DNA methylation in mammalian development and disease

DNA Methylation in Mammalian Development andDisease

Wendy Dean,* Diana Lucifero, and Fatima Santos

INTRODUCTIONThe unfolding of development ofany organism requires a series ofprogressive restrictions leading tocellular differentiation. These re-strictions must be heritable and cu-mulative, such that earlier cell fatesare difficult or impossible to returnto. In mammals, the considerableregulatory task of progressive generepression has been aided by thecovalent addition of a methyl groupin position 5 of the cytosine base ofCpG dinucleotides. Extensive in-vestigation into the activities re-sponsible for DNA methylation andtheir roles in a wide number of bio-logical processes reinforces the im-portance of this extra layer ofgenomic complexity (Robertsonand Wolffe, 2000; Li, 2002).

DNA methylation occupies up to70% of the CpG dinucleotides in thegenome, and represents one of themajor epigenetic modifications inmammals (Robertson and Wolffe,2000). The organization of themammalian genome is such thatthere is a high density of CpG in theupstream promoter regions in mostof the approximately 30,000 genes,as well as within gene introns andexons (Bird, 2002). This organiza-tional feature and the capacity forDNA methylation in mammals leadto the early speculation that generegulation might be highly sensitiveto the methylation status of theseso-called CpG islands (Holliday andPugh, 1975). However, this provednot to be the case, as these poten-tial targets for methylation werefound to be unmethylated, at least

for most genes under normal cir-cumstances (Antequera and Bird,1993; reviewed in Meehan andStancheva, 2001). Notable excep-tions to this general rule includeimprinted genes and those of theinactive X-chromosome. Furthergenomic characterization identifiedother classes of sequence familieswith significant levels of CpG, lead-ing to alternative proposals for thesignificance of this stable and heri-table modification. The mammaliangenome contains an extremely highburden of sequences that havearisen due to integration of retro-transposons. Uncontrolled expres-sion of these sequences from theirviral promoters would result intranscriptional chaos, were it notfor the susceptibility of these pro-moters to be repressed by DNAmethylation (Yoder et al., 1997).This regulatory role for DNA meth-ylation has been embodied in thegenome defense hypothesis, andremains one of the significant func-tions of DNA methylation (Walsh etal., 1998; Bestor, 2000).

One of the most comprehensivelystudied roles of DNA methylation isthe marking of parental alleles bygenomic imprinting. Imprintedgenes are expressed in a non-Men-delian fashion, in which parent-of-origin specifies the active allele(Reik and Walter, 2001). Thesegenes are essential for fetal growthand development, and have beenshown to also influence postnatal

Wendy Dean, Diana Lucifero, and Fatima Santos are from the Laboratory of Developmental Genetics and Imprinting, The BabrahamInstitute, Babraham Research Campus, Cambridge, UK.

Grant sponsor: Biotechnology and Biological Sciences Research Council (to W.D. and W.R.); Medical Research Council (to W.D. andW.R.); Grant sponsor: European Molecular Biology Organisation Long-Term Fellowship (to D.L.).

*Correspondence to: Wendy Dean, Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Babraham ResearchCampus, Cambridge, CB2 4AT, UK. E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20037

Epigenetic modification of the cytosine base of DNA by its methylationintroduced the possibility that beyond the inherent information containedwithin the nucleotide sequence there was an additional layer ofinformation added to the underlying genetic code. DNA methylation hasbeen implicated in a wide range of biological functions, including anessential developmental role in the reprogramming of germ cells and earlyembryos, the repression of endogenous retrotransposons, and ageneralized role in gene expression. Special functions of DNA methylationinclude the marking of one of the parental alleles of many imprintedgenes, a group of genes essential for growth and development inmammals with a unique parent-of-origin expression pattern, a role instabilizing X-chromosome inactivation, and centromere function. In thisregard, it is not surprising that errors in establishing or maintainingpatterns of methylation are associated with a diverse group of humandiseases and syndromes. Birth Defects Research (Part C) 75:98–111,2005. © 2005 Wiley-Liss, Inc.

Key words: DNA methylation; epigenetic reprogramming; mammals;early mammalian development

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WBirth Defects Research (Part C) 75:98–111 (2005)

© 2005 Wiley-Liss, Inc.

growth trajectories and diverse bi-ological processes, for example, af-fecting thermogenesis in offspring,maternal care and suckling behav-ior, and adult behavior and cogni-tion (Li et al., 1999; Reik et al.,2001; Plagge et al., 2004). Themarking of the active and inactivealleles is achieved though differen-tial DNA methylation in critical reg-ulatory regions. These differentiallymethylated regions (DMRs), are es-sential for expression or repres-sion. It is interesting to note that adisproportionately high number ofimprinted genes are found to bemethylated on the maternal allele(Reik and Walter, 2001).

During development in mam-mals, there are at least two periodsof genome-wide DNA methylationreprogramming (Reik and Walter,2001). The two periods best char-acterized include a time during pri-mordial germ cell differentiationand one during preimplantation de-velopment. The extent of this re-programming and whether, in fact,it is required for normal develop-ment in all mammalian species re-main unknown (Bestor, 2000). Inthis regard, the details of DNAmethylation reprogramming in hu-mans are only just beginning to beappreciated. The degree to whichthis important regulatory mecha-nism operates in early developmentand germ cell differentiation in hu-mans will influence our under-standing of the potential impact ofDNA methylation in human healthand disease.

A growing body of evidence indi-cates that DNA methylation, to-gether with chromatin modifica-tions, are competent to specifytranscriptional states and perhapsmore importantly, mutually rein-force transcriptionally repressivestates (Tamaru and Selker, 2001;Jackson et al., 2002; Fuks et al.,2003; Tamaru et al., 2003).

In this review, we will focus onDNA methylation, explore theknown enzymatic activities respon-sible for both establishment andmaintenance of DNA methylationpatterns, compare the reprogram-ming dynamics of methylation be-tween mammals, and consider theconsequences of inappropriate reg-ulation of this epigenetic mark on

genome stability and reproductivebiology.

DNA METHYLATION:ENZYMATIC ACTIVITIESAND SUBSTRATES

Introduction of the methyl groupinto the symmetrical dinucleotide5�-CpG-3� results in its positioninginto the major groove of the DNAwithout interference with the base-pairing of nucleotides. Methylationof cytosine residues imposes agreater level of risk to the stabilityof the genome as deamination ofmethyl-cytosine results in the tran-sition of meC-T, a nucleotide basechange less easily repaired and rec-ognized than the deamination ofcytosine to uracil (Hermann et al.,2004). The lack of high fidelity re-pair capacity for the meC-T transi-tion explains the observation thatCG sites are a major mutationalhotspot, accounting for up to 30%of point mutations in the germline,and are decidedly underrepre-sented in mammalian genomes(Bird et al., 1985). This situationsuggests that the maintenance ofthis modification and its functionshave been the result of consider-able evolutionary pressure, andthat the continued maintenance ofcytosine methylation must confer asignificant advantage given thesubstantial costs. The complexity ofDNA methylation in the genomesuggests that there must be a num-ber of activities responsible for itsestablishment and maintenance,able to operate in both specializedand generalized functions (Chen etal., 2003).

Mammalian DNAMethyltransferases

DNA methyltransferases (MTases)represent a collection of three fam-ily groups numbered in order oftheir discovery (Bestor, 2000).These enzymes serve the two dis-tinctive processes of DNA methyl-ation, the establishment of DNAmethylation state by de novo meth-ylation and, thereafter, the mainte-nance of those states by templatingthis information to daughter

strands arising from replication (Leiet al., 1996; Okano et al., 1999).Despite some sequence similari-ties, the divisions of labor amongstthis group can, in part, be inferredby their functional organization.Broadly, their organization can beresolved into two functional do-mains; the N-terminal domaincomprising regulatory functionsand the C-terminal catalytic do-main (Bestor, 2000; Robertson,2002).

Dnmt1

This was the first of the group ofMTases identified, and is the largestof these activities with an extensiveN-terminal regulatory domain anda smaller C-terminal domain (Be-stor et al., 1988). This large regu-latory domain contains a wide vari-ety of functional motifs, including anuclear localization signal, a prolif-erating cell nuclear antigen (PCNA)(Chuang et al., 1997) interactingdomain, and a replicating foci tar-geting region (Leonhardt et al.,1992). Dnmt1 does not function inthe cell in an isolated manner, andis capable of interacting with manyproteins via the N-terminus. Thisinteraction is likely to be facilitatedby the polybromo domain, a hall-mark of protein–protein interaction(Bestor and Verdine, 1994). Fur-thermore, this domain has been im-plicated in a transport role forDnmt1 to the replication foci (Liu etal., 1998).

Dnmt1 methylates DNA specifi-cally at CG sites, with a strict pref-erence for hemimethylated overunmethylated substrates in vitro(Pradhan et al., 1997, 1999). It isthis preference for hemimethylatedsubstrates that forms the basis forits function as a maintenancemethylase. In this respect, it is notsurprising that Dnmt1 expression istightly coordinated with DNA repli-cation. In addition to its function asa MTase, Dnmt1 has been shown tobe associated with a wide variety ofchromatin modifying activities, in-cluding histone methyltransferases,methyl CpG binding proteins, andheterochromatin binding proteinHP1 (reviewed in Hermann et al.,2004). Collectively, these associa-

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tions share in common the proper-ties of transcriptional repressorsleading to the understanding thatDnmt1 and, hence, DNA methyl-ation stably reinforces chromatinsilencing (Bird, 2002).

Dnmt2

The Dnmt2 gene is the most highlyconserved of the MTases in eu-karyotes, found in both organismsthat show methylation and thosethat have no detectable DNA meth-ylation. Although it is ubiquitouslyexpressed at low levels in most hu-man and mouse tissues as well asmouse embryonic stem cells, micehomozygous for a Dnmt2 null mu-tation are viable, and display nor-mal levels of methylation at endog-enous sequences (Okano et al.,1998b). Introduction of an induc-ible transgene containing Dnmt2indicated that a genuine methylaseactivity could be demonstrated, al-beit on CpT and CpA targets(Kunert et al., 2003). A weak butreproducible in vivo methyltrans-ferase activity was recently demon-strated for the recombinant proteinin mammalian cell lines (Liu et al.,2003).

Dnmt3 family

The highly related enzymes,Dnmt3a and Dnmt3b, are encodedby different genes but share thepreference for methylation of un-methylated CG dinucleotides. Thissubstrate preference identifiesthem as de novo DNA methylases(Okano et al., 1998a). Dnmt3a andDnmt3b are thought to differ mech-anistically due to inherent differ-ences in the catalytic domains, sug-gesting that Dnmt3a is distributivewhile Dnmt3b is processive(Gowher and Jeltsch, 2002). Theseintrinsic differences allow for an ef-fective division of labor betweenthese related de novo methylases.The processive Dnmt3b is moresuited to methylation of CG-rich re-gions of the genome, such as theCG-rich pericentromeric repeats(reviewed in Hermann et al., 2004).The distributive nature of Dnmt3arequires that it adds back methyl-

ation to dinucleotides on a target bytarget basis, and is thus implicatedin de novo methylation at single ge-netic loci (Hata et al., 2002).Dnmt3a is the major form in adulttissues, where it colocalizes withheterochromatin. In contrast, theisozyme Dnmt3a2 is the major formduring embryogenesis, and hasbeen shown to localize with euchro-matin (Chen et al., 2002).

The third significant member ofthe Dnmt3 family of enzymes isDnmt3L. This highly degenerateprotein shows clear homology toDnmt3a and Dnmt3b, but despiteconserved folding like MTases,Dnmt3L lacks any catalytic activitybut is expressed together withDnmt3a and 3b during gametogen-esis and embryogenesis (Bourc’hiset al., 2001b; Hata et al., 2002).Dnmt3a and Dnmt3L are essentialfor establishment of imprinted re-gions in oocytes (Hata et al., 2002).The exact mechanism of this pro-cess is not fully worked out, butthere is the suggestion that the se-quence specific function of Dnmt3amay require an activator to enforcethe accuracy of this targeting. Inthis regard, Dnmt3L may functionas an activator protein in the meth-ylation of single copy genes.

DEVELOPMENTALREGULATION OF DNAMETHYLTRANSFERASES

DNA MethyltransferaseFunctions duringDevelopment

The essential requirement for DNAmethyltransferases during devel-opment is confirmed by gene tar-geting experiments disrupting theDnmt loci. With the exception ofDnmt2, all Dnmt knockouts showsevere developmental defects. Em-bryos homozygous for a Dnmt1 nullmutation targeting two highly-con-served C-terminal domain motifs(IV and VI) including the enzymaticactive site do not survive pastmidgestation, and show severe de-velopmental abnormalities as earlyas ED 8.5 (Lei et al., 1996). Dnmt1inactivation results in genomewideloss of methylation, with only verylow residual levels of methylation

detectable in mutant embryonicstem (ES) cells (Lei et al., 1996).Specifically, its deletion results inhypomethylation at centromericminor satellite repeats, retroviralrepeats such as intracisternal Aparticles (IAPs) and Moloney mu-rine leukaemia virus (MoMuLV),and single-copy sequences includ-ing imprinted genes and Xist (Li etal., 1993; Beard et al., 1995; Lei etal., 1996).

In addition to the ubiquitously ex-pressed full-length somatic form ofDnmt1, sex-specific promotersdownstream of the 1s exon giverise to truncated oocyte andpachytene spermatocyte specificDnmt1 transcripts (Mertineit et al.,1998). While the function of theDnmt1p transcript in male germcells remains unknown, the oocytespecific isoform of Dnmt1, Dnmt1o,is implicated in maintaining DNAmethylation at imprinted loci at theeight-cell stage of preimplantationdevelopment (Howell et al., 2001).The methylation of other sequencessuch as major satellite repeats, IAPretrotransposons, as well as single-copy genes do not appear to be af-fected in heterozygous Dnmt1o ED9.5 embryos from homozygous oo-cytes (Howell et al., 2001). Theseembryos show a spectrum of devel-opmental defects and die betweenED 14.5 and postnatal day 1.5 (PND1.5), presumably due to somaticmosaicism resulting from randomsegregation of hypomethylated im-printed alleles (Howell et al., 2001).While these results point to a highlyspecific role for Dnmt1o in cell-cy-cle restricted maintenance of meth-ylation at imprinted loci, the possi-bility that other sequences aredemethylated and subsequentlyremethylated cannot be formallyexcluded.

Dnmt2 knockout mice are viableand fertile (unpublished observa-tion by E. Li, reported in Hermannet al., 2003), and Dnmt2 null EScells also appear normal withoutany overt de novo or maintenanceDNA methylation defect (Okano etal., 1998b). Unlike Dnmt1,Dnmt3a, and Dnmt3b, which havelarge regulatory N-terminal do-mains, Dnmt2 lacks any similar do-mains but shares all the conserved

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motifs required for methyltrans-ferase activity. Perhaps paradoxi-cally, Dnmt2 appears to be themost evolutionarily conserved andwidespread of the Dnmt families,with closely related enzymes inseveral species of plants, fungi,metazoans, and protozoa (Pongerand Li, 2005). To date a biologicalfunction for Dnmt2 has not beenidentified in mammals (Ponger andLi, 2005).

Residual de novo MTase activityobserved in Dnmt1 null ES cellspointed to the existence of de novoMTases that we now attribute toDnmt3a and Dnmt3b, the two func-tional members of the Dnmt3 fam-ily. Gene targeting studies revealedde novo methylation by Dnmt3aand Dnmt3b to be essential for nor-mal development, with Dnmt3a nullmice surviving until four weeks ofage, and Dnmt3b homozygous em-bryos showing developmental ar-rest between ED 14.5 and ED 18.5(Okano et al., 1999). Compoundhomozygotes of Dnmt3a/3b weremore severely affected, and dis-played developmental defects be-tween ED 8.5 and ED 9.5 with noembryos surviving beyond ED 11.5,thereby suggesting overlappingfunctions of the two closely relatedenzymes. Analysis of DNA methyl-ation patterns in mutant ES cellsrevealed both enzymes to be nec-essary for de novo methylation,and also showed a specific require-ment for Dnmt3b in the methyl-ation of centromeric minor satelliterepeats (see discussion of the ICFsyndrome below) (Okano et al.,1999).

During germ cell development,erasure of imprinted methylation isfollowed by temporally asynchro-nous reacquisition of sex-specificimprints. This restoration of meth-ylation marks requires the activityof de novo MTases. However, in-vestigation into a possible role forDnmt3a and Dnmt3b in the de novomethylation of imprinted genesduring gametogenesis was pre-cluded by the lethality of bothknockouts. The hypomethylatedstatus of imprinted genes in em-bryos derived from transplantedDnmt3a–/– Dnmt3b�/– ovaries sug-gested a possible role for these

MTases in de novo imprint methyl-ation (Hata et al., 2002). Conclu-sive evidence for Dnmt3a’s role inestablishing methylation at im-printed loci was achieved using aconditional targeting strategy thatallowed for its selective deletion ingerm cells (Kaneda et al., 2004).Methylation analysis of these em-bryos revealed severely hypo-methylated imprinted genes, sug-gesting an essential role forDnmt3a in the establishment ofmaternal methylation imprints dur-ing female germ cell development.The methylation of paternallymethylated imprinted genes H19and Rasgrf1, minor satellite se-quences, and IAP repeats appearednormal in these embryos. Likewise,spermatogonia from mutant malesalso showed hypomethylation ofimprinted target sequences. Thus,Dnmt3a is required by both maleand female germ cells for the denovo methylation of imprintedgenes during gametogenesis.

Like Dnmt1o and the conditionaltargeting of Dnmt3a, a lethal ma-ternal effect mutation was ob-served in Dnmt3L knockout micesuch that heterozygous embryosborn from homozygous Dnmt3L de-ficient oocytes failed to develop be-yond ED 10.5 (Bourc’his et al.,2001b; Hata et al., 2002). Themethylation targets of Dnmt3L areidentical to those of Dnmt3a, withmaternally imprinted genes beingcompletely hypomethylated in het-erozygous embryos derived fromDnmt3L–/– females (Bourc’his et al.,2001b; Hata et al., 2002). Sper-matogenesis was also perturbed inDnmt3L null males, and germ cellsshowed impaired de novo methyl-ation of the imprinted gene H19 aswell as IAP and LINE1 retrotrans-posons (Bourc’his and Bestor,2004; Kaneda et al., 2004). Thishypomethylation of IAP and LINE1repeats results in their transcrip-tional reactivation. Interestingly,Dnmt3L-deficient spermatocytesdisplay severe meiotic defects atsynapsis, presumably as a result ofthe failure to establish methylationat interspersed repeat elements(Bourc’his and Bestor, 2004). Thisreinforces the critical role for main-tenance of DNA methylation in

overt chromosome stability. WhileDnmt3L lacks the critical MTasemotifs, recent in vitro studies haveindicated that Dnmt3L is capable ofincreasing the catalytic activity ofDnmt3a and Dnmt3b 15-fold by in-ducing a more open conformationin the de novo MTases and acceler-ating the binding of the methyl do-nor Ado Met and the target se-quences (Gowher et al., 2005).

Expression Profile of DNAMethyltransferases duringDevelopment

A strict division of functions be-tween the maintenance and denovo MTases may prove to be toonaıve and simplistic (Robertson andWolffe, 2000). This likelihood ismost apparent on consideration ofthe phenotypic outcomes of dele-tion of the MTases alone and in con-cert. Suggestions that Dnmt1 canalso behave as a de novo methylasehave long persisted (reviewed inBestor, 2000), and may even leadto de novo methylation spreading.In contrast, Dnmt3a and 3b havebeen shown to be required for site-specific maintenance of methyl-ation. The maintenance of thedensely methylated regions of het-erochromatin may pose a problemfor Dnmt1, yet silencing of activeretrotransposons in this region re-mains essential after each replica-tion cycle. Several studies suggestthat Dnmt1 remains active follow-ing replication, and may achievethis postreplication methylation inconcert with Dnmt3a and/orDnmt3b. (Liang et al., 2002; Rheeet al., 2002). Together, these re-sults suggest a mutual interrelated-ness of the MTases to create andmaintain distinctive patterns andlevels of DNA methylation that de-fine specific genomic regions.

Preimplantation Embryos

In all instances, the full magnitudeof the deletion of MTases results ina lethal phenotype. The importanceof Dnmt1o has been well estab-lished but what, if anything, isknown about the transcriptionalregulation of the other isoforms of



Dnmt1 and other MTase familymembers during early develop-ment? Dnmt1o is actively tran-scribed and translated during oo-cyte growth and maturation, aperiod when no replication takesplace. This observation suggested ade novo methylase function forDnmt1o (Mertineit et al., 1998). Bythe time the MII oocyte matures, itinherits a vast store of Dnmt 1oprotein. The unique restricted dis-tribution pattern of the protein inthe MII oocyte, and two- and four-cell stage embryo, in a subcorticalzone has been the subject of muchinterest. No less so is the singulartranslocation to the nucleus at theeight-cell stage only to be excludedthereafter (Carlson et al., 1992;Cardoso and Leonhardt, 1999;Howell et al., 2001). However,there is only very limited detectionof the Dnmt1o transcript followingfertilization (Ratnam et al., 2002;Ko et al., 2005). It is very interest-ing to note that the somatic form ofDnmt1 transcript can be detectedthroughout this period, with the ex-ception of the fertilized oocyte (Fig.1). Analysis of the Dnmt3 family ofproteins and activities has not beencompleted; however, expression ofthe de novo methylase Dnmt3a isdetected throughout preimplanta-tion. Although Dnmt1s is alsopresent throughout this period, theabsence of maintenance methyl-ation (described in the next sec-

tion) and the observation that im-printed methylation remains intact,suggests that Dnmt3a, in its target-ing role, acts as a maintenancemethylase but only on imprintedtarget sequences. Clearly, a full ex-pression profile of Dnmt3L duringmouse preimplantation develop-ment remains an important piece ofthis puzzle (Fig. 1).

DNA METHYLATION ISREPROGRAMMED DURINGDEVELOPMENT

Primordial Germ Cells andGametogenesis

Dynamic reprogramming of DNAmethylation patterns takes placeduring at least two key periods ofdevelopment in the mouse (Fig. 2).The first is associated with the de-velopment of primordial germ cells(PGCs) during gametogenesis.These cells arise in the posteriorprimitive streak at the base of theallantois on E 7.5 (Ginsburg et al.,1990). They begin to proliferateand migrate along the hindgutwhere they take up residence in thegenital ridge about E 10.5. Herethey remain, exponentially increas-ing in number, during the differen-tiation of the definitive gonads at E12.5 (Ginsburg et al., 1990). Dur-ing this early phase of differentia-tion, PGCs appear to be methylatedas measured by indirect immuno-

fluorescence using a highly specificantibody against 5 methyl cytidine(Seki et al., 2005). In the transi-tional phase between establish-ment and migration, the genome-wide methylation is no longerobserved (Seki et al., 2005). Thisrapid decline suggests that the lossof DNA methylation results from anactive targeted process of DNA de-methylation. However, not all lociare demethylated at this stage ofdifferentiation. Methylation-sensi-tive bisulfite analysis of PGCsisolated at day E 11.5 indicate thatimprinted genes retain their meth-ylation marks at this stage but un-dergo a rapid demethylation within24 hr, suggesting yet another pe-riod of gene-specific demethylation(Hajkova et al., 2002). Investiga-tion into some single-copy genes,members of an long-term terminalrepeats (LTR)-like family of retro-transposons, IAPs, and LINE1 ele-ments, indicates a variable degreeof demethylation throughout thissame period of germ cell develop-ment (Hajkova et al., 2002; Lane etal., 2003). This period of profoundmethylation erasure is associatedwith the essential resetting of par-ent-of-origin specific methylationmarks, which must later match thesex of the developing embryo. Byday E 12.5, most sequences havebecome maximally demethylated(Hajkova et al., 2002; Sato et al.,2003). Thereafter, both male and

Figure 1. Developmental dynamics of DNA methyltransferase expression profiles during germ cell and embryo development. Thepresence or absence of transcripts for the different MTases is indicated below the depicted time points. Transcript levels were chosenfor this summary due to the lack of protein data available for most time points and enzymes. Dnmt1 isoforms 1s, 1o, and 1p areconsidered separately in an effort to highlight their respective roles during gametogenesis and embryogenesis. (1) denotes a time pointat which there appears to be a discrepancy in the literature (Ratnam et al., 2002; Ko et al., 2005). PGCs � primordial germ cells.

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female gametes are arrested, thefemale in meiotic prophase and themale in mitosis.

During DNA methylation erasurein the male germline, a testis-spe-cific CCCTC-binding factor-paraloggene, BORIS (Brother of the Regu-lator of Imprinted Sites) is dramat-ically upregulated in a mutually ex-clusive manner from CTCF inprimary spermatocytes. Expressionof BORIS takes place in associationwith erasure, while reestablish-ment of methylation marks is curi-ously associated with the silencingof BORIS and the reexpression ofCTCF. This has led to the sugges-tion that BORIS may participate to-gether with demethylases, and thatBORIS-CTCF switching may belinked to CTCF targeting of paternalmethylation marks (Loukinov et al.,2002).

The re-establishment of newlyrestored imprints occurs at very dif-ferent times in males and females.The reacquisition of DNA methyl-ation requires the activity of a de

novo MTase(s), which, togetherwith Dnmt3L, restores the imprintedmethylation to the female germlineduring the postnatal growth phase ofoogenesis (Bourc’his et al., 2001b).Elegantly executed studies, involv-ing reconstituted parthenogeneticembryos derived from haploid com-ponents of postnatal-stage growingoocytes and fully-grown oocytes,provided the first evidence that ma-ternal methylation in imprintedgenes is acquired at different stagesof oogenesis (Obata and Kono,2002). These studies have been con-firmed and extended for other im-printed loci, using methylation sensi-tive bisulfite analysis (Lucifero et al.,2004b). In males, the acquisition ofDNA methylation patterns begins be-fore birth in prospermatogonia and iscompleted for many sequences afterbirth, prior to the end of thepachytene phase of meiosis. In themale mouse, the initial acquisition ofmethylation occurs between ED 15.5and E 18.5 (Davis et al., 1999,2000). Studies using an antibody

against 5 methyl cytidine indicatethat male germ cells always possesshypomethylated heterochromatin,whereas their euchromatin passesfrom a demethylated to a stronglymethylated status between ED 16and E 17 (Coffigny et al., 1999). Thishypermethylation occurs in the ab-sence of DNA replication, germ cellsbeing blocked in the G0-G1 phasefrom E 15 to birth. The DNA hyper-methylation of germ cells is main-tained until birth, and can be visual-ized on both chromatids ofmetaphase chromosomes at the firstpostpartum cell division (Coffigny etal., 1999).

PreimplantationDevelopment

A second phase of methylation re-programming has been described,initiated upon fertilization (Fig. 3).Highly specialized gametes pos-sessing genome-specific matern-ally- and paternally-directed epige-

Figure 2. DNA methylation reprogramming cycle. The cycles of DNA methylation reprogramming in the mouse can be divided into twodistinct time periods: one associated with gametogenesis, and the other with preimplantation development. By the primitive streakstage of embryonic development (E 7.5), the primordial germ cells (PGCs) are assembled at the posterior end of the primitive streakat the base of the allantois. These cells are initially highly methylated but rapidly lose this methylation around ED 8 at the time theybegin to migrate toward their final destination in the genital ridge between E 10.5 and E 11.5. Here PGCs continue to proliferate andundergo differentiation into distinct male and female germlines (approximately E 12.5). By ED 12.5, most sequences in both gendershave become maximally demethylated. This loss of methylation is associated with the erasure of germline imprints (black line).Thereafter, both male (blue) and female (red) gametes are arrested, the female in meiotic prophase and the male in mitosis. In males,de novo methylation of the genome, including imprinted loci, takes place prenatally, while in the female germline, de novo methylationoccurs postnatally in growing oocytes. The reacquisition of DNA methylation requires the activity of a de novo MTase that, together withDnmt3L, restores the imprinted information content to both germlines. Fertilization signals the second round of methylation repro-gramming when the maturing paternal pronucleus is demethylated in a replication independent or active manner. Thereafter, thezygote and embryo are demethylated by a replication dependent or passive process owing to the general exclusion of the maintenancemethylase Dnmt1o from the nucleus. Despite genomewide demethylation, imprinted methylation is maintained during this period. Denovo methylation roughly coincides with the differentiation of the first two lineages of the blastocyst stage. These lineages areasymmetrically modified epigenetically with the inner cell mass (ICM) being hypermethylated in comparison to the trophectoderm (TE),reflecting the DNA methylation status of their somatic and placental derivatives, respectively. Imprinted genes are depicted with a blackline throughout. Maternal and paternal alleles of nonimprinted genes and genomic sequences are depicted in red and blue, respectively.



netic marks, including a fullcomplement of appropriately im-printed genes, are remodeled to re-store a uniform diploid nucleus tothe newly formed zygote. In orderfor this to happen, the sperm nu-cleus in its highly specialized prota-mine form of chromatin must be-come rapidly reorganized into anucleosomal chromatin conforma-tion (reviewed in Perreault, 1992;McLay and Clarke, 2003). Almostimmediately upon decondensationand nucleoprotamine exchange,the paternally inherited sperm nu-cleus begins to undergo paternalspecific demethylation (Santos etal., 2002). This process initiates thepreimplantation stage of DNAmethylation reprogramming in themouse. This change in the methyl-ation content of the developingmale pronucleus occurs in the ab-sence of DNA replication and henceis called active demethylation(Mayer et al., 2000). Within 6 hr offertilization, the fully-formed malepronucleus is devoid of DNA meth-ylation, except for the perinucleolarregions known to contain the minorsatellites sequences of the centro-mere (Santos et al., 2002). Thisepigenetic asymmetry is particu-larly evident at syngamy after thecompletion of the first S phasewhen the highly condensed chro-mosomes align along the firstmetaphase plate (Santos et al.,2002). This is the first and only timethat maternal and paternal chro-mosomes are exclusively segre-gated in the life of the organism.

The first cell cycle is largely charac-terized by transcriptional silencing(Aoki et al., 1997). Yet the prefer-ential demethylation of the malepronucleus that might ensure theearliest minor transcriptional burst,at the one-cell stage, can occurfrom paternal alleles (Ram andSchultz, 1993). Active demethyl-ation has been suggested to be aresponse of maternal interests, inevolutionary terms, to mediate par-ent–offspring conflict in which pa-rental alleles vie to exert their inter-ests to maximize their lifelongreproductive fitness (Moore andHaig, 1991; Moore and Reik, 1996).This epigenetic conflict is focusedaround imprinted loci, with theircritical reliance on methylationmarks to specify expression ofgenes vital for fetal growth and de-velopment.

To date, the mechanism of thisactive demethylation remains un-known. Active demethylation couldbe achieved through a number ofpossible mechanisms. The moststraightforward form of cytosinedemethylation would be the re-moval of the methyl group from theC-5 position in the heterocyclicring. Another possibility would bethe removal of the methylcytosinebase from the CpG dinucleotide andits replacement with cytosine. Asdouble-stranded nicks to the sugar-phosphate backbone are requiredin this mechanism, it could bethought of as a form of base exci-sion repair (BER). A third possibilityinvolves an initial deamination step

rather than the removal of the 5-methyl-cytidine base, and results ina transition of 5 MeC to T, a modi-fication for a BER mechanism to re-store. While hydrolytic deaminationis mechanistically possible, it is en-ergetically unfavorable, and a num-ber of enzymatic deaminase candi-dates have been proposed. Adetailed consideration of mecha-nisms for active demethylation andpossible candidate activities hasbeen suggested elsewhere (re-viewed in Santos and Dean, 2004;Morgan et al., 2005).

Between the two-cell and themorula stage, the remaining DNAmethylation, largely of maternal or-igin, declines in a step-wise pro-gression, presumably due to theactive exclusion from the nucleus ofthe maintenance methylase Dnmt1(Monk et al., 1987; Rougier et al.,1998; Santos et al., 2002). Thisreplication-dependent loss of DNAmethylation is defined as passivedemethylation. Despite genome-wide demethylation, imprintedgenes are exempt from this processand remain methylated, as do cer-tain retrotransposons such as IAPs(Olek and Walter, 1997; Warneckeet al., 1998; Lane et al., 2003). Thisapparent protection of retrotrans-posons and imprinted genes hasimplied a mechanistic link betweenthese diverse sequences. The oneexception to this replication-depen-dant methylation reduction is theeight-cell stage when Dnmt1o en-ters the nucleus. Deletion of thisDnmt1 isoform is associated with

Figure 3. Quantitative and qualitative aspects of genomewide DNA methylation reprogramming in the mouse. Indirect immunofluo-rescence using an antibody to 5-methyl-cytidine (5MeC) is presented from the germinal vesicle (GV) stage in oogenesis throughfertilization and preimplantation development. Prior to the completion of meiosis, the female gamete is highly methylated. Thismethylation is depicted here in GV oocytes and ovulated MII oocytes in which the signal is confined to the highly condensedchromosomes. Upon fertilization the decondensation of the sperm in association with protamine–histone exchange coincides with theinitiation of paternal specific active demethylation (PNo). Pronuclear formation continues with both pronuclei still positive for DNAmethylation (PN2), although the male (arrowhead) is already less methylated than the female. Maximum size of pronuclei is achievedby PN5 after DNA replication is completed. Overexpose of the image is necessary to illustrate the residual methylation in theperinucleolar region. Between the two-cell stage and the morula, DNA methylation is diminished passively in a step-wise manner owingto the exclusion of Dnmt1o. De novo methylation at the late morula stage results in asymmetric hypermethylation of the ICM andhypomethylation of the trophectoderm. Note the intensely staining heterochromatin foci that are first apparent by the blastocyst stage.

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failed maintenance of methylationspecifically at imprinted loci, al-though this has yet to be shown inpreimplantation embryos (Howellet al., 2001). This observationhighlights one of the outstandingquestions of epigenetic regulationduring preimplantation develop-ment. If Dnmt1o is critical for main-taining methylation of imprintedloci in a stage-specific manner,what are the activity(ies) that sharethis responsibility both prior to andfollowing the eight-cell stage?

De novo methylation at the latemorula–early blastocyst transitionbegins to restore methylation toboth of the newly defined lineagesof the blastocyst. However, oncethe blastocyst is fully expanded, itis apparent that this de novo meth-ylation results in an asymmetricamount of methylation in differen-tiated lineages (Dean et al., 2001;Santos et al., 2002). The inner cellmass (ICM), which gives rise to allof the tissues of the adult, becomeshypermethylated in comparison tothe trophectoderm (TE), whichgives rise to the placenta and mostof the extraembryonic tissues.These early distinctions remain inplace throughout the lifetime of thelineages. Hence, fully-differenti-ated somatic derivatives are hyper-methylated compared to those ofthe placenta and extraembryonicmembranes. Among the somatictissues that derive from the ICM arethe highly methylated PGCs, whicharise as a distinct population at ap-proximately ED 7.0 in the extraem-bryonic mesoderm of the develop-ing embryo (Ginsburg et al., 1990).Their migration via the allantois tothe developing germinal ridges,where they will eventually differen-tiate into mature gametes, com-pletes the cycle of epigenetic repro-gramming.

Active DNA Demethylationin Mammalian Zygotes

The precision of the timing of activedemethylation, the genome-widereduction of the remaining mater-nal methylation, and the asymmet-ric lineage-specific reacquisition ofmethylation at the blastocyst stagesuggest that modulation of the pat-

terns and level of DNA methylationis functionally significant for earlymammalian development. As such,there is a tacit expectation thatDNA methylation reprogrammingmight be assiduously conservedthroughout Mammalia. Speciescomparisons have produced inter-esting and unexpected results. Per-haps most comprehensively inves-tigated is the conservation of thepaternal specific-active demethyl-ation. Using an antibody approach,Dean et al. (2001) found that activedemethylation was conservedamong widely divergent mamma-lian species that included rat, bo-vine, and pig. These results havealso been independently verified inmouse, bovine, and pig throughmethylation-sensitive molecularanalyses, albeit at a restrictednumber of target loci (Oswald et al.,2000; Kang et al., 2001b, 2002). Inthe mouse, active demethylationhas been demonstrated for a di-verse selection of sequences in-cluding the high copy number L1family, single-copy genes, and cen-tromeric satellites (Oswald et al.,2000; Lane et al., 2003; Kim et al.,2004). More recently, analyses ofother species accentuate the diver-sity of methylation reprogramming.Investigations in the sheep suggestthat there is a partial reduction ofDNA methylation in the zygote(Beaujean et al., 2004a). In therabbit, there is no apparent reduc-tion of DNA methylation at thisstage (Beaujean et al., 2004a) al-though technical problems relatedto overexposure during image cap-ture, and hence saturation of theimages, preclude a definite confir-mation of this result (Shi et al.,2004). In humans, a similar analy-sis using an antibody approach sug-gests that, like the majority of spe-cies surveyed to date, the putativemale pronucleus undergoes de-methylation in the zygote (Beau-jean et al., 2004a; Santos andDean, 2005), although it is appar-ently much more variable than inthe mouse (Fulka et al., 2004; Xu etal., 2005). Experimental manipula-tion generating interspecies hy-brids and staining using 5-methyl-cytidine antibody clearly indicatesthat ovine sperm are demethylated

in mouse oocytes and, conversely,that mouse sperm is demethylatedin ovine oocytes (Beaujean et al.,2004b). These results establish theintrinsic capacity for demethylationin the sheep oocyte, and suggestthat the partial demethylation ofconspecific sperm may be relatedto the absolute amount, and orga-nization, of genomic DNA methyl-ation in the sheep compared toother mammals (Jabbari et al.,1997).

Dynamic DNA MethylationReprogramming inMammals

Comparisons of DNA methylation ofembryonic stages during preim-plantation indicated that there is anapparent functional conservation ofboth passive and de novo methyl-ation, although the absolute timingand the extent of methylation varyconsiderably (Fig. 4). Indirect im-munofluorescence studies in bo-vine revealed a much more re-stricted passive demethylationphase, with de novo methylationinitiated at the 10–16-cell stage(Bourc’his et al., 2001a; Dean etal., 2001). This passive demethyl-ation is most elegantly depicted byasymmetrically methylated chro-mosomes. By the blastocyst stage,these differentials are no longer ap-parent and the heterochromaticcentromeric regions become un-dermethylated (Bourc’his et al.,2001a). Analysis of the centromericsatellite sequences in bovine and pigembryos (satellite 1) using a com-bined restriction enzyme analysis af-ter bisulfite treatment of embryoDNA confirmed that these centro-meric sequences were hypomethyl-ated during preimplantation devel-opment (Kang et al., 2001a, 2001b,2002). Despite the results suggest-ing that DNA methylation is not dy-namically regulated during preim-plantation in the rabbit, bisulfite-treated DNA clearly indicates thatpassive demethylation of both a sin-gle copy gene as well as a centro-meric satellite sequence (RsatIIE)does occur, albeit not until theeight- to 16-cell stage (Chen et al.,2004).This result agrees with amuch earlier report of uniform hy-



permethylation up to the eight-cellstage in the rabbit (Manes and Men-zel, 1981).

De novo methylation in mostmammals occurs around the timeof the first lineage decisions, whereblastomeres positioned more exte-riorly will become allocated to theTE lineage and those most interiorlyto the ICM of the blastocyst. In themouse, there is a distinctive orga-nizational change in the pattern ofDNA methylation between the four-and eight-cell stage, which mayprecede the epigenetic asymmetry

that becomes formally apparent atthe blastocyst stage. In most spe-cies analyzed to date, there is aDNA methylation difference seenbetween the hypermethylated ICMand the hypomethylated TE (Fig.4). These differences may well indi-cate that regulation of lineage-spe-cific gene expression relies on DNAmethylation to a greater or lesserextent. In support of this sugges-tion is the very interesting reportthat many placenta-specific im-printed genes do not rely on DMR-based organization for their allelic

marking. Furthermore these genesfrequently have a leaky low level ofexpression from the “silenced” al-lele, reinforcing the importance ofmethylation for complete silencingof imprinted genes (Lewis et al.,2004). This observation has inter-esting implications for regulation oftranscriptional repression and si-lencing of single-copy genes, in ad-dition to the numerous LTR retro-transposons in the mammaliangenome.

Insights and Concepts:DNA Methylation in Healthand Disease

It has been clear for some time thaterrors in the regulatory or codingsequences of genes can result indisease. However, epigenetic er-rors that alter chromatin structureor DNA methylation without anychange to the underlying geneticcode may also result in disease. Es-tablishing the importance of DNAmethylation and its regulation toensure normal mammalian devel-opment naturally forces us to con-sider the possibilities and repercus-sions of its failure. It is notsurprising that the misregulation ofgene expression and silencingmechanisms results in a broadspectrum of diseases and syn-dromes. Many of these epigeneticdiseases find the targets of theirpathology in the failure to appropri-ately establish or maintain im-printed loci, DMRs, or imprintingcenters (ICs). Others are associ-ated with DNA methylation require-ments of specific non-imprinted lociin association with DNA methylbinding proteins and their targets(Rett syndrome). Yet others mayinfluence the primary methylationmachinery of the genome and re-sult in chromosome instability(Dnmt3b and ICF) (Egger et al.,2004).

Epigenetic changes can also havea major role in the development ofhuman cancers. It is a consensus ofobservation that in human carcino-mas there is a generalized and sub-stantial reduction in genomewideDNA methylation, with the excep-tion of tumor suppressor genes thatbecome de novo hypermethylated

Figure 4. Comparative DNA methylation reprogramming in mammals. Analysis of DNAmethylation was investigated in several mammalian species using a 5MeC antibody.Included in this figure are two representative stages from the mouse and bovine, inwhich passive demethylation has been demonstrated, and the sheep, which does notapparently undergo passive demethylation. The intensity of the staining between thedifferent species provides an indication of overall genomic methylation content. Notealso that the distribution of DNA methylation intensity within individual nuclei of blas-tomeres suggests differences in the organization of the CpG-dense sequences through-out the respective genomes. De novo methylation becomes apparent at the 10–16-cellstage in bovine and by the blastocyst stage in the mouse, defining an asymmetricdifference between the inner cell mass and the trophectoderm (Dean et al., 2001). Incontrast, in the sheep there is no apparent asymmetry at this stage. However, fullyexpanded sheep blastocysts also display this characteristic DNA methylation difference(Beaujean et al., 2004a).

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leading to gene silencing. Most re-cently, there has been a growingawareness that children conceivedthrough the varied procedures ofassisted reproductive technologies(ART) have been found to have in-creased incidences of epigeneticdiseases.

Excellent detailed reviews on theinfluence of epigenetic factors incancer (Feinberg and Tycko, 2004)and diseases arising from imprintedgene associated syndromes havebeen produced elsewhere. Thereare also detailed considerations ofART on human health and disease(Maher et al., 2003; Lucifero et al.,2004a; Niemitz and Feinberg,2004). Here we identify a few ex-amples of epigenetic errors thatmay arise during early embryonicdevelopment in humans, especiallythose that have bearing on repro-ductive success and arise from ARTprocedures in which there is suffi-cient knowledge to attribute an ac-tivity or mechanism.

ART and ImprintingDisorders

Beckwith-Wiedemann andAngelman syndromes

In 2003, three independent studiesreported that children conceived byART were found to have increasedfrequencies of Beckwith-Wiede-mann syndrome (BWS), a prenataland postnatal overgrowth syn-drome associated to errors at theimprinting cluster on 11p15.5 (De-Baun et al., 2003; Gicquel et al.,2003; Maher et al., 2003). Beyondthe non–life-threatening features ofthis syndrome is the association ofincreased risk of developing embry-onal tumors, in particular Wilms tu-mor of the kidney. Typically, BSW isthought to be a result of the failureof appropriate imprinting at specificloci on the maternal allele, such asinactivation of the cyclin-depen-dent kinase CDKN1C or defects in-volving the H19 DMR, resulting inbiallelic expression of insulin-likegrowth factor 2.

The reported cases of BWS arosethrough a variety of ART proce-dures which included intracytoplas-mic sperm injection (ISCI), in vitro

fertilization (IVF), and embryocryopreservation. A more recentcase-control study in Australiasought to analyze the exact risk ofBSW for ART-conceived childrencompared to normal births. In thisstudy, Halliday et al. (2004) foundthat the increase in risk for ARTconceptions of having BWS wasnine times greater than in the gen-eral population.

Angelman Syndrome (AS) iscaused by loss of function in thebrain of the maternal copy ofUbe3a, which encodes a ubiquitinprotein ligase. The vast majority ofAS patients have an interstitial mi-crodeletion of the maternal homo-logue of chromosome 15q11–13.Ordinarily, roughly 4% of AS casesarise due to a maternally-inheritedIC microdeletion or epigenetic al-teration to this locus, which is nor-mally methylated on the maternalallele. Two reports (Cox et al.,2002; Orstavik et al., 2003), collec-tively reported three ICSI-con-ceived patients with AS in which theIC was hypomethylated. Furtheranalysis ruled out microdeletions,confirming that all three cases weresporadic imprinting defects associ-ated to hypomethylation, whichusually accounts for no more than3% of all AS cases.

Although other underlying ge-netic causes for the observed fre-quency of imprinting disorders re-sulting from ART procedures cannotbe entirely ruled out, evidence froma number of studies in the mousesupport the idea that embryo cul-ture and the use of immature oo-cytes may contribute significantlyto epigenetic alterations. Dohertyet al. (2000) studied the effect ofculture on H19 and showed that theloss of methylation from the nor-mally-methylated paternal alleleoccurred in Whitten’s medium butnot in potassium simplex optimisedmedium. Khosla et al. (2001) con-firmed this result, showing that lim-ited culture in M16 medium con-taining serum resulted in alteredexpression and methylation of im-printed genes.

Analysis of the acquisition of im-printed methylation in growing oo-cytes suggests that immature oo-cytes may not yet be properly

methylated, and hence, nonviableor more susceptible to error (Lucif-ero et al., 2004b). Owing to the ma-jority of cases arising due to failuresof maternal methylation imprintingat the KCNQ1OT1 locus in the caseof BWS and the maternal SNRPN ICin AS, a review of the practice anduse of immature human oocytesand the details of culture media inART may be entirely justified (Hal-liday et al., 2004; Lucifero et al.,2004a; Niemitz and Feinberg,2004).

ART and Retinoblastoma

Moll et al. (2003) reported fivecases of retinoblastoma (RB) inchildren born in the Netherlands asa result of IVF procedures. Al-though RB arises from a mutation ina tumor suppressor gene, epige-netic mechanisms through interac-tions with DNMT1 have been asso-ciated to hypermethylation of thepromoter region of RB. This rein-forces the generality of the poten-tial for inappropriate methylationmodification leading to both hyper-and hypomethylation of gene tar-gets that, in turn, result in epige-netic diseases. Although the initialerror is likely to have arisen in thematernal environment of the oo-cyte, a number of different enzy-matic activities are probably in-volved.

DNA Methylation,Chromosome Stability,and ART

In mammalian chromosomes, thebulk of heterochromatin is charac-teristically organized with highly-methylated DNA in and around cen-tromeric regions. This structuralportion of the chromosome is madeup of the pericentromeric majorand centromeric minor satellite re-gions that contain an overwhelmingnumber of repeats (700,000 and10,000–50,000, respectively) witheach repeat unit containing a num-ber of CpG sites. The importance ofDNA methylation in maintainingchromosome integrity is reinforcedby the observation that all mousemodels null for MTases show some



degree of chromosome instabilityand abnormal chromosomal rear-rangements (Bestor, 2000).

A number of diseases in humanshave been found to arise owing tofailures of appropriate levels ofmethylation, resulting in widespreadchromosome instability. Mutations inthe DNMT3B gene in humans isthought to be responsible for ICFsyndrome, a rare recessive autoso-mal disorder characterized by immu-nodeficiency, centromere instability,and facial anomalies (Hansen et al.,1999; Okano et al., 1999; Xu et al.,1999). ICF syndrome patients have aconstitutional methylation defi-ciency, mainly affecting heterochro-matin (Miniou et al., 1994), makinginstability of chromosomes 1, 9, and16 the most typical feature of the dis-ease. Ordinarily, classical satelliteDNA is normally hypermethylated atCpG residues, but in ICF syndrome itis almost completely unmethylatedin all tissues. De novo MTases suchas DNMT3B do not show sequencespecificity per se, although theyclearly target minor satellites in mice(Okano et al., 1999). Therefore, in-teractions with other protein part-ners may be necessary to mediatethe positioning of the DNMT3B en-zyme to achieve sequence specificmethylation.

Targeting of DNA methylationmay rely on chromatin remodelingactivities. In this regard, it is inter-esting that aberrant DNA methyl-ation has been reported in muta-tions for a family of ATP dependenthelicases of the SNF2 family of re-modeling activities (Li, 2002). Inhumans, ATR-X syndrome is an X-linked syndrome characterized bysevere mental retardation, reducedor absent speech, delayed develop-mental milestones, and also facialdysmorphism, �-thalassemia, andsexual dysgenesis (Gibbons andHiggs, 2000) associated with DNAmethylation defects in specific re-gions of the genome (Gibbons etal., 2000). ATRX binds to pericen-tric heterochromatin regions inmouse and to the short arms of hu-man acrocentric chromosomes(McDowell et al., 1999). ATRX mu-tations result in both loss and gainof genomic DNA methylation, im-plying that aberrant chromatin

structures, the result of an improp-erly functioning or improperly tar-geted chromatin remodeling pro-tein, may be able to target DNAmethylation to regions where itnormally would not occur (Robert-son, 2002).

More recently, the ATRX proteinhas been suggested to play an im-portant role during mouse oogene-sis. De La Fuente et al. (2004) sug-gest that genomewide epigeneticmodifications, such as histonedeacetylation, are essential forATRX binding to centromeric het-erochromatin, and that this bindingis essential for correct chromosomealignment and organization of thebipolar meiotic metaphase II spin-dle during mouse oogenesis. Fur-ther investigations including the lo-calization of DNA methylationrelative to ARTX should provide in-valuable information on whetherthis metaphase II failure is directlyor indirectly correlated to the fail-ure to methylate centromeric het-erochromatin.

Chromosome instability also hasspecific and significant repercus-sions for human fertility. Studies onDNA methylation patterns of testic-ular cells during mouse perinatalspermatogenesis have shown thatDNA methylation patterns changeextensively during this period(Coffigny et al., 1999; Bernardinoet al., 2000). A latter study in bothmouse and rat has further shownthat the DNA methylation status ofjuxtacentromeric heterochromatinmay be an important factor that in-fluences the centromere splittingdynamic. In both species, hypo-methylation was associated withlack of cohesion or premature split-ting of either sister chromatids orsister centromeres, and hyper-methylation correlated with strongand/or prolonged cohesion (Ber-nardino-Sgherri et al., 2002). Thisfinding has interesting implicationsin human infertility, and a recentinvestigation on global DNA meth-ylation levels of human sperm usedfor IVF has uncovered a significantcorrelation between DNA methyl-ation levels and pregnancy rate af-ter IVF (Benchaib et al., 2005).

These results point to the possi-bility of a common mechanism able

to cause infertility and epigeneticerrors leading to disease. Targetingof DNA methylation to achievestructurally stable configurations atthe centromere may be one of themost important roles for DNAmethylation in mammals. In thisregard, it is interesting that the fail-ure of appropriate targeting of denovo MTases to centromeric satel-lites in mice with a null mutation forDnmt3L results in chromosome in-stability leading to meiotic failureand sterility (Bourc’his and Bestor,2004).

CONCLUSIONS

The critical importance of epige-netic information and its impact onhuman health has received muchattention of late due to the ever-greater numbers of ART births.Continued practices involving hor-monal stimulation for the produc-tion of oocytes and their subse-quent in vitro maturation, thecomposition of oocyte and embryoculture medium, and their time inculture all conceivably have somebearing on the fidelity of epigeneticmethylation marks. We will in thenext few years begin to see the useof human ES cells derived solely fortherapeutic purposes. These cellshold unprecedented promise intheir ability to be instructed to dif-ferentiate into a wide variety of celltypes for medical therapeutic appli-cations to disease such as diabetesand neurological degenerative dis-eases such as Parkinson’s andamyotrophic lateral sclerosis. In-formation learned now about theestablishment and maintenance ofthe epigenetic information contentof the genome will be essential toensure that this possibility realizesits full potential.

ACKNOWLEDGEMENTSWe thank Alain Niveleau for thekind gift of the 5-methyl-cytidineantibody. Drs. Lino Loi and GrazynaPtak, Department of ComparativeBiomedical Sciences, Teramo Uni-versity, provided the valuable ovineembryo materials included in thisstudy. We acknowledge the valu-able comments and suggestions

108 DEAN ET AL.


made by Wolf Reik (W.R.) in thepreparation of this review.

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DNA methylation in mammalian development and disease