second, do changes in DNA methylationvary in response to physiological and environmental signals?Third, can we activate methylated genes by drugs that act onchromatin modifying enzymes?

Epigenetic silencing cross-talk

The genetic evidence for a possible causal role of chro-matin in delineating DNA methylation patterns came fromTamaru and Selker, who showed that the histone methyl-transferase DIM-5 from Neurospora crassa is important fornormal DNA methylation (Tamaru et al. 2003). Similarly,kryptonite, a histone methyltransferase for Lys9 of H3, con-trols CpNpG DNA methylation in Arabidopsis thaliana(Jackson et al. 2002; Johnson et al. 2002). There is geneticevidence in mammals that chromatin disruption influencesDNA methylation. In mouse, the targeted deletion of theLsh gene, which encodes a SNF2 helicase involved in chro-matin remodeling, produces a substantial loss of CpG meth-ylation throughout the genome (Yan et al. 2003). Thissuggests that the presumed chromatin remodeling activity ofLsh is crucial for setting DNA methylation patterns. Interest-ingly, Lsh is involved in mediating de novo methylation pat-terns by interacting with DNMT3a, as well as DNMT3b.Lsh does not affect the maintenance of previously methy-lated episomes, and interestingly, the Lsh protein was notshown to interact with DNMT1 (Zhu et al. 2006). In hu-mans, a mutation in the ATRX gene encoding a member ofthe SWI/SNF chromatin remodeling family of proteins leadsto certain DNA methylation deficits. A knockout of histone

Suv39h H3K9 methyltransferase in murine embryonic stemcells lead to a decrease in DNMT3b-dependent CpG methyl-ation at major centromeric satellites (Lehnertz et al. 2003).

What are the mechanisms by which chromatin mightdirect DNA methylation? An attractive model is thatchromatin-modifying enzymes recruit DNMTs to specificgenes. The first line of data that is consistent with thishypothesis came from studies that examined the interac-tion between HDAC1 and DNMT1 (Fuks et al. 2000). Ithas been subsequently shown that DNMT1 interacts withHDAC2 as well (Rountree et al. 2000). More recent ex-periments showed that DNMTs interact with the histonemethyltransferases SUV39 (Fuks et al. 2003), whichmethylates H3 histone at K9, and EZH2, a member ofthe multiprotein Polycomb complex PRC2, which methyl-ates H3 histone at the K27 residue (Vire et al. 2006).Both modifications mark inactive chromatin (Cao et al.2002). These studies highlight the significance of target-ing of DNMTs to promoters for establishing and main-taining a specific DNA methylation pattern. A secondline of data shows that EZH2 not only serves to recruitDNMT to chromatin, but also targets the DNA methylation– histone modification multiprotein complexes to specificsequences in DNA (Vire et al. 2006). Since the knock-down of EZH2 results in the loss of DNA methylation ofa methylated target gene, the targeting of DNMTs is re-quired not merely for initiating de novo methylation, butalso for the safeguarding of this pattern. These studies sup-port the hypothesis that this chromatin mark precedesDNA methylation and predisposes DNA to methylation.

One example of how DNA methylation patterns can betargeted and actively defined in somatic cells by cis-actingfactors is the role of PML-RAR fusion in aberrant methyla-tion in certain leukemias (Di Croce et al. 2002). The PML-RAR fusion protein engages HDACs and DNMTs to its tar-get binding sequences and produces de novo DNA methyla-tion of adjacent genes (Di Croce et al. 2002). Although it isnot yet known whether histone deacetylation precedes DNAmethylation, this case demonstrates that a cis-acting re-pressor, which targets genes for chromatin inactivation, alsocauses de novo DNA methylation. This is an excellent illus-tration of targeting DNMT to a gene as an essential require-ment for generating a specific methylation pattern.Moreover, this study also illustrates the dynamic nature ofDNA methylation in somatic cells, since treatment with reti-noic acid, which reverses the fusion protein from a repressorto an activator, results in chromatin activation and DNA de-methylation (Di Croce et al. 2002). Thus, targeting is re-quired not only for the establishment of this pattern ofmethylation, but also for its maintenance.

A recent study provided further support for the targetedrecruitment of DNMT and DNA methylation by chromatin-modifying enzymes in Drosophila, which was believed for along time to be devoid of methylation (Urieli-Shoval et al.1982). Drosophila was recently found to posses DNMT andmethylation of DNA at certain developmental stages (Lykoet al. 2000). By studying tumorigenesis in the Drosophilaeye, Ferres-Marco et al. identified 2 Polycomb group epige-netic silencers, Pipsqueak and Lola, which induce metastatictumors when deregulated (Ferres-Marco et al. 2006). Thisphenotype is dependent on the HDAC Rpb3 in association

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with E(z) (the Drosophila homolog of EZH2), which meth-ylates histone lysine 27, and Sur(var)3–9 (the homolog forSUV39h), which methylates histone H3 at lysine 9. The au-thors found that the metastatic tumors silenced the retino-blastoma tumor suppressor gene by DNA methylationexactly as they do in mammals (Ferres-Marco et al. 2006).This work opens interesting possibilities for using well de-veloped Drosophila genetic models to study the relation-ships between chromatin-modifying enzymes, trans-actingfactors, nodal-signaling pathways, and DNA methylation.

These studies explain the observed correlation betweenchromatin structure and DNA methylation. Moreover, thenotion that DNMTs can be targeted to specific sequencesprovides a mechanism for the generation of new patterns ofDNA methylation throughout the life cycle. According tothis hypothesis, the maintenance of certain DNA methyla-tion patterns is a directed process, which requires the pres-ence of targeted chromatin-inactivation complexes. Thismodel leads to a more dynamic concept of DNA methyla-tion and removes the fundamental difference between main-tenance DNA methylation, catalyzed by DNMT1, and denovo methylation catalyzed by DNMT3a and DNMT3b.There is now evidence that classic de novo methyltransfer-ases, such as DNMT3a and DNMT3b, are required for themaintenance of certain methylation patterns (Chen et al.2003; Kim et al. 2002; Liang et al. 2002), and that DNMT1could catalyze de novo methylation, particularly on CpG is-lands (Jair et al. 2006).

One question that must be addressed in future studies iswhether the majority of the genome’s methylation pattern iscopied by a semiconservative DNA methyltransferase of thetemplate strand according to the classic model, or whetherthe entire DNA methylation pattern is a mirror image of thechromatin state.

The existence of chromatin-based gene expression pro-gramming in organisms whose genome does not contain me-thylated cytosines suggests that, from an evolutionaryperspective, chromatin modification precedes DNA methyla-tion. For example, there is no evidence for DNA methyla-tion in Saccharomyces cerevisiae; nevertheless, thisorganism has very sophisticated chromatin-based gene si-lencing homologous to that found in vertebrates.

This notion that chromatin inactivation is upstream ofDNA methylation is also supported by a time course ofgene inactivation in mammalian systems. For example, atime course study has demonstrated that methylation of thehprt gene on the inactive X chromosome occurs after chro-mosome inactivation (Lock et al. 1987). Similarly, the si-lencing of fetal g-globin precedes DNA methylation (Enveret al. 1988). A study of the epigenetic inactivation of theRASSF1A tumor suppressor in proliferating human mam-mary epithelial cells demonstrated that, with increasing pas-saging, RASSF1A was dramatically silenced. Histone H3K9trimethylation occurred in the same time window as gene in-activation and preceded DNA methylation (Strunnikova etal. 2005). Bachman et al. have studied the silencing of p16in culture following the elimination of DNA methylation,and showed that the methylation of histone H3 at the K9 po-sition, which corresponded to the silencing of the gene, oc-curred prior to DNA methylation (Bachman et al. 2003).

What, then, is the advantage of possessing 2 layers of epi-

genetic information? A possible model is that gene silencingis a self-reinforcing mechanism in which histone H3 K9methylation leads to the binding of adaptor molecules, suchas HP1, which in turn recruit DNA methylases (Fig. 2).DNA methylation directs the binding of methylated DNAbinding proteins, such as MeCp2, which in turn recruit

Fig. 2. Inactive chromatin engages DNA methyltransferases(DNMTs). Transcriptional repressors recruit histone methylases(e.g., SUV39) to methylate K9 of histone H3, which stabilizes theinactive state by recruiting HP1 and DNMTs. The resulting methy-lated DNA serves to guard the inactive state by engaging methy-lated DNA binding proteins, such as MeCp2, which in turn recruithistone deacetylases to further condense and inactivate the chroma-tin.

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HDACs to deacetylate histones and further precipitate theclosed chromatin state. Epigenetic silencing informationwould flow from chromatin to DNA and back. This self-reinforcing mechanism may have evolved to prevent theepigenetic information from drifting. The inadvertent lossof chromatin modifications associated with methylatedDNA would quickly be corrected by recruitment of chro-matin modifying enzymes (Nan et al. 1998a, 1998b), whileaberrant loss of DNA methylation would be corrected byrecruitment of DNMTs by the modified chromatin.

DNA demethylation

Chromatin structure changes are not limited to develop-ment and they take place throughout life. If the correlationbetween DNA methylation and chromatin structure is main-tained, DNA methylation must change in adulthood. Theprevious discussion described the mechanisms of DNAmethylation following the inactivation of chromatin. How-ever, when chromatin is activated later in life, there mustbe mechanisms to demethylate DNA in response to the acti-vation of chromatin.

The central dogma in the field has been that DNA meth-ylation is an irreversible reaction. The idea of the irreversi-bility of DNA methylation and the concept of passivemaintenance of DNA methylation patterns in somatic cellshas entrenched the notion that DNA methylation is fixedpostdevelopment and that it plays no significant role inadaptive responses. Although it is possible to reverse DNAmethylation in replicating cells by passive demethylationbrought on by blocking DNMT1 during DNA synthesis(Fig. 1), this mechanism could not work in postmitotic, fullydifferentiated cells. Like phosphorylation and acetylation, ifDNA methylation patterns act as biological signals in post-development cells, these patterns must be reversible in away that is independent of DNA replication (Ramchandaniet al.1999).

A similar discussion has evolved around the question ofwhether histone methylation is a reversible process. Therewas a reluctance to accept the idea that histone methylationis a reversible process and alternative passive mechanismsof histone demethylation by histone turnover in the absenceof methylation were proposed. However, this discussion wasresolved recently by the discovery of histone demethylasesand unraveling the mechanism of histone demethylation asan oxidative process. Moreover, it was shown that histonedemethylases, like histone methyltransferases, are targetedto specific genes. Histone demethylases that can remove ei-ther repressive or activating histone methylation marks havebeen identified (Metzger et al. 2005; Shi et al. 2004). Twoclasses of histone demethylases were recently discovered:FAD-dependent amine monooxygenases, such as LSD1,which demethylate methyl-K4 H3 via an oxidation reactionthat releases formaldehyde as the leaving group, and JmjCdomain demethylases, which also release formadelhyde butrequire �-ketoglutarate and Fe(II). LSD1 was found in acomplex with the CoREST complex, which represses neuro-nal genes in nonneuronal cells (Lee et al. 2005). A represen-tative of the second class of histone demethylases, JHDM1(JmjC domain-containing histone demethylase 1), which

specifically demethylates histone H3 at lysine 36 (H3-K36),was recently discovered (Tsukada et al. 2006).

There are now convincing examples of active, replication-independent DNA demethylation during development. Genesthat are highly methylated in the paternal genome are rap-idly demethylated in the zygote only hours after fertilization,before the first round of DNA replication commences (Os-wald et al. 2000). In contrast, the oocyte-derived maternalalleles are protected from this reprogramming (Oswald etal. 2000). Other studies have shown that the process of dif-ferentiation of erythroleukemia cells (Razin et al. 1984) andEpstein-Barr virus producing cell lines were associated withgenome-wide replication-independent demethylation (Szyf etal. 1985). Examples of gene-specific active DNA demethy-lation in differentiating cells include the following: the Iggene locus during B cell maturation (Frank et al. 1990; Kir-illov et al. 1996), the muscle specific alpha-actin gene (Pa-roush et al. 1990), and the vitellogenin genes in chickentissues upon estrogen stimulation (Wilks et al. 1982). Activedemethylation was reported for the myosin gene in differen-tiating myoblats cells (Lucarelli et al. 2001), for the Il2 geneupon T cell activation (Bruniquel and Schwartz 2003), andfor the interferon � gene upon antigen exposure of memoryCD8 T cells (Kersh et al. 2006). However, these data, whichsupport the existence of active demethylation, do not by anymeans exclude the possibility that passive demethylationplays an important role in shaping the methylation pattern.Passive demethylation also occurs in some instances of celldifferentiation in vitro, and actually as a major mechanismin development up to the gastrulation stage.

Although the concept of active DNA demethylation isslowly being accepted, the identity of the DNA demethy-lase(s) responsible for this reaction remains a subject of con-troversy. Different activities could explain this DNAdemethylation process. The first possible mechanism en-gages a glycosidase that recognizes methylated CpGs andcleaves the bond between the methylated cytosine and thedeoxyribose. The apyrimidine deoxyribophosphate is re-moved and replaced with an unmethylated cytidylate byDNA repair enzymes. Two mismatch-repair glycosidasewere shown to possess methyl-CG-DNA-glycosidase (5-MCDG) activity that results in demethylation in vitro: a G/T mismatch repair enzyme (Zhu et al. 2000) and the methy-lated binding protein MBD4 (Zhu et al. 2001). Expression ofthe 5-MCDG results in demethylation of a stably integratedecodysone – retinoic acid promoter (Jost et al. 2001). 5-MCDG participates in global demethylation, because trans-fection with an antisense oligonucleotide against this en-zyme produces genome-wide methylation of C2C12 duringdifferentiation (Jost et al. 2001). During development, how-ever, a global DNA repair to achieve hypomethylation mightcreate a serious threat to the genome’s integrity.

A second mechanism proposes a direct removal of themethyl moiety from DNA. Although the cleavage of thisbond has been considered to be improbable because of thestrength of the C-C bond between the methyl residue andthe cytosine ring, in 1999, a bona fide demethylase purifiedfrom human lung cancer A549 cells (Ramchandani et al.1999) was able to produce this reaction, releasing the methylgroup in the form of methanol. The demethylase activity isprocessive (Cervoni et al. 1999), which is possibly critical

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for the global hypomethylation observed during early devel-opment.

In addition, the methyl-CpG-binding protein MBD2 wasfound to possess demethylase activity. MBD2 was found toactively demethylate DNA both in vitro (Bhattacharya et al.1999) and in vivo (Cervoni and Szyf 2001; Detich et al.2002). Furthermore, its expression is associated with the de-methylation of endogenous genes (Hattori et al. 2001a). TheDrosophila homolog of MBD2 formed foci that associatedwith DNA at the cellular blastoderm stage, concurrent withthe activation of the embryonic genome, and also associatedwith the active Y chromosome (Marhold et al. 2002).

The assignment of a demethylase function to MBD2,which was independently discovered to recruit repressorcomplexes (Zhang et al. 1999), has triggered controversy inthe field since Ng et al. were unable to show that their puri-fied MBD2 catalyzed DNA demethylation in vitro (Ng et al.1999). In addition, mbd2 knock out mice did not suffer themassive problems that would be expected if MBD2 were in-deed a major DNA demethylase (Hendrich et al. 2001). Theknockout data suggest that, even if MBD2 is indeed a deme-thylase, other demethylases must exist to explain the deme-thylation events that occur during development.

However, other studies showed that MBD2 could functionas a transcriptional activator interacting with transcriptionfactor TAX to activate transcription from the methylatedHTLV-1 LTR (Ego et al. 2005). In addition, Fujita et al.showed that MBD2 is able to induce CRE-dependant ex-pression of an unmethylated somatostatin promoter by inter-acting with RNA helicase A and RNA polymerase II (Pol II)(Fujita et al. 2003).

Thus, it is possible that, under certain cellular conditionsand within certain promoters, MBD2 can act as a transcrip-tional repressor by recruiting the NuRD complex. However,it is equally possible that, in different cellular environmentsor within different promoters, MBD2 may act independentlyof NuRD as an activator (Detich et al. 2002). A recentstudy showed that the histone methylase PRMT5 stably andspecifically associates with and methylates the RG-rich N-terminus of MBD2 (Le Guezennec et al. 2006). It would beinteresting to see if different post-translational modificationson MBD2 regulate the ability of MBD2 to interact with ei-ther activator or repressive complexes. In support of the lat-ter, a novel MBD2 interactor (MBDin) has the capacity toreactivate transcription from MBD2-repressed methylatedpromoters (Lembo et al. 2003). In addition, TACC3 dis-plays a similar activity on methylated genes (Angrisano etal. 2006). MBD2/TACC3 forms a complex in vivo with thehistone acetyltransferase (HAT) pCAF. MBD2 could alsoassociate with HDAC2, a component of the MeCP1 repres-sion complex (Angrisano et al. 2006; Feng and Zhang2001). However, both complexes were mutually exclusive.

Recent data suggest that MBD2 could interact withchromatin-activating complexes. HAT enzymatic assaysdemonstrated that HAT activity associates with MBD2 invivo (Angrisano et al. 2006). Our recent data show thatMBD2 colocalizes with the HAT CBP (J.-N. Ou, S. Ten-dulkar, and M. Szyf, unpublished data) and that MBD2may act as a demethylase, since over-expression of MBD2in nontransformed NIH 3T3 cells (S.D. Andrews and M.Szyf, unpublished data), as well as cancer cell lines (J.-N.

Ou, S. Tendulkar, and M. Szyf, unpublished data; S. Ten-dulkar, J.-N. Ou, and M. Szyf, unpublished data) results inboth gene-specific and global demethylation, as well ashistone acetylation.

Epigenetic histone acetylation and DNAdemethylation cross-talk

Early observations by Cedar and Razin demonstrated atight correlation between chromatin structure and DNAmethylation. DNA packaged in tightly packed chromatin ishypermethylated, while the DNA associated with open chro-matin is hypometylated (Razin and Cedar 1977). An indica-tion that active chromatin structure can trigger active DNAdemethylation in somatic cells came from studies showingthat the histone deacetylase inhibitor sodium butyrate cantrigger replication-independent DNA demethylation in theEBV producing cell line P3HR-1 (Szyf et al. 1985).

To test the hypothesis that activation of chromatin couldtrigger active demethylation, we transiently introduced an invitro methylated nonreplicating plasmid into a human cellline, HEK 293 (Cervoni and Szyf 2001). Demethylation ofthe plasmid is followed at different time points after intro-duction into the cells using either HpaII restriction enzymeanalysis or bisulfite mapping of DNA methylation at singlenucleotide resolution. We validated that the plasmid did notreplicate in the cells by showing that the bacterial methyla-tion pattern at GATC sites is preserved following transfec-tion, as judged by the sensitivity to restriction enzymeDpnI, which only cleaves DNA that is methylated at the Es-cherichia coli methylated sequence GATC (Cervoni andSzyf 2001). If the plasmid had replicated in mammaliancells it would not be methylated at these sites, since humancells do not bear the methyltransferase required to methylatethese sites.

Using this assay, we studied the mechanisms of activedemethylation in human cells. Ectopically methylated genes,which are controlled by highly active promoters, becomedemethylated when introduced into HEK293 cells, whereasweak promoters remain methylated (Cervoni and Szyf2001). Histone acetylation induced by HDAC inhibitors,such as trichostatin A (TSA), triggers demethylation ofthese ectopically methylated genes, suggesting that the crit-ical event dictating DNA demethylation is histone acetyla-tion (Cervoni and Szyf 2001; Detich et al. 2003). The over-expression of InHAT proteins, which inhibit HATs, re-presses TSA-induced demethylation (Cervoni et al. 2002),which suggests that the main mechanism by which TSA ac-tivates DNA demethylation is through histone acetylation.There is also evidence that TSA causes the demethylationof genomic sequences. In Neurospora crassa, TSA can alsocause selective loss of DNA methylation (Selker 1998);however, this study did not determine whether the loss ofmethylation was a result of active demethylation. Strongevidence that TSA could induce demethylation in nonrepli-cating cells in the adult comes from experiments on the glu-cocorticoid receptor in the hippocampus of adult rats(Weaver et al. 2004). TSA is not a chemical demethylatingagent. Therefore, these experiments imply the presence ofactive demethylation machinery in somatic cells and post-mitotic neurons. The activity is masked by deacetylated his-

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tones and is revealed once histones are acetylated. This isconsistent with the idea that the DNA methylation patternis a balance of methylation and demethylation reactions(Szyf and Detich 2001).

These studies demonstrate that the state of chromatin candirect demethylation and might provide an explanation forthe unmethylated state of active genes (Cervoni and Szyf2001). Although TSA is a pharmacological agent causingacetylation, the cell bears many transcription factors that tar-get HATs to specific genes. There is data to suggest thatcertain transcription factors can cause site-specific demethy-lation, which is dependent on the presence of their cognatecis-acting signals. For example, the intronic kappa chain en-hancer and the transcription factor NF-kappaB are requiredfor B-cell-specific demethylation of the kappa gene (Lich-tenstein et al. 1994). Heritable epigenetic inactivation of themaize Suppressor-mutator (Spm) transposon is associatedwith promoter methylation, and its demethylation is medi-ated by the transposon-encoded transcriptional activatorTnpA protein (Bruniquel and Schwartz 2003).

What is the mechanism through which histone acetylationbrings about DNA demethylation? One possible model isthat the tight interaction between nonacetylated histone tailssimply blocks the access of DNA demethylases to con-densed chromatin (Cervoni and Szyf 2001; Detich et al.2003) (Fig. 3). However, our recent unpublished observa-tions (A.C. D’Alessio, I.C. Weaver, and M. Szyf) suggestthat other members of the cast list play an important role.We performed a time-course analysis of the events leadingto replication-independent active DNA demethylation usinga combination of chromatin immunoprecipitation with thepertinent epigenomic status antibodies and bisulfite mappingto follow the time course of demethylation of DNA in rela-tion to changes in the chromatin state. We found that deme-thylation did not occur immediately after histone acetylationbut rather, it followed the interaction of RNA Pol II with themethylated promoter (Fig. 4). Following demethylation, bothRNA Pol II interaction and histone acetylation are enhanced.Our data are consistent with the hypothesis that RNA tran-scription is required for DNA demethylation, since the in-hibition of expression with actinomycin D inhibited DNAdemethylation. These data are consistent with a biphasic re-lation between RNA Pol II and the epigenetic state. The firststage involves a weak primary interaction of RNA Pol IIand the transcription machinery with the methylated andpartially acetylated promoter, resulting in the movement ofRNA Pol II along the coding regions of the gene. Theseevents in turn enable full epigenetic reprogramming involv-ing further histone acetylation and DNA demethylation. In-terestingly, histone marks of actively transcribed genes,such as the Ser5 phosphorylated RNA Pol II and histone H3trimethyl lysine 4, followed the demethylation of the pro-moter, suggesting that these mark the later stages of epige-netic reprogramming of the promoter. We found thatMBD2, which, as discussed above, is a highly disputed de-methylase, is a rate-limiting step in DNA demethylation fol-lowing chromatin activation. A small interfering RNAknockdown of MBD2 inhibited TSA-induced DNA deme-thylation, and the overexpression of MBD2 increased TSAinduced DNA demethyation.

The fact that RNA Pol II interaction was required for de-

methylation was surprising and unexpected. The basic no-tion in the field is that the essential raison d’etre ofepigenetic activation is to enable RNA Pol II interactionwith a promoter and gene transcription. The fact that RNAPol II interaction is required to allow RNA Pol II interactionseems tautological. What is the role of RNA Pol II in deme-thylation? It is possible that the movement of RNA Pol IIalong the gene opens up the chromatin and prepares thegene for demethylation. Alternatively, RNA Pol II mightphysically recruitDNA demethylases, such as MBD2, to thegene.

DNA demethylation triggered by activation of chromatinby TSA is inconsistent with the commonly accepted viewthat TSA acts only on chromatin and that HDAC inhibitorsand DNA methylation inhibitors target different processes(Cameron et al. 1999). The idea that HDAC inhibitors andclassic DNA demethylation agents might have overlappingtargets and mechanisms is supported by a comparative mi-croarray analysis of the effects of 5-aza-CdR, a classic DNAmethylation inhibitor, and TSA treatment on colorectal can-cer cells (Gius et al. 2004). Since the effects of 5-aza-CdRon gene expression were the same after one or five days, the

Fig. 3. Active chromatin enlists DNA demethylation. A silencedgene is maintained by the action of histone deacetylases (HDACs),histone methylases, and DNMTs and is inaccessible to DNA de-methylases. Gene activation requires either the recruitment of astrong transcriptional activator, which brings histone demethylases(HdMTases) and HATs to the gene, or pharmacological inhibitorsof HDACs (e.g., trichostatin A). These open chromatin to DNA de-methylases, resulting in a stable activation of the gene.

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authors concluded that the changes in gene expression pro-duced by 5-aza-CdR were caused by active DNA demethy-lation. If it is true that DNA methylation patterns are only

maintained by DNMTs in the absence of DNA demethy-lases to oppose the reaction, then 5-aza-CdR should onlyproduce passive DNA demethylation. However, the factthat 5-aza-CdR can induce active DNA demethylation isconsistent with the working hypothesis that DNA methyla-tion patterns constitute a steady-state balance of methyla-tion and demethylation reactions. The inhibition of DNMTby 5-aza-CdR leaves demethylation reactions unopposed,leading DNA demethylases to induce a new unmethylatedstate.

The partial overlap between HDAC inhibitor and DNAmethylation inhibitor target mechanisms has important phar-macological and therapeutic implications (Szyf 2001). First,if HDAC inhibitors induce DNA demethylation, they mightbe used to alter DNA methylation in nondividing tissues,such as the brain (Weaver et al. 2004). A pertinent exampleis the reversal of maternally programmed methylation ofglucocorticoid receptor in the adult rat brain. The demethy-lation resulted in distinct behavioral changes, such as re-duced stress responsivity and anxiety (Weaver et al. 2004).One can envisage the future use of different HDAC inhibi-tors to modulate DNA methylation as a therapeutic approachto certain mental disorders. Indeed, this approach has beenrecently proposed and specific HDAC inhibitors are in theearly stages of development for treating psychiatric disor-ders (Costa et al. 2003; Goffin and Eisenhauer 2002; Simo-nini et al. 2006). In contrast, all DNA methylation inhibitorscurrently in clinical trials target DNA methyltransferase in-hibition at the time of DNA synthesis and are effective onlyin dividing cells (Goffin and Eisenhauer 2002). In additionto treating nondividing somatic tissues, HDAC inhibitorsthat are not S-phase specific DNA demethylating agentsmight be of interest in treating slow-growing low-mitotic tu-mors.

A second pharmacological implication is that one mustconsider the potential impact of HDAC inhibitors on theDNA methylation pattern both in dividing and nondividingtissues. When assessing the mechanism of action of thesedrugs, as well as their potential positive and adverse clinicaleffects on the state of methylation in target and other tis-sues, must be evaluated. This is rarely done today, as it isgenerally assumed that HDAC inhibitors target chromatinexclusively.

Epigenetics and cancer

Histone modifications and cancerDifferent histone-modifying enzymes are involved in can-

cer. The dynamic equilibrium of histone acetylation is gov-erned in vivo by the opposing reactions of deacetylases. Thebalance of these reactions is critical for the regulation of cellproliferation.

On the one hand, mutations and chromosomal transloca-tions involving HATs result in the development of cancers(reviewed in Iyer et al. 2004; Pakneshan et al. 2004). Thesetranslocations produce a gain of function by deregulating theHAT activity or targeting lysine acetylation to new sub-strates. In addition to catalyzing histone acetylation, a num-ber of HAT proteins, including CBP/p300 and PCAF,acetylate transcription factors, such as p53, ELKF, NF-kB,MyoD, GATA1, and E2F1, which alters their DNA-binding

Fig. 4. RNA Polymerase II (RNA Pol II) sets the stage for activeDNA demethylation. Pharmacological induction of a methylatedgene with the histone deacetylase inhibitor trichostatin A enableshistone acetylation through the enzymatic activity of HATs. Fol-lowing histone acetylation, RNA Pol II binds to the methylatedpromoter. RNA transcription is required for DNA demethylation,which later brings histone methylases to methylate lysine 4 at thecoding region.

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activity (reviewed in Santos-Rosa and Caldas 2005). Acetyl-tranferase activity targeted to nonhistone substrates, such ashuman or viral oncoproteins, also contributes to the develop-ment of malignancies. The acetylation of adenovirus E1A byCBP/p300 disrupts its interaction with repressive complexes,thus promoting aberrant gene activation (Zhang et al. 2000).

On the other hand, an abnormal increase in HDAC activ-ity can also result in the transcriptional inactivation of tumorsuppressor genes. Studies have shown that HDACs are ex-pressed to a greater extent in tumors than in normal tissues(de Ruijter et al. 2003). Moreover, HDAC2 is overexpressedin mice lacking the APC tumor suppressor (Zhu et al. 2004),and a recent study has shown a truncating mutation ofHDAC2 in human cancers (Ropero et al. 2006). However,since the tumor suppressors RB (Frolov and Dyson 2004)and p53 (Sengupta et al. 2005) require HDACs to inactivatetumor genes, mutations that inactivate HDACs may alsocontribute to the disease.

Another histone modification deregulated in cancer is ly-sine methylation. Histone methylation can be either an acti-vation or a repressive signal. Methylation on histone H3lysine 4 is activating, whereas a methylation on either lysine9 or lysine 27 is repressive. The human histone H3 K4methyltranferase MLL1 is fused and translocated with doz-ens of other genes, resulting in acute leukemia. The overex-pression, mistargeting, and deregulation of histone H3 K4methylation results in an aberrant regulation of gene expres-sion and cellular transformation (Ayton and Cleary 2001;Hamamoto et al. 2004; Hess 2004; Milne et al. 2005). Micelacking the histone H3 K9 methylatranferase genes suv39h1and suv39h2 exhibit telomere abnormalities, chromosomeinstability, and increased rates of lymphomas (Peters et al.2001). In addition, the histone K27 methylase EZH2 ishighly expressed in metastatic prostate cancers, lymphomas,and breast cancer (Kleer et al. 2003; Varambally et al.2002), and the overexpression of EZH2 confers a prolifera-tive advantage in primary cells (Bracken et al. 2003).

DNA methylation and cancerIn addition to changes in chromatin structure, cancer cells

display aberrant DNA methylation patterns. The first attemptto correlate aberrant DNA methylation and cancer focusedon determining the total levels of 5mC (Feinberg and Vogel-stein 1983; Feinberg et al. 1988; Lu et al. 1983). The levelsof global DNA methylation observed in tumors were lowerthan those in normal tissues. A loss of methylation was ob-served in oncogenes (Feinberg and Vogelstein 1983), as wellas in prometastatic genes, such as S100P (Sato et al. 2004),S100A4 (Rosty et al. 2002), and uPA (Guo et al. 2002), es-pecially in metastatic cells. Later studies showed global hy-pomethylation of repetitive elements, such as LINE1(Chalitchagorn et al. 2004), as well as a loss of methylationin imprinted genes (LOI). On the other hand, the hyperme-thylation of tumor suppressor genes, adhesion molecules,DNA repair, and inhibitors of metastasis has also receivedintense attention as an important step in tumorigenesis(Baylin et al. 2001).

What is the mechanism that allows a coexistence ofglobal hypomethylation with regional hypomethylation? Per-haps the answer resides in the idea that both methylationand demethylation are targeted processes and are defined by

the presence of the targeting factors and the sequences thatthey recognize. The hypomethylation of specific genes maytherefore be secondary to local chromatin changes targetedby transcription factors recognizing specific sequences. Asmentioned previously, global chromatin changes do occur incancer due to HAT activation, as well as histone H3 K4methyltransferase overexpression. These events can triggerthe global DNA demethylation observed in cancer cells.However, a global rise in DNA demethylase may also ex-plain the global DNA demethylation change (Szyf 2001;Szyf et al. 2004a).

Recent studies demonstrated that DNA hypomethylationleads to genomic instability in mice and in human cancercells (Eden et al. 2003; Ehrlich 2003; Gaudet et al. 2003).It has also been recently proposed that hypomethylationserves as a mechanism for the coordinate activation ofprometastatic genes in late-stage cancer, while hypermethy-lation serves as a mechanism for uncontrolled growth (Szyfet al. 2004b). These are 2 separate mechanisms which arerequired at different stages of tumor growth. In support ofthis hypothesis, it was shown that the pro-metastatic geneuPA is methylated in the less invasive human breast cancercell line MCF-7 and is totally unmethylated in highly meta-static MDA-MB-231 cells (Guo et al. 2002; Pakneshan et al.2003). The DNA methylation inhibitor 5-aza-CdR inducesuPA and breast cancer invasiveness in MCF-7 cells, whilehypermethylating agents, such as the methyl donor SAM,trigger hypermethylation and suppression of uPA and inhib-ition of metastasis in vitro and in vivo (Pakneshan et al.2004). The overexpression of Ras, which promotes growthin metastatic MDA-MB-231 cells, results in the methylationof uPA and reduced metastasis (Pakneshan et al. 2005). Thislatter study illustrates a dissociation of tumor promoting andmetastasis promoting pathways in cancer.

One candidate for promoting hypomethylation in meta-static cancer cells is the demethylase MBD2 (Szyf et al.2004a). Knockdowns of MBD2 in a number of cancer celllines resulted in the inhibition of anchorage-independentgrowth and the growth of human tumor cell lines in mice invivo (Slack et al. 2001). Pharmacological inhibition ofMBD2 in vivo using antisense oligonucleotides againstMBD2 decreased tumor growth (Campbell et al. 2004).MBD2 levels correlate with hypomethylation in ovarian(Hattori et al. 2001b) and lung (Hattori et al. 2001a) cancer.However, the inverse correlation is observed in colon cancer(Kanai et al. 1999). An antisense oligo MBD2 knockdownin metastatic breast cancer cell line MDA-MB-231 blockedthe prometastatic uPA gene expression, caused its hyperme-thylation, and blocked invasiveness in vitro and metastasisto different organs in vivo (Pakneshan et al. 2004). MBD2depletion was shown to inhibit tumorigenesis by a genetictest as well. Mbd2 knockouts were shown to suppress spon-taneous adenomatous polyps when crossed to the Min mouse(Sansom et al. 2003). Overexpression of MBD2 in both tu-mor and nontransformed cells resulted in global hypomethy-lation, the hypomethylation of metastasis-promoting genes,and enhanced invasiveness (S.D. Andrews, S. Tendulkar, J.-N. Ou, and M. Szyf, unpublished results).

The hypermethylation observed in cancer is most prob-ably a consequence of regional inactivation of the chromatinassociated with tumor suppressor genes. Such inactivation

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might be triggered by the activation of a transcriptional re-pressor by either tumorigenic signaling pathways or by a re-arrangement such as the PML/RAR fusion protein (Di Croceet al. 2002). These suppressors in turn recruit HDACs andother histone methylases, which result in the recruitment ofDNMTs (Fig. 2) to specific genes.

Concluding remarks

The chromatin field has reached a turning point with anew understanding of the relationship between histone mod-ification and DNA methylation. Chromatin can affect DNAmethylation in both directions, triggering either de novoDNA methylation or demethylation. This connection is ex-tremely important for establishing different epigenetic pro-grams not only in cellular transformation, but also inphysiological, behavioral, and pathological areas. However,While DNA methylation and chromatin modification inter-act with each other, DNA methylation does act as a long-term lock-in mechanism in many situations, not the least ofwhich is the definitive repression of retroelements and trans-posons and in the formation of facultative heterochromatin,which was not discussed in this minireview (Henikoff2000). New avenues for epigenetic cross-talk are openingup in fields other than cancer and we are far from the endof the story. Exciting recent data point to a role of epige-netic programming in behavior and adaptation to differentbehavioral interactions (Meaney and Szyf 2005). Whole ge-nome approaches combining methylation arrays and chroma-tin arrays are elucidating the dynamics of DNA methylationin different organs in different responses to the environmentand in different pathologies. Understanding these processeswill require knowledge of how chromatin activation leads tothe reprogramming of DNA methylation patterns. Thesearch for additional DNA demethylases, as well as theirrole in cellular transformation, should become a priority inthe next few years.

AcknowledgementsA.C.D. thanks Dr. Kalle Gehring and Shelley Brown for

critical comments on the manuscript. This work is supportedby a National Cancer Institute of Canada grant to M.S.A.C.D. has a McGill Faculty of Medicine scholarship.

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