De novo DNA methylation:a germ cell perspective
modulateSe´bastien A.Smallwood and Gavin Kelsey
Epigenetics Programme,The Babraham Institute,Cambridge CB223AT;and Centre for Trophoblast Research,University of Cambridge,Cambridge CB23EG,UK
DNA methylation is a fundamentally important epigenet-ic modification of the mammalian genome that has wide-spread influences on gene expression.During germ-cell specification and maturation,epigenetic reprogramming occurs and the DNA methylation landscape is profoundly remodelled.Defects in this process have major conse-quences for embryonic development and are associated with several genetic disorders.In this review we report our current understanding of the molecular mechanisms associated with de novo DNA methylation in germ cells. We discuss recent discoveries connecting histone mod-ifications,transcription and the DNA methylation machin-ery,and consider how these newfindings could lead to a model for methylation establishment.Elucidating how DNA methylation marks are established in the germline has been a challenge for nearly20years,but represents a key step towards a full understanding of several biological processes including genomic imprinting,epigenetic reprogramming and the establishment of the pluripotent state in early embryos.
Remodelling of DNA methylation marks in germ cells is essential
Epigenetic marks(Glossary)are covalent modifications of the DNA(DNA methylation)or post-translational modifi-cations of the histone proteins(histone modifications)that make up the chromatin into which our DNA is packaged. These epigenetic marks participate in the regulation of gene expression and,broadly speaking,different sets of marks are present depending on whether a gene is active or inactive.DNA methylation of promoter regions of genes, for example,is generally associated with transcriptional silencing.Epigenetic marks can be conserved on genes as cells divide,and are therefore a key component of cellular identity and the maintenance of differentiated states.The zygote is the only totipotent cell of the organism because it will give rise to all cells of the embryo.Male and female gametes in mammals originate from embryonic cells marked with their own landscape of DNA methylation and histone modifications.As a consequence,these pre-existing epigenetic marks must be reset during germ-cell specification and new ones established to guarantee re-newal of totipotency at each generation.This process is termed epigenetic reprogramming[3].The highly coordi-nated manner in which DNA methylation is laid down in germ cells makes them an important model system for studying the principles of DNA methylation.From a gen-eral point of view,understanding how DNA methylation marks are established in germ cells is fun
damentally important in reproductive biology and embryology(includ-ing stem cell science)because defects in gametic DNA methylation will result in embryonic lethality or deficien-cies,with drastic repercussions in adults.
It is almost twenty years since the discovery of the biological importance of germline DNA methylation in the context of imprinted genes[4]and ten years since the identification of the key enzymes responsible for de novo DNA methylation in mammals[5,6].Even so,what specifies why specific DNA sequences become epigenetical-ly distinguished in germ cells is still only partially under-stood.In no small part,this is because of the difficulty in Glossary
CpG islands(CGIs):regions in the genome in which CpG dinucleotides are present at high density(elsewhere,CpGs are under-represented in vertebrate genomes).CGIs are generally between a few hundred and a few thousand bp in length.CGIs can be identified in genome sequences using appropriate algorithms or biochemically by binding to CG-binding domains of specific proteins(CAP-Seq[1]).CGIs are associated with key regulatory elements and, for example,in mice around60%of annotated promoters are CGIs.
DNA methylation:covalent modification of DNA characterised in mammals by the addition of a methyl
group to position C5of the pyrimidine ring of cytosines, predominantly in CpG dinucleotides.DNA methylation is an epigenetic mark and DNA methylation of gene promoters is usually associated with promoter inactivity/gene silencing.
Epigenetic marks:molecular determinants regulating gene expression,and genome function in general,that can lead to heritable(through cell division) changes in gene expression without changes in DNA sequence.The major epigenetic marks are post-translational modifications of histone proteins and DNA methylation[2].
Epigenetic reprogramming:the global remodelling of epigenetic marks associ-ated with major transitions of cellular identity.In mammals,epigenetic repro-gramming usually refers to the remodelling of epigenetic marks during germ-cell specification and following fertilisation in the early embryo.
Histones and histone modifications:histones are the protein component of nucleosomes which,together with DNA and additional proteins,form chroma-tin.Specific amino acid residues in histones can be modified post-translation-ally in chromatin by the addition or removal of acetylation,methylation, phosphorylation,ubiquitination or sumoylation.Histone modifications modu-la
te the interaction of histone-binding proteins and are associated with different regions of the genome,correlating with and regulating chromatin state and gene expression.For example,H3K4me3refers to tri-methylation on lysine residue4in the amino-terminal tail of histone H3and is associated with active or potentially active promoter regions.
Imprinted gDMRs(germline differentially methylated regions):sequences with differences in DNA methylation between male and female gametes which retain parental-allele-specific DNA methylation throughout development and control genomic imprinting.Imprinted gDMRs coincide with the imprinting control regions(ICRs)of imprinted genes or imprinted gene clusters.Maternal gDMRs are methylated in oocytes,paternal gDMRs in sperm.Imprinted gDMRs are CGIs according to the definition used in this review.
Imprinted genes:genes expressed predominantly from one parental allele and whose allelic expression is controlled by imprinted gDMRs.
Corresponding author:Kelsey,G.(gavin.kelsey@babraham.ac.uk).
0168-9525/$–see front matterß2011Elsevier Ltd.All rights reserved.doi:10.1016/j.tig.2011.09.004Trends in Genetics,January2012,Vol.28,No.133
conducting standard molecular studies in germ cells and especially oocytes,which can only be obtained in small numbers.In this review we report our current understand-ing of the molecular mechanisms associated with the es-tablishment of DNA methylation patterns in germ cells,especially oocytes.We describe which sequences become methylated,their intrinsic characteristics,and what pro-tein factors are known to be involved.We shall focus particularly on specific regions of the genome termed CpG islands (CGIs)that are associated with key regulatory elements of gene expression,including many promoters.We shall discuss recent major advances concerning the link between histone modifications,transcription and the de novo DNA methylation machinery.Lastly,we shall con-sider what the future holds in this important and challeng-ing research area,particularly with the development of high-throughput sequencing techniques.
The dynamic nature of DNA methylation in germ cells and its fate in the embryo
Erasure of epigenetic marks in primordial germ cells Soon after the onset of gastrulation in the mouse embryo,the precursors of germ cells,or primordial germ cells (PGCs),emerge from the epiblast (embryonic day E7.25)as a founder population of <50cells.They proliferate,migrate to and colonise the genital ridge,from which the gonads develop (E10.5-E11.5).Because PGCs originate from embryonic cells that have started to adopt a somatic fate,extensive remodelling of histone modi
fications and
DNA methylation marks towards the requirements of a germ cell is essential ([3,7–10]for more details).Pre-exist-ing DNA methylation patterns are comprehensively erased during PGC migration,such that by E13.5the overall methylation level is <10%[11](Figure 1);for comparison,the same study measured the methylation level of the entire embryo to be >70%.However,some regions escape epigenetic remodelling in PGCs and retain DNA methyla-tion marks;for example,retrotransposons of the intracis-ternal A-particle class (IAPs)remain highly methylated [11,12].The consequence of wholesale DNA methylation erasure in PGCs is that de novo DNA methylation during germ-cell development takes place on a largely blank slate.Establishment of specific DNA methylation landscape in germ cells
Following sex-determination of the embryo ($E12.5),new DNA methylation patterns are established,differently in male and female germ cells,resulting in distinct methyla-tion profiles of mature oocytes and sperm.This asymmetry is related to the fact that de novo DNA methylation takes place in distinct cellular contexts in male and female germ cells (Figure 1).In the female germline de novo methyla-tion takes place during the postnatal growth phase of oocytes arrested in meiotic prophase I.In the male germ-line it initiates before birth in mitotically arrested prosper-matogonia,bef
ore the onset of meiosis (Box 1).
Various sequences are the target of the de novo DNA methylation machinery in germ cells (Box 2).DNA
PGCs
Male Key:
P r o l i
f e r a t i o n  & m i
g r a t i o n
D N A  m e t h y l a t i o n
Embryo
PGCs
P r o s p e r m
a t o g o n
i a
G r o w i n g
o o c y t e s
Puberty  fertilisation
Implantation
Blastocyst
8-cell
16-cell
Birth
Primary oocyte E12.5
E7.25
Mitotic arrest
Meiotic arrest
Proliferation
Meiosis
4-cell
2-cell
TE
ICM
Imprinted gDMRs
Zygote
MII
GV
Female
TRENDS in Genetics
Figure 1.DNA methylation changes during developmental epigenetic reprogramming.Primordial germ cells (PGCs)emerge in embryos at E7.5and,concomitant with their proliferation and migration towards the genital ridge,DNA methylation is globally erased (black line).Following sex-determination,new DNA-methylation landscapes are established in germ-cell precursors in an asymmetrical fashion in male and female embryos.In the male embryo (blue line),de novo methylation takes place before meiosis in mitotically arrested cells (G1-phase;prospermatogonia)and is completed before birth.In the female embryo (red line),primary oocytes enter meiosis and arrest in prophase-I (diplotene stage);DNA methylation is established after birth during the follicular/oocyte growth phase.At puberty,under specific endocrine triggers,fully-grown germinal vesicle (GV)oocytes resume the first meiotic division.After extrusion of the first polar body,oocytes arrest in metaphase of the second meiotic division (MII oocytes)and meiosis is completed only upon fertilisation.Following fe
rtilisation,a new wave of DNA demethylation takes place that is distinct on the parental genomes.In the zygote,DNA methylation of the paternal genome is rapidly erased by an active mechanism (blue line).Demethylation of the maternal genome is slower (red line)and is dependent on DNA replication (passive demethylation).These post-fertilisation demethylation events do not include imprinted gDMRs (green dotted line),resulting in parental-allele-specific methylation of these elements in early embryos and consequent parental-allele-specific expression of associated imprinted genes.Concomitant with blastocyst implantation and cell-lineage determination,new methylation landscapes become established,associated with cellular differentiation.
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methylation in mammals occurs almost exclusively at CpG dinucleotides(CpGs).CpGs are generally under-repre-sented and isolated in the genome,and are mostly meth-ylated by default.However,in some specific regions of the genome CpGs can be present at high density,thereby forming CGIs.CGIs are DNA elements ranging from $200bp to several kb in length and are generally associ-ated with gene promoters;there are$23000CGIs in the mouse genome(Box2).Within CGIs,CpGs are
usually unmethylated(unmethylated CGIs),and it is likely that there are general mechanisms to protect CGIs from meth-ylation(see below).Nevertheless,there are some hylated CGIs)and this is where the greatest difference in methylation between male and female germ cells is found.Recent genome-wide analysis identified $900CGIs specifically methylated in mature oocytes and$60specifically methylated in sperm[13].Included among these methylated CGIs,although in a small minor-ity[21],are the germline differentially methylated regions of imprinted genes(imprinted gDMRs),which control the parent-of-origin specific monoallelic expression of imprinted genes[14].These are the CGIs for which meth-ylation in gametes has been most extensively studied and is best understood.In this review we shall refer either to methylated CGIs in general or imprinted gDMRs.
Post-fertilisation outcome of germ-cell DNA methylation and its biological role
Epigenetic reprogramming does not only take place during germ-cell specification,but also after fertilisation,result-ing again in remodelling and erasure of DNA methylation marks(Figure1).This demethylation does not affect the maternally or paternally contributed genomes in the same way–DNA methylation marks originally established in male germ cells are quickly erased in the zygote by an active demethylation mechanism,probably involving oxi-dation by Tet proteins[15];maternal DNA
methylation marks are thought to be lost‘passively’by lack of DNA methylation maintenance at replication,resulting in pro-gressive loss of methylation at each cell division.
Box2.What sequences become methylated in germ cells?
DNA methylation is distributed throughout the genome,at repetitive elements(REs),and single-copy sequences.With the recent develop-ment of genome-wide methylation profiling techniques employing next-generation sequencing,the full pattern of DNA methylation in gametes,and how it is laid down during germ-cell development,is beginning to emerge.
Methylation in mammals occurs on cytosine residues(5-methyl-cytosine,5mC)predominantly in CpG dinucleotides.A high proportion of CpGs are methylated in germ cells.Methylation in other contexts (mCHG or mCHH,where H is A,C or T)has also been found,including in ES cells and oocytes,but not in sperm[18,49,87].The function of non-CpG methylation in oocytes is unclear but,because it occurs specifically in regions in which CpG-methylation is present,non-CpG methylation could reflect a degree of non-specificity of the de novo DNA methyltransferase DNMT3A[18,38,39].Methylation at non-CpG sites cannot be restored at DNA replication by the maintenance DNA methyltransferase DNMT1,which is active only at CpG sites.
CpG dinucleotides are under-represented in the mammalian genome, except in specific regions termed CpG islands(CGIs)where they are present at high density.CGIs are associated with a high proportion of gene promoters:$23000CGIs have been identified biochemically in the mouse genome,$60%at transcription start-sites,whereas the remain-der(‘orphan’CGIs)could correspond to uncharacterised promoters or enhancers[2,72].Whereas‘isolated’CpGs are usually methylated,CpGs within CGIs are,with some exceptions,unmethylated.Methylation of CGIs is expected to silence their associated promoters,either by impeding the binding of sequence-specific transcription factors or via the recruitment of repressor complexes that specifically bind to methylated CpGs.
Exceptions to the general rule that CGIs are unmethylated include imprinted gDMRs.Twenty-one imprinted gDMRs have been de-scribed,17maternal gDMRs(methylated in oocytes)and four paternal gDMRs(methylated in sperm).In addition to imprinted gDMRs, genome-wide analysis has identified a number of CGIs methylated in oocytes or sperm that are unrelated to genomic imprinting because they are not completely protected from demethylation after fertilisa-tion[13,17].The function of methylation at these CGIs is unclear at this time,but could influence gene expression patterns in the preimplan-tation embryo.
DNA methylation is also associated with repression of repetitive elements(REs)such as retrotranspos
ons[short interspersed nuclear elements(SINEs),long interspersed nuclear elements(LINEs),and long terminal repeats,(LTRs)].Retrotransposon silencing is especially important in germ cells,which transmit genetic information between generations.Reactivation of retrotransposons in germ cells causes meiotic failure[88,89].
Box1.Male and female germline asymmetry and de novo DNA methylation
De novo DNA methylation takes place in distinct cellular contexts in male and female germ cells and this is an important notion to consider from a mechanistic point of view.In male embryos,DNA methylation is initiated in prospermatogonia arrested in the G1-phase of mitosis,and is completed before birth.It thus occurs before the onset of meiosis,and multiple cell divisions separate DNA methyla-tion establishment and the development of mature sperm.In female embryos,primary oocytes enter meiosis and arrest in the diplotene stage of prophase of the first meiotic division(prophase-I);DNA methylation will only initiate after birth,during the follicular growth phase[10].
The fact that growing oocytes are in meiotic arrest during de novo DNA methylation has several important mechanistic implications.(i) Oocytes are diploid(having a set of maternal and paternal chro
mo-somes)but have a4N DNA content(DNA replication has occurred but the first meiotic division has not).Thus,genomic sequences must be methylated on four chromatids to assure fidelity of DNA methylation patterns in ovulated haploid oocytes.(ii)In somatic cells,DNA methylation marks are the combination of de novo events and the degree to which these marks have been propagated during DNA replication and cell division by the DNA methylation-maintenance machinery.In oocytes,DNA methylation takes place in non-dividing cells(in meiotic arrest)from a state in which the vast majority of CpGs/CGIs are unmethylated[13].Therefore,oocytes represent a true and highly informative model for de novo DNA methylation because DNA methylation patterns detected in oocytes have not been modified by maintenance events.(iii)During de novo DNA methyla-tion,the oocyte genome may be organised in a specialised chromatin environment because methylation directly follows homologous recombination between non-sister chromatids.This is likely to represent an important factor from a mechanistic point of view. Some information on chromatin organisation and histone modifica-tions in growing oocytes is available,but is largely limited to global studies due to the technical difficulty in determining chromatin organisation at a single gene level in very small numbers of cells [82–86].Elucidating the local chromatin state of loci receptive for DNA methylation represents a major challenge in fully understanding DNA methylation-establishment mechanisms.
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Not all regions of the genome are affected by this second wave of demethylation.Indeed,methylation of imprinted gDMRs is faithfully maintained after fertilisation as a lifelong memory of parental origin of the allele in the new generation,leading to monoallelic expression of the associated imprinted genes.However,because a majority of the CGIs methylated in germ cells are apparently not imprinted gDMRs[13],what is the biological function(s)of their methylation?Global lack of DNA methylation marks in oocytes does not result in infertility per se[16],and recent transcriptomic analysis suggests that DNA methyl-ation at CGIs does not significantly affect gene expression in oocytes[13],probably because of the presence of unmethylated alternative promoters for the corresponding genes.These observations argue against a prime role of CGI methylation in oocyte biology itself.Instead,two recent genome-wide studies have revealed that many CGIs methylated in oocytes do not become completely demethy-lated after fertilisation but retain a considerable degree of methylation.This persistence of methylation could affect the level of expression of the associated genes during preimplantation development and influence early line-age-specification events.Interestingly,non-imprinted methylated CGIs are also highly prone to becoming meth-ylated in early embryos,suggesting that CGI methylation in oocytes could be a strong factor determining embryo CGI methylation[13,17].
Mechanisms of DNA methylation establishment:the DNA methylation machinery
De novo DNA methylation at imprinted gDMRs,and prob-ably at methylated CGIs in general,results in the complete methylation of the hundreds of CpGs that comprise the imprinted gDMR or CGI[13,18].The process is therefore comprehensive,specific,and of highfidelity,and this must be considered in any model that explains DNA methylation in germ cells.Over the years several proteins involved in DNA methylation of every,or a restricted number of, imprinted gDMRs have been identified,predominantly from genetic studies in mice or human.Importantly,the fact that some factors are required for methylation of some imprinted gDMRs but not others indicates that there may not be a single,universal mechanism of de novo DNA methylation,although elements of the mechanism are probably shared.
The central role of the DNMT3family members Members of the DNA methyltransferase3(DNMT3)family are essential factors in de novo DNA methylation(Box2). DNMT3A and DNMT3B are the active enzymes,and although DNMT3L lacks a catalytic domain it interacts with DNMT3A or DNMT3B,acting as a cofactor to stimu-late their methyltransferase activities[19–21].All three DNMT3proteins are expressed in both male and female germ cells[13,22,23],but genetic studies have revealed that in oocytes only DNMT3A and DNMT3L are involved in methylation of impr
inted gDMRs and CGIs,as well as of repetitive elements(REs)[5,13,16,24].In the male germ-line,in addition to DNMT3A and DNMT3L,DNMT3B also plays an important role,being responsible for methylation of the gDMR of the imprinted locus Rasgrf1and REs[25].The locus-specific involvement of ZFP57
ZFP57,a member of the KRAB zinc-finger protein family, has recently been identified for its role both in gametic establishment and embryonic maintenance of DNA meth-ylation[26].KRAB proteins are DNA-binding transcrip-tional repressors that interact with the KAP-1corepressor complex,which can recruit factors associated with DNA methylation and repressive chromatin formation.Al-though the precise function of ZFP57remains unclear, in oocytes it is specifically required for de novo DNA methylation of the gDMR of the imprinted gene Snrpn, but dispensable for methylation of the Nnat,Peg3and Mest imprinted gDMRs[26].In parallel,ZFP57is also necessary after fertilisation for maintenance of methylation at sev-eral imprinted gDMRs[26],and mutation of ZFP57in human is associated with hypomethylation at several imprinted loci[27],most probably owing to impaired DNA methylation maintenance.
What makes ZFP57such an interesting factor is that, despite absence of Snrpn imprinted gDMR methylation in oocytes lacking ZFP57,this gDMR can become appropri-ately methylated on the mate
rnal allele in some embryos generated from ZFP57-null females.One possibility to explain thisfinding is that an additional as yet unidenti-fied epigenetic mark(perhaps a specific histone modifica-tion)is present at the Snrpn imprinted gDMR in oocytes, and that this additional mark is preserved after fertilisa-tion and recognised in the early embryo by the somatic DNA methylation machinery to restore parental-allele-specific methylation.Interestingly,reacquisition of Snrpn imprinted gDMR methylation depends upon zygotic ex-pression of ZFP57,suggesting that ZFP57can interact with this mark and promote de novo DNA methylation either in the oocyte or early embryo.A similar possibility of transmitting an epigenetic memory from oocytes indepen-dently of DNA methylation emerges from the observation that normal methylation of the Snrpn and Peg3imprinted gDMRs can be found in some progeny of females with homozygous depletion of DNMT3L[28,29].The role of this potential and unknown additional mark needs to be stud-ied in more detail,and future genome-wide analysis of methylation in ZFP57-depleted oocytes or embryos might reveal additional CGIs behaving similarly to the imprinted gene Snrpn gDMR.
Others trans-acting factors implicated in de novo DNA methylation
Others trans-acting factors involved in imprinted gDMR methylation have emerged from the study of human ge-netic disorders,notably a gestational abnormality termed familial biparental hydatidiform m
ole(BHM).BHMs are conceptuses that lack methylation at many or all maternal gDMRs[30–32].Familial BHM displays maternal-effect autosomal recessive inheritance,suggesting that the gene(s)involved normally act in the maternal germline on gDMR methylation.Genetic studies have identified mutations in familial BHM mothers in NLRP7,a protein of the Nod-like receptor(NLR)pyrin domain-containing family[30–32],and C6orf221,a poorly defined member of a family of K homology(KH)domain proteins[33].Mutation in the related protein,NLRP2,has been found in cases of
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the imprinting disorder Beckwith–Wiedemann syndrome [34].Several NLRP and KH domain proteins are abun-dantly expressed in oocytes,and it is suggested that NLRP7and C6orf221could function in a complex.Howev-er,whether they act in the oocyte or early embryo is unclear at this stage,and the possible mechanisms by which these proteins contribute to DNA methylation es-tablishment or maintenance are largely obscure and may have to await knockout studies in mice.A role for histone remodellers in DNA methylation establishment at some imprinted gDMRs has also been described and is discussed in a subsequent section.
Proteins implicated in the genesis of small RNAs,spe-cifically of the piRNA pathway(PIWI protein interacting RNAs),also play a role in de novo DNA methylation in mammalian germ cells;but this appears to be specific to the male germline.Deletion of the MILI and MIWI2pro-teins causes impaired methylation at retrotransposons [35,36].In addition,methylation of the Rasgrf1imprinted gDMR,but not of other paternal imprinted gDMRs,has been shown to depend on piRNAs[37],but whether this pathway is involved in methylation of non-imprinted CGIs in the male germline is unknown.
Mechanisms of DNA methylation establishment:the target sequence
Following the discovery of the role of DNA methylation in genomic imprinting[4]it was soon suggested that DNA sequence could be an important component in defining which CGIs become methylated in germ cells.Methylated CGIs would possess particular sequence characteristics, absent from unmethylated CGIs,allowing the specific re-cruitment of the DNA methylation machinery(DNMTs and other trans-factors).For example,DNMT3A and DNMT3B have been reported to have preferential and distinct target sequence specificities[38–40].However,the addition of their cofactor DNMT3L relaxes this selectively to allow targets to be methylated more uniformly[40].
As described in Box2,repetitive elements(REs)are methylated in germ cells,and this gave rise to the
notion that imprinted gDMR methylation may be a‘by-product’of a host defence mechanism that silences mobile genetic REs)by DNA methylation.More specifically, several reports have described an association between REs of the tandem repeat(TR)class and imprinted gDMRs [41,42].TRs are reiterated sequence features in which the repeat units are of various lengths and repetition and there appears to be little sequence similarity between TRs at different loci.However,where tested,TRs do not seem to be involved functionally in DNA methylation establishment at endogenous imprinted loci[43–45],except at the Rasgrf1 imprinted gDMR[46].In this case,however,the require-ment for TRs in DNA methylation establishment may be linked to the piRNA pathway(see above),this TR unit serving as a promoter for a transcript crossing the gDMR [37].Similarly,no differences in enrichment of REs,in-cluding TRs,were detected between unmethylated and methylated CGIs identified in oocytes by genome-wide DNA methylation profiling[13].
The spacing between CpGs within a given CGI(or CpG periodicity)has also been proposed as a sequence determinant of CGI DNA methylation.This notion emerged from structural studies of the DNMT3A:DNMT3L complex,which exists as a tetramer with the two catalytic sites of DNMT3A separated by a distance corresponding to 8–10bp of DNA[47].Following this idea,CGIs with CpGs loc
ated8–10bp apart would be preferential targets for DNMT3A:DNMT3L.The predicted8–10bp periodicity was indeed found to be associated with DNA methylation in several contexts,including imprinted gDMRs methylat-ed in oocytes[47],non-CpG and CpG methylation in sev-eral human cells,including embryonic stem cells(ES cells) [48,49],and in the Arabidopsis thaliana genome[50]. Nonetheless,such CpG periodicity can be found through-out the genome and,for example,LINE elements that depend on DNMT3A:DNMT3L for DNA methylation do not possess a particular CpG periodicity[51].Moreover,no differences were observed in the CpG periodicities of imprinted gDMRs,or CGIs methylated or unmethylated in mouse oocytes identified by genome-wide DNA methyl-ation profiling[13,18].
In conclusion,whereas several observations have im-plied a role for DNA sequence in targeting DNA methyla-tion in germ cells,its actual significance is still open to question.Although we cannot exclude an influence of DNA sequence(there may be a sequence component in the function of factors such as ZFP57),on present evidence it seems unlikely that it is a strong determinant of whether a CGI becomes methylated or not.
Mechanisms of DNA methylation establishment:a link with histone modifications
DNA methylation in vivo does not occur on‘naked’DNA but within a chromatin environment,and biochemical studies and genetic evidence have indicated that this has a major impact on the ability of de novo DNA methyl-ation complexes to interact with their genomic targets. Histone modifications,CpG islands,and the DNA methylation machinery
Specific domains of the DNMT3proteins interact with histones(the protein components of the nucleosome), and this interaction is regulated by specific modifications of the amino terminal tails of histones(Box3).For exam-ple,binding of both DNMT3A and DNMT3L is inhibited by methylation of lysine residue4of histone3(H3K4),where-as DNMT3A binding is promoted by trimethylation of lysine36of histone3(H3K36me3).These interactions have been determined in vitro,and whether they occur in vivo in growing oocytes has not been directly confirmed owing to obvious technical difficulties.
Recent evidence suggests that unmethylated CGIs adopt a unique set of histone modifications that may help maintain a state of DNA hypomethylation.For example, the H3K36me2demethylase KDM2A(also known as JHDM1A or CXXC8)binds to unmethylated CGIs via its CxxC domain,resulting in depletion of H3K36me2at these regions in comparison with the surrounding DNA[52,53]. CGIs that gain DNA methylation during ES cell differen-tiation lose KDM2A binding and
acquire H3K36me2[52]. The majority of unmethylated CGIs are also enriched in H3K4me3,which inhibits the interaction of the DNMT3s
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with histones,therefore protecting CGIs against de novo DNA methylation[2,54–57].H3K4me3enrichment at unmethylated CGIs depends on the CxxCfinger protein 1(Cfp1,also known as CxxC1),which interacts with the H3K4methyltransferase Setd1complex.Cfp1and mem-bers of the Setd1complex are expressed in growing oocytes.Therefore,because de novo DNA methylation takes place upon a blank slate of unmethylated CGIs in immature oocytes[13],it may be that CGIs are pro-tected by default from DNA methylation in germ cells and,for the DNA methylation machinery to access target CGIs,major remodelling of histone modifications,or destabilisation of factors protecting against DNA meth-ylation,is required.
The possibility that remodelling of H3K4methylation is necessary for DNA methylation establishment at CGIs in germ cells is supported by recent genetic evidence.In oocytes,deficiency of KDM1B(also known as AOF1or LSD2),an H3K4me1/2demethylase,results in DNA meth-ylation establishment def
ects at several imprinted gDMRs [58].Why only a subset of imprinted gDMRs is affected is unclear,but this could indicate a redundancy in H3K4me2 demethylases.Interestingly,maternal imprinted gDMRs are enriched in H3K4me2in male germ cells,implying a link between this modification and their protection from DNA methylation during spermatogenesis[59].Genome-wide analysis of KDM1B mutant oocytes will be necessary to ascertain its role in methylation of non-imprinted CGIs. Furthermore,it is likely that additional H3K4demethy-lases,active against H3K4me3(the KDM5/JARID1fami-ly),will be found to be implicated in DNA methylation establishment in germ cells.
CpG island methylation and nucleosome remodelling Independently of the regulation by histone modifications of the interaction of the DNMT3A:DNMT3L tetrameric com-plex with nucleosomes,it remains unclear from a tridimen-sional perspective how all the CpGs of a given CGI can be accessible in vivo to the DNA methylation machinery. Indeed,this complex is relatively large compared to a nucleosome,and3D simulation has revealed that a single DNMT3A:DNMT3L tetramer can interact with a single nucleosome,each DNMT3L binding to one H3amino-ter-minal tail[60].If one were to consider a static organisation of nucleosomes at a CGI,in such a3D configuration only a minority of the CpGs within a CGI would be accessible by the DNMT3A:DNMT3L tetramer because1
46bp of DNA are wrapped around the nucleosome.In addition,de novo DNA methylation of CGIs in oocytes appears to be a progressive and protracted process because intermediates towards full methylation can be detected during oocyte growth,indicating that not all CpGs of a given CGI acquire DNA methylation at the same time[23,61,62].Moreover, DNMT3A is not a processive enzyme(it cannot move along the DNA strand)[63],suggesting that multiple rounds of engagement of the DNMT3A:DNMT3L complex with ge-nomic targets are required to completely methylate all CpGs of a CGI.
These observations suggest that nucleosome remodel-ling(a change of nucleosome position along the DNA)may be required to facilitate de novo DNA methylation of CGIs by making all CpGs accessible to the DNA methylation complex.Determining nucleosome occupancy at specific loci on DNA requires many more cells than the number of growing oocytes that can be readily obtained,and this possibility therefore remains untested.However,a role for nucleosome remodelling in DNA methylation in oocytes is suggested by work on the lymphoid-specific helicase LSH (or Hells),a member of the SNF2/helicase family of chro-matin ATP-remodelling proteins.Analysis in mouse em-bryonicfibroblasts(MEFs)and ES cells demonstrates that LSH influences CpG methylation globally and at multiple single copy sequences,including promoter CGIs[64–67]. Mice lacking LSH die soon a
fter birth,precluding investi-gation of this factor in growing oocytes when CGI de novo methylation initiates,but immature oocytes obtained from E18embryos exhibit reductions in methylation at several REs,including IAPs[68].A conditional deletion approach will be useful to investigate fully the function of LSH in de novo DNA methylation in oocytes.
Box3.Interaction of DNMT3proteins with chromatin is modulated by histone tail modifications
The de novo DNA methyltransferases DNMT3A and DNMT3B have a similar structure,comprising a PWWP domain,an ADD domain (ATRX–Dnmt3–Dnmt3L)that contains a PHD(plant homeo domain)-like domain,and a carboxy-terminal catalytic domain(CD)[60].The non-active isoform DNMT3L is a truncated protein with a degraded CD and lacking a PWWP domain.
Interaction of DNMT3proteins with chromatin is mediated and regulated via their PHD and PWWP domains.Transfection experi-ments showed that the PWWP domains of DNMT3A and DNMT3B are required for targeting to chromatin but the ADD domains appear to be dispensable[90,91].Peptide-interaction studies have shown that the ADD domains of all three proteins bind to the amino-terminal ends of histone H3,but that this interaction is sensitive to the methylation state of lysine residue4(K4).Binding by DNMT3L is inhibited by mono-,di-and trimethylation of H3K4[92],and binding
by DNMT3A and DNMT3B is inhibited by di-and trimethylation[93].This reflects the negative correlation between H3K4methylation and DNA methylation observed in genome-wide studies[94,95].Binding of DNMT3A to the histone tail of H3is also inhibited by phosphorylation at threonine3,serine10or threonine11,and acetylation at K4,but not by K9methylation[93].DNMT3A has also been reported to interact with H4R3me2via the ADD domain,a repressive mark established by the protein arginine methyltransferase PRMT5(expressed in oocytes [13])[96],but this was not seen in another study[93].The PWWP domain of DNMT3A,but not of DNMT3B,specifically binds to H3K36me3,a mark associated with transcription elongation[76]. Binding to appropriately modified histone tails is considered to constitute a docking platform to target DNMT3s to specific chromatin domains,and multiple interactions may act synergistically to specify preferred targets.In addition to a structural role,H3amino-terminal tail binding by DNMT3A,specifically when unmethylated at K4, allosterically stimulates its methyltransferase activity[97].DNMT3A is also reported to interact with non-histone proteins,such as the H3K9 methyltransferase G9a[98,99],the DNMT1chaperone protein,nuclear protein Np95(also known as ubiquitin-like protein with PHD and ring finger domains1,UHRF1)[100].These interactions may further delineate genomic targets.
These observations should provide a basis for understanding de novo DNA methylation in oocytes,e
specially why a subset of CGIs become methylated.However,the involvement of a given histone modification in helping to target DNMT3As will have to be studied in the context of the growing oocyte.
Trends in Genetics January2012,Vol.28,No.1
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