parallel mechanisms of epigenetic reprogramming in the germline

Parallel mechanisms of epigeneticreprogramming in the germlineJamie A. Hackett, Jan J. Zylicz and M. Azim Surani

Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Physiology, Development and Neuroscience,

University of Cambridge, Cambridge, CB2 1QN, UK

Review

Glossary

5-hydroxymethylcytosine (5hmC): oxidation of methylated cytosines (5mC) by

TET proteins generates 5hmC, which may be an intermediate during DNA

demethylation and can be further converted to 5caC and 5fC. 5hmC is enriched

in pluripotent and some neuronal cell types, but its precise functional

consequences in the genome and its role in DNA demethylation are unclear.

Base excision repair (BER): a cellular mechanism for repair of nonhelix-

distorting base mutations or lesions in the genome. BER is initiated by a DNA

glycosylase (e.g. TDG) that removes inappropriate bases and forms an

apurinic/apyrimidinic (AP) site, which is then cleaved by an AP endonuclease

and repaired by specific lyases and polymerases. BER may function to remove

downstream derivatives of 5mC, such as 5caC, and mediate repair to

unmodified C [60].

Basal epigenetic state: the unique epigenetic state of PGCs following

reprogramming. By E13.5, the PGC epigenome has undergone extensive

reorganisation of histone modifications and is stripped of genome-wide DNA

methylation, rendering it at its most basal level during the mammalian life

cycle.

Bisulfite sequencing: a technique used to determine the pattern of allelic DNA

methylation (5mC) at specific genomic regions. Bisulfite sequencing cannot

distinguish 5mC from 5hmC [33]. Additionally, 5caC is indistinguishable from

unmodified C by bisulfite sequencing [60].

DNA demethylation: the removal of a methyl group from position 5 of a

cytosine base (5mC), which usually resides within a CpG genomic context, to

generate an unmodified C. DNA demethylation may occur through either a

‘passive’ mechanism that relies on replication-dependent dilution or an ‘active’

process driven by enzymatic replacement independently of DNA replication. As

DNA methylation is associated with transcriptional silencing, DNA demethyla-

tion can generate a transcriptionally competent state.

Epigenetic reprogramming: genome-wide reorganisation of epigenetic mod-

ifications that overcomes stable epigenetic barriers and enables acquisition of

genomic potential. During the mammalian life cycle, epigenetic reprogram-

ming occurs in PGCs and in early zygotic development.

Genomic imprints: genomic sequences that exhibit differences in CpG methyla-

tion according to the parent of origin. These differentially methylated regions

(DMRs) can influence the allele-specific expression of one or more genes.

Primordial germ cell (PGC): the precursors of mature germ cells that are

specified during post-implantation development. In vivo, PGCs are restricted

as a unipotent lineage and only give rise to gametes, which generate the

totipotent state upon fertilisation. Early PGCs also possess an underlying

genomic plasticity, as evidenced through their capacity to form pluripotent EG

cells upon in vitro culture.

Totipotency: the ability of a cell to give rise to all the cell types of the

Germ cells possess the extraordinary and unique capac-ity to give rise to a new organism and create an enduringlink between all generations. To acquire this property,primordial germ cells (PGCs) transit through an unprec-edented programme of sequential epigenetic events thatculminates in an epigenomic basal state that is thefoundation of totipotency. This process is underpinnedby genome-wide DNA demethylation, which may occurthrough several overlapping pathways, including con-version to 5-hydroxymethylcytosine. We propose thatthe epigenetic programme in PGCs operates throughmultiple parallel mechanisms to ensure robustness atthe level of individual cells while also being flexiblethrough functional redundancy to guarantee high fideli-ty of the process. Gaining a better understanding of themolecular mechanisms that direct epigenetic repro-gramming in PGCs will enhance our ability to manipu-late epigenetic memory, cell-fate decisions andapplications in regenerative medicine.

Reprogramming PGCs towards totipotencyDevelopment from the zygote to adulthood is characterisedby a progressive restriction of cellular potential that givesrise to all the differentiated somatic cell types. A uniqueexception to this unidirectional process occurs in the germ-line, where an unprecedented reprogramming event inPGCs (see Glossary) reverses epigenetic barriers to plas-ticity and resets genomic potential. Reprogramming inPGCs results in chromatin remodelling, erasure of genomicimprints and extensive DNA demethylation [1]. This pro-cess represents the most comprehensive erasure of epige-netic information in the mammalian life cycle andunderpins the totipotent state. Therefore, unravellingthe mechanisms that drive reprogramming, particularlyDNA demethylation, in the unique context of PGCs is ofgreat interest.

In mice, PGCs are specified from a subset of posteriorproximal epiblast cells at approximately embryonic day (E)6.25, resulting in the establishment of a founder populationof PGCs at E7.25 [1,2]. These nascent PGCs subsequentlymigrate to the genital ridges by approximately E10.5 and,from E12.5 onwards, they undergo sex-specific developmentin the gonads [3,4]. Because mammalian PGCs are specifiedfrom cells that are already primed towards a somatic fate,nascent PGCs must both repress the ongoing somatic pro-gramme and activate the germ cell transcriptional network

Corresponding author: Surani, M.A. ([email protected]).

164 0168-9525/$ – see front matter � 2012 Elsevier Ltd. All rig

[5,6]. This process is initiated in response to localised sig-nals, including BMP4 and WNT3, which direct activation ofthe key transcriptional regulators B-lymphocyte-inducedmaturation protein 1 (Blimp1) and PR domain containing14 (Prdm14) in competent epiblast cells [1,7–9]. Lineage-restricted PGCs then embark on an orchestrated sequenceof reprogramming that culminates in a basal epigeneticstate. The number of early PGCs is highly restricted (ap-proximately 40 by E7.25) so it is crucial that the complexseries of epigenetic events be robust to ensure that most, ifnot all, cells efficiently transit through the process [10].Reprogramming must also proceed rapidly because of strict

embryonic and extra-embryonic lineages. By contrast, pluripotency refers to

the capacity of a cell to generate all the cell types of the embryo.

hts reserved. doi:10.1016/j.tig.2012.01.005 Trends in Genetics, April 2012, Vol. 28, No. 4

mailto:[email protected]

http://dx.doi.org/10.1016/j.tig.2012.01.005

Review Trends in Genetics April 2012, Vol. 28, No. 4

temporal constraints imposed by the entry of male germcells into mitotic arrest and female germ cells into meioticarrest at approximately E13.5. To overcome these con-straints, epigenetic reprogramming in PGCs is probablydirected by multiple parallel mechanisms that ensure thefidelity, flexibility and efficiency of this fundamental pro-cess. As such, events like genome-wide erasure of DNAmethylation in PGCs may occur through several intercon-nected mechanisms, including both active and passive path-ways, that collectively confer redundancy and hencerobustness to PGC reprogramming and development.

Here, we discuss the epigenetic events in PGCs andthe mechanisms that may operate to drive epigeneticreprogramming. We suggest that an integrated processinvolving parallel systems is at play and consider thepotential pathways of DNA demethylation. We also high-light some of the potential roles and developmentalprocesses that epigenetic reprogramming contributes toin PGCs.

Epigenetic events in PGCsAt the point at which PGCs are specified from post-im-plantation epiblast cells, they are epigenetically indistin-guishable at the global level from their neighbours, whichare destined for a somatic fate [9,11]. Therefore, nascentPGCs inherit stable epigenetic states, including DNAmethylation and X-inactivation, which constitute an epi-genetic barrier against the eventual acquisition of totipo-tency [1,12]. It is thus an important early step in PGCdevelopment to initiate a process of reprogramming thaterases these stable epigenetic blocks. The first gross epi-genetic changes in PGCs entail a reciprocal loss of histoneH3 lysine 9 dimethylation (H3K9me2) from E7.75 and, inmost PGCs, a global increase of H3 lysine 27 trimethyla-tion (H3K27me3) by E9.5 (Figure 1) [11,13,14]. The ge-nome-wide depletion of H3K9me2 is potentially aconsequence of the downregulation of GLP, a methyltrans-ferase that forms a heteromeric complex with G9a (alsoknown as EHMT2) that is required for deposition of H3K9mono- and dimethylation, and the parallel upregulation ofspecific lysine demethylases ([15] and unpublished obser-vations). By contrast, the mechanisms responsible for theincreased H3K27me3 levels in PGCs remain unclear, al-though notably enhancer of zeste homologue 1 (Ezh1),which has H3K27me3 methyltransferase activity, is upre-gulated in PGCs [16]. Because H3K9me2 and H3K27me3marks are both associated with transcriptional repression,it has been postulated that the loss of H3K9me2 is com-plemented by the gain of H3K27me3 to maintain a repres-sive chromatin state in PGCs [17]. However, the precisegenomic location and relationship between these epigenet-ic changes remains to be determined. Nonetheless, theglobal enrichment of H3K27me3 and loss of H3K9me2establishes a chromatin environment in PGCs that isgrossly similar to that in pluripotent embryonic stem(ES) cells and is coupled to upregulation of pluripotencygenes, such as Nanog and SRY Sox2 [1]. Additionally,unlike global H3K27me3 levels, the inactive X-chromo-some (Xi) exhibits a protracted decline in H3K27me3 infemale PGCs, which is linked to initiation of X-reactivation[18]. Thus, while PGCs are unipotent and only form either

sperm or oocytes in vivo, during migration (E8.5–E11.5),PGCs show multiple transcriptional and epigenetic simi-larities to pluripotent ES cells. Indeed, E8.5–E11.5 PGCscan form pluripotent embryonic germ (EG) cells in vitro,which resemble ES cells rather than the post-implantationepiblast cells from which PGCs were originally specified[19,20]. It is possible that this epigenetic state of PGCs isan underlying requirement for the initiation of meiosisand the eventual acquisition of totipotency in the zygote.Interestingly, migrating PGCs additionally exhibit upre-gulation of histone H2A/H4 arginine 3 symmetrical meth-ylation (H2A/H4R3me2 s), which is catalysed by proteinarginine methyltransferase 5 (PRMT5) [21]. This modifi-cation may contribute to maintaining PGCs in a unipotentstate in vivo and to repression of the somatic programme[22].

Changes in histone modifications occur in parallel witha reported reduction in global levels of DNA methylation(5mC) in migrating PGCs from approximately E8.0 [11].Any loss of 5mC might reflect the effects of BLIMP1 andPRDM14, which repress both DNA (cytosine-5)-methyl-transferase 3a and 3b (Dnmt3a and Dnmt3b) and ubiqui-tin-like, containing PHD and RING finger domains 1(Uhrf1), which are essential components of the de novoand maintenance methylation machinery, respectively(Figure 1) [7,8,23–26]. Additionally, repression of GLPmay directly affect DNA methylation through a parallelmechanism that is both dependent and independent ofH3K9me2 [27,28]. However, most analysed genomicregions, including transposable elements, imprinted lociand single-copy genes, apparently retain DNA methylationat CpG sites in PGCs until at least E10.5 [29–32], althoughthis does not exclude the possibility that there is conver-sion of 5mC to 5-hydroxymethylcytosine (5hmC) [33].Therefore, it is unclear to what extent DNA demethylationcontributes to the early stages of epigenetic reprogram-ming (from E8.0) in PGCs and whether conversion to 5hmCplays a role. Conversely, it is unclear how 5mC (or 5hmC) ismaintained at analysed genomic regions in migratingPGCs given that essential components of the de novoand maintenance machinery, particularly UHRF1, areabsent [7]. One possibility is that UHRF2, which is con-served with UHRF1 at the sequence level and preferen-tially binds hemi-methylated DNA associated withH3K9me3, can compensate for UHRF1 to maintain DNAmethylation in PGCs, either globally or at specific loci [34].In support of this, UHRF2 is specifically upregulatedduring PGC specification, at least in vitro [7,35]. Addition-ally, unlike H3K9me2, global H3K9me3 levels are main-tained in migrating PGCs [14]. The in vitro PGC-like cells(PGCLC) generated in an elegant recent study may enablefurther mechanistic insights into epigenetic events in na-scent PGCs at a higher resolution [36].

The early stages of epigenomic reorganisation in PGCsare followed by, and are probably a prerequisite for, thedramatic genome-wide erasure of DNA methylation andextensive chromatin remodelling subsequent to entry intothe genital ridges at approximately E10.5. Because grossDNA demethylation seems to occur rapidly at a distincttime point, while most PGCs are in G2 phase, it is held thatthis process is an ‘active’ event occurring independently

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Tet3

Figure 1. Chromatin and transcriptional changes in primordial germ cells (PGCs). Following specification (Spec.), PGCs transit through two sequential phases of

reprogramming during migration (R1) and subsequent to entry into the genital ridge (R2). (a) Epigenomic reorganisation in PGCs. At R1, PGCs exhibit global erasure of

histone H3 lysine 9 dimethylation (H3K9me2), upregulation of H3 lysine 27 trimethylation (H3K27me3) and some loss of the DNA methylation signal. At R2, there is a further

dramatic loss of DNA methylation, which includes erasure of imprints. This correlates with a transient reorganisation of H3K27me3, loss of heterochromatic chromocentres

and remodelling of other chromatin modifications, including histone H2A/H4 arginine 3 symmetrical methylation (H2A/H4R3me2; not shown). (b–d) Transcriptional changes

during PGC development [13]. (b) Specification and chromatin-modifying factors. Upregulation of B-lymphocyte-induced maturation protein 1 (Blimp1) and PR domain

containing 14 (Prdm14) is necessary for specification of PGC fate, whereas the parallel downregulation of glucagon-like peptide (Glp)/G9a and upregulation of lysine

demethylases (not shown) contribute to erasure of H3K9me2 at R1. (c) DNA methylation proteins. Downregulation of DNA (cytosine-5)-methyltransferase 3b (Dnmt3b) and

ubiquitin-like, containing PHD and RING finger domains 1 (Uhrf1) may account for DNA demethylation at R1. UHRF2 may partially compensate for the absence of UHRF1 to

maintain DNA methylation until R2. (d) Ten-eleven translocation gene Tet dioxygenases. Tet1 and Tet2 are expressed in PGCs by R2, whereas Tet3 cannot be detected [50].

Dashed line indicates putative levels. Abbreviation: E, embryonic day.


from DNA replication, although the precise mechanism isyet to be elucidated [14]. Reprogramming in PGCs at thisstage results in almost full erasure of DNA methylation byE13.5 [37], with complete stripping of parental imprintsand promoter CpG methylation at germline-specific genes

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[29,32]. The process of DNA methylation erasure alsoinitiates a cascade of chromatin remodelling in PGCs atapproximately E11.5. This includes a transient reorgani-sation of linker histone H1, H3K27me3 and H3K9me3 andstable remodelling of global H3K9ac and H2A/H4R3me2 s


[14]. Some sequences, including intracisternal A particle(IAP) retrotransposons, partially evade DNA demethyla-tion in PGCs, although the mechanism that protects theseelements remains to be determined [31]. Nevertheless, theerasure of DNA methylation and extensive chromatinreorganisation at approximately E11.5 renders PGCs ina basal epigenetic state that represents an epigenomicnadir during mammalian development. It is of note thatthe apparent biphasic nature of demethylation in PGCs (atapproximately E8.0 and approximately E11.5) may poten-tially represent a continuous process of 5mC erasure. Inany case, reprogramming of the PGC genome as a whole isdistinguished from reprogramming during zygotic devel-opment, where imprints, DNA methylation at multiple lociand polycomb-based chromatin modifications remain; thisimplies that reprogramming in PGCs represents a moreextensive process [32]. Although both reprogrammingevents probably share some mechanistic similarities, itis also likely that both employ unique systems to achievedistinct levels of epigenomic resetting.

Erasure of DNA methylation and cellular identityDNA methylation (5mC) within a CpG context is a highlyheritable epigenetic mark that is associated with tran-scriptional repression and that contributes to stable line-age commitment [38–41]. In this respect, global erasure ofDNA methylation during PGC development is a fundamen-tal event towards the acquisition of totipotency. The ca-pacity of global DNA demethylation to alter cellularidentity, for example in the course of derivation of inducedpluripotent stem cells (iPS) from somatic cells, makesunravelling the mechanisms that mediate this process ofgreat importance [42,43]. Although extensive studies haveestablished how and where 5mC is and can be introduced,it remains enigmatic precisely how DNA methylation isremoved from the genome [44]. The global DNA demethyl-ation during PGC development represents a unique in vivoevent in that it results in near-complete stripping of CpGmethylation at almost all genomic loci, including imprints.The magnitude of DNA demethylation makes PGCs anunparalleled system to understand the process of 5mCerasure in an in vivo context.

Active demethylation in PGCsThe recent identification of three 5mC-dioxygenases [ten-eleven translocation gene 1, 2 and 3 (TET1, TET2 andTET3)], which can convert 5mC to 5-hydroxymethylcyto-sine, presented a potential solution to the longstandingdebate regarding the mechanism of DNA demethylation[45,46]. Conversion of 5mC to 5hmC enables several alter-native but partially overlapping routes to generate anunmodified cytosine (C) residue independently of, or de-pendent on, DNA replication (Figure 2). In the zygote, therapid loss of 5mC from the male pronucleus correlates witha concomitant gain in 5hmC, implying that 5mC is con-verted to 5hmC in this context and has a fundamental rolein reprogramming [47,48]. Indeed, loss of TET3, which isthe only 5mC-dioxigenase significantly expressed inzygotes, led to a failure to erase 5mC and neonatal lethality[49]. Although TET3 is not detectable in PGCs, TET1 andTET2 are present in these cells, suggesting that 5hmC may

replace 5mC during global DNA demethylation in PGCs[50].

Mechanistically, conversion of 5mC to 5hmC could leadto unmodified cytosine through several routes in PGCs,including through providing a substrate for base excisionrepair (BER)-mediated active demethylation [40]. Severalrecent studies have linked BER components to demethyl-ation during zygotic reprogramming and at specific loci insomatic contexts [50–52]. As BER components are alsoupregulated in PGCs relative to somatic neighbours duringepigenetic erasure, genome-wide demethylation in PGCsmay also occur, at least partially through BER. Indeed, thepresence of chromatin-associated XRCC1 and active poly[-ADP-ribose] polymerase 1 (PARP1) in PGCs at approxi-mately E11.5, which signify single-strand DNA breaksassociated with BER, further support this possibility [50].

One potential involvement of BER-mediated DNA de-methylation in PGCs is that TET-generated 5hmC is furtherprocessed by deamination to 5-hydroxymethyluridine(5hmU) by the activation-induced cytidine deaminase/apo-lipoprotein B mRNA-editing, enzyme-catalytic, polypeptide(AID/APOBEC) family of deaminases, and subsequentlyexcised by a glycosylase and repaired to unmodified C.Consistent with this, loss of AID impairs global demethyla-tion in PGCs by E13.5, indicating that 5mC erasure in PGCsdepends, at least in part, on AID [37]. Indeed, AID alsoenhances locus-specific active demethylation mediated byTET1 in somatic cells and has been reported to interact withthymine DNA glycosylase (TDG), which can excise thedeamination product 5hmU [52,53]. Moreover, AID hasbeen proposed to be required for DNA demethylation ofoctamer-binding transcription factor 4 (Oct4) and Nanogduring somatic cell reprogramming induced by heterokary-on formation with ES cells as well as in demethylation inzebrafish embryos [54,55]. However, murine PGCs stillundergo extensive demethylation by E13.5 in the absenceof AID; global 5mC is reduced to 22% and 20% in male andfemale mutant PGCs, respectively, as opposed to 16% and8% in wild-type PGCs and compared with approximately74% in E13.5 embryonic soma. Moreover, AID-mutant miceare both viable and fertile, as are APOBEC 1- and APOBEC2/3-null mice [56–58]. It is also unclear whether AID isexpressed to any significant degree in PGCs at the time ofgross demethylation (approximately E11.5), as it has onlybeen detected from E12.5 in PGCs, suggesting that AID hasa role in PGC demethylation either after global 5mC erasure(approximately E12.5) or perhaps at an earlier stage (ap-proximately E8.5), when the expression of AID is unknown[50,59]. Likewise, TDG is not detectable in PGCs betweenE10.5 and E13.5, as judged by immunofluorescence studies[50]. However, TDG-null PGCs accumulate biallelic CpGmethylation by E11.0 at the insulin-like growth factor 2receptor (Igf2r)-imprinted differentially methylated region(DMR), suggesting that TDG has a role in maintaininga methylation-free state at this locus, presumably by de-methylation, although this event may occur prior to PGCspecification [52]. Although further clarification of TDGfunction and expression in PGCs is necessary, its putativeabsence at E11.5 argues against a direct AID- and/or TDG-mediated active demethylation reaction in PGCs, as hasbeen reported in other contexts [52]. Therefore, alternative

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(a)

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Figure 2. Multiple parallel mechanisms of DNA demethylation. (a) Methylated cytosine (5mC) can be demethylated through several overlapping pathways, including

passive and active mechanisms, which may occur in parallel. Passive demethylation (right) can occur through direct replication-dependent depletion of 5mC owing to an

absence or reduction of DNA methyltransferase activity. Alternatively, TET oncogene (TET) proteins can catalyse oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) or

further conversion to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which may all lead to passive demethylation. Active erasure of 5mC (left) can occur through

deamination of either 5mC or 5hmC to thymidine (T) or 5-hydroxymethyluridine (5hmU), respectively, which can act as a substrate for base excision repair (BER) to

unmodified C. Further conversion of 5hmC to 5fC or 5caC may also be actively removed by BER or 5caC can putatively be removed via a direct decarboxylation reaction

(centre). (b) Putative temporal pattern of 5mC conversion to 5hmC during primordial germ cell (PGC) development. The depletion of the 5mC signal at R1 may occur as a

result of conversion to 5hmC or through other mechanisms. Similarly, 5mC may be converted to 5hmC at R2, which may subsequently be removed through passive and/or

active mechanisms. Abbreviations: AID/APOBEC, activation-induced cytidine deaminase/apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide; R1 and R2, the two

sequential phases of reprogramming during migration (R1) and subsequent to entry into the genital ridge (R2); Spec. specification.


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deaminase–glycosylase complexes may operate down-stream of 5hmC (or 5mC) in PGCs. Investigation of potentialcandidates, such as other members of the APOBEC family,and methyl-CpG-binding domain protein 4 (MBD4), single-strand selective monofunctional uracil DNA glycosylase(SMUG1), nth endonuclease III-like 1 (NTH1) or nei endo-nuclease VIII-like 1 (NEIL1) glycosylases, may shed light onthe issue.

An alternative possibility is that TET-mediated hydro-xymethylation has a deamination-independent role in ac-tive demethylation in PGCs, as TET proteins can furtheroxidise 5hmC to 5-formylcytosine (5fC) and subsequently to5-carboxylcytosine (5caC) [60,61]. Interestingly, these newC derivatives could also be targets for BER excision [60].Indeed, 5fC and 5caC are substrates for TDG, and poten-tially other glycosylases [60,62]. 5caC could also theoreti-cally be ‘actively’ removed from the genome by a BER-independent pathway that would involve decarboxylationby an as yet unknown enzyme. It will be important todetermine the prevalence of 5fC and 5caC in PGCs duringthe key stages of reprogramming to establish whether theycontribute to demethylation through any mechanism. Afurther possibility is that 5hmC is directly targeted forBER-mediated excision by 5hmC-specific glycosylases with-out the requirement for processing by deamination or fur-ther oxidation. Indeed, 5hmC glycosylase activity has beenreported in calf thymus extract [63]. Taken together, amechanism based on BER may play at least a partial rolein PGC demethylation, although it is currently unclearwhich protein complexes direct the process or whether thereis a degree of redundancy. Likewise, it is not clear which5hmC or 5mC derivative substrate [5hmC, 5hmU, 5fC, 5caCor thymidine (T)] might be targeted by the BER machineryfor active DNA demethylation in PGCs.

It is also possible that alternative mechanisms, indepen-dent of 5hmC, contribute to, or drive, active demethylationin PGCs. In Arabidopsis thaliana, demethylation occurs viadirect excision of 5mC by the specific bifunctional DNAglycosylase/lyases, DEMETER (DME) and REPRESSOROF SILENCING 1 (ROS1), followed by recruitment ofBER machinery [64,65]. Although no mammalian orthologsof DME–ROS1 or 5mC-specific glycosylases have been de-scribed, it remains a formal possibility that 5mC is directlyexcised from the genome in PGCs. Another possibility is that5mC is deaminated directly to T followed by excision by aglycosylase that recognises the T–G mismatch, such as TDGor MBD4, and repaired to unmodified cytosine by BER. It isadditionally possible that a demethylation route based onradical sterile alpha motif (SAM) enzymes operates, al-though experimental evidence for this is lacking in PGCs[66]. Similarly, active 5mC erasure has been reported to belinked to the nucleotide excision repair (NER) pathway, andthis could contribute to DNA demethylation during PGCmigration (at approximately E8.5–E10.5) [67]. However, itremains unclear whether such a mechanism could operatein PGCs at approximately E11.5 owing to the absence ofseveral key NER components between E10.5 and E12.5 [50].Thus, the consensus of available data points towards aputative active DNA demethylation mechanism in PGCsat approximately E11.5 being based on initial TET-mediat-ed hydroxylation of 5mC and either deamination towards

5hmU or further oxidation to 5fC and 5caC, all of which areBER substrates, ultimately leading to repair-driven de-methylation.

Passive demethylation in PGCsThere is accumulating evidence that genome-wide DNAdemethylation events include at least a partial ‘passive’component. Conversion of 5mC to 5hmC in the zygote hasrecently been shown to lead to replication-dependent pas-sive demethylation, rather than to active removal of 5hmCor its derivatives [68]. Thus, chromosomes originating inthe paternal pronucleus retain 5hmC through successivecleavage divisions, whereas newly synthesised sister chro-matids are devoid of 5mC and 5hmC, leading to a progres-sive dilution of DNA modifications during development.The passive loss of DNA modification is consistent with thefact that the human maintenance DNA (cytosine-5)-methyltransferase 1 (DNMT1) cannot recognise 5hmCand there are no other known 5hmC maintenance mecha-nisms (although notably UHRF1 does efficiently recognise5hmC) [69,70]. Nevertheless, bisulfite sequencing has in-dicated there may be an additional active mechanism inzygotes that is dependent on BER and operates prior toDNA replication, which may target specific genomic land-marks [51]. Additionally, the high sensitivity of the 5hmCantibody may mask partial or locus-specific active erasureof 5hmC on the paternally derived pronuclear chromatids[71]. However, it seems probable that the bulk of paternalpronuclear demethylation occurs passively following5hmC conversion. It remains unclear how the maternalpronucleus, which does not undergo 5mC to 5hmC conver-sion, undergoes concomitant passive demethylation, asDNMT1 is present and sufficient to maintain maternalimprints [68]. Nonetheless, these studies indicate thatconversion of 5mC to 5hmC coupled with passive demeth-ylation is feasible and could operate in PGCs.

In murine PGCs, although there is an overall loss ofDNA methylation over a relatively short period at approx-imately E11.5, it is important to reconsider whether era-sure of imprinted genes can occur over a protracted periodof 2 days [30], which would not be compatible with a singlegenome-wide wave of ‘active’ demethylation model [14].Indeed, erasure of DNA methylation from imprinted lociand repeat elements in porcine PGCs occurs over an ex-tended period of up to 20 days, which suggests that ‘active’DNA demethylation does not apply in all mammalianPGCs, possibly including those in mice [72]. Similarly tozygotes, conversion of 5mC to 5hmC (or 5fC and 5caC) inPGCs would facilitate passive replication-dependent de-methylation (Figure 2). Such a conversion of 5mC to 5hmCwould account for the dramatic loss of 5mC staining ob-served in PGCs at E11.5 and the protracted nature ofimprint erasure, given that bisulfite conversion wouldidentify hydroxymethylated loci as methylated but 5mCantibodies would not [14,30,33]. Interestingly, conversionof 5mC to 5hmC may also skew bisulfite sequencingresults, as polymerases appear to amplify unmethylatedalleles preferentially over 5hmC-modified alleles after bi-sulfite treatment, which may falsely imply demethylationhas occurred rather than conversion to 5hmC [73]. Addi-tionally, TET-mediated conversion to 5hmC may proceed

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to 5caC in PGCs, which is read as unmethylated by bisul-fite sequencing, erroneously indicating the occurrence ofactive demethylation [60]. Although functional conse-quences of 5hmC, 5caC or other C derivatives are as yetunclear, the inherent biases and lack of derivative speci-ficity of bisulfite sequencing mean that observing anunmethylated allele via this technique does not necessarilysignify the presence of unmodified C or the implied activedemethylation. As such, conversion to 5hmC or down-stream C derivatives could at least partially account forthe apparent active ‘replication-independent’ demethyla-tion that has been reported in PGCs.

An alternative to passive erasure through 5hmC con-version is that PGCs undergo direct passive dilution of5mC, based on an absence or exclusion of maintenancecomponents, such as DNMT1 or UHRF1. As the doublingtime of PGCs at this stage is approximately 16 hr [13,74],direct replication-dependent dilution could account for asignificant reduction in global 5mC in PGCs between E10.5and E11.5. Because the 5mC antibody (33D3) exhibitsrelatively weak avidity (e.g. as compared to the 5hmCantibody [71]) and 5mC levels at E10.5 may already bepartially depleted relative to somatic neighbours, thispassive reduction of 5mC may appear as a largely completeand, therefore, active erasure of global DNA methylation,while there would also be a significant loss of methylationby bisulfite sequencing. Although it is not clear preciselyhow a passive demethylation mechanism might be trig-gered, the existence of such a system would circumvent thepotential genetic damage that may result from genome-wide BER.

Parallel mechanisms of epigenetic reprogrammingAlthough cumulative evidence indicates that several mo-lecular pathways of DNA demethylation may operate,the precise mechanism(s) that mediate the comprehen-sive methylation erasure in PGCs remain to be clarified[44,75,76]. Indeed, DNA demethylation and chromatinremodelling in PGCs may occur through several comple-mentary parallel pathways, including active and passivesystems, which would provide a degree of redundancyand confer robustness and flexibility to the programme(Figure 3). This seems necessary because of the funda-mental importance of epigenetic reprogramming to germcell development. Furthermore, there are very limitednumbers of PGCs and most, if not all, of them musttransit through the process efficiently and prior to theentry of female germ cells into meiosis shortly afterreprogramming.

A robust system based on multiple parallel mechanismsand inbuilt redundancy may account for the observationthat Tet1-null and Tet2-null mice are viable and fertile,despite a probable role for 5hmC in demethylation in PGCs[77–80]. Although functional redundancy coupled withgenetic background effects may explain this observation,it is also possible that impeded DNA demethylation inTET-deficient PGCs would not cause complete loss of thecells but rather a reduction in their numbers. In support ofthis, impeded demethylation of the paternal genome inTET3-deficient zygotes is only associated with a severedevelopmental phenotype in a subset of mutant embryos

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[49]. This suggests that rapid epigenetic reprogramming isinvolved in diminishing developmental barriers that somecells can nevertheless still overcome in a stochastic man-ner. Similarly, Tet1-null mice sire reduced litter sizes andmutant pups have a smaller body size at birth, potentiallyowing to a delay in overcoming epigenetically imposedbarriers [77]. The presence of multiple means of 5mCerasure could compensate for the loss of function of Tet1through the use of alternative routes in a stochastic man-ner. For example, given that key maintenance-methylationenzymes, including UHRF1, are absent in PGCs, it ispossible that direct passive demethylation of 5mC canpartly offset the absence of any putative 5hmC-mediatedmechanisms, a possibility that might also affect interpre-tation of the demethylation that still occurs in AID-defi-cient PGCs by E13.5 [7,37].

In addition to contributing to a robust system, parallelmechanisms of 5mC erasure could function to target spe-cific genomic loci in PGCs. For example, passive demeth-ylation could account for the bulk of demethylation,analogously to the zygote, whereas erasure of imprintedregions may be targeted by specific active mechanisms thatare absent in the zygote, perhaps based on BER. Interest-ingly, targeting specific loci with distinct demethylationmechanisms may also lead to particular functional out-comes, perhaps based on the specific C derivative (5hmC,5fC, 5caC, etc.) that directed demethylation. This couldhave an important functional role during meiosis, similarlyto the role of inherited histone modifications at develop-mental loci in the zygote [81]. Active DNA demethylationmechanisms could also operate in parallel to a passivesystem to initiate DNA repair-driven chromatin remodel-ling [14]. Notably, other phases of epigenetic reprogram-ming in PGCs also utilise parallel systems. For example,erasure of H3K9me2 in early PGCs may occur throughboth downregulation of the methyltransferase GLP andupregulation of specific lysine demethylases at approxi-mately E8.5, which potentially contribute to erasure of thismodification in a redundant manner. Similarly, the histonereplacement that is associated with reorganisation of glob-al H3K27me3 and H3K9me3 marks in PGCs at approxi-mately E11.5 also occurs in parallel with upregulation ofspecific lysine demethylases, which may contribute to theefficient erasure. Indeed, both Tet1 and Tet2 are alsoupregulated at this stage, indicating a possible parallelrole [50]. Thus, we propose that the unique importance ofreprogramming in PGCs necessitates a system of multipleepigenetic erasure mechanisms to confer efficiency, fidelityand robustness to the process.

Potential roles of epigenetic reprogramming in PGCsThe fundamental role of epigenetic reprogramming inPGCs is to overcome multiple epigenomic barriers to theeventual acquisition of totipotency acquired by epiblastcells during early development. This is necessary becausemammals utilise an inductive mechanism of germ cellspecification (they specify the germline from cells primedtowards a somatic fate) rather than acquire germ cell fatethrough an inherited germplasm. This essential role ofreprogramming in mammals is evident in mice lacking thetranscriptional regulator PRDM14. Nascent PGCs in

TET1/2 activity

Deamination BE

R

Passive

DN

A dem

ethylation

Me

Ac

Me

Histone exchange Histone mark reprogramming

Progression towardsBasal state

Primed state

Reprogram

ming

Me

Ac

HD

M/H

DA

C upregulation

HM

T/H

AT downregulation

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Ac

Me

5mC 5mC 5mC

Me

Ac

Me

Dnmt3a/b, Uhrf1 downregulation

Figure 3. Parallel routes towards the basal epigenetic state. Post-implantation epiblast cells acquire epigenetic modifications that constitute a barrier against their reversion

to a pluripotent state but remain primed to respond to signals that specify a primordial germ cell (PGC) fate. Upon specification, PGCs embark on a process of

reprogramming that overcomes the epigenetic barrier and establishes a basal epigenetic environment that underpins totipotency. Multiple parallel mechanisms contribute

to epigenetic reprogramming in PGCs to ensure robustness and redundancy to the process. Global erasure of DNA methylation (upper routes) can occur through passive or

active mechanisms directed through several interconnected processes. Likewise, reorganisation of chromatin architecture (lower routes) can occur through the parallel

upregulation of histone demethylases (HDM)/deacetylases (HDAC), downregulation of chromatin-modifying enzymes, including histone methyltransferases (HMT)/

acetyltransferases (HAT), and dynamic histone exchange. Abbreviations: 5mC, methylated cytosine; Ac, acetylation; BER, base excision repair; Dnmt3a/b, DNA (cytosine-5)-

methyltransferase 3a/b; Me, methylation; TET, ten-eleven translocation gene; Uhrf1, ubiquitin-like, containing PHD and RING finger domains 1.


Prdm14-mutant mice fail to undergo early reprogrammingof chromatin and DNA methylation or to activate the germcell genetic programme and consequently exhibit severelyimpaired PGC development and are sterile [8]. Evidenceshows that PGC specification and epigenetic reprogram-ming are intimately linked. Furthermore, within the broadremit of establishing a state of ‘underlying totipotency’,epigenomic reprogramming has many distinct roles thatcollectively contribute to a continuous process of PGCdevelopment (Figure 4).

An important event driven by reprogramming is reacti-vation of Xi in female PGCs. This requires the integrationof multiple genetic and epigenetic systems; with earlychromatin reorganisation initiating X-reactivation andDNA demethylation and repression of the long noncoding

RNA Xist necessary to complete the process [18,82]. Thewave of global DNA demethylation in PGCs at approxi-mately E11.5 seems to be a trigger for several otherimportant processes, including the erasure of parentalimprints to enable establishment of new methylationmarks according to the sex of the embryo [17,29]. Thesignificant impact that aberrant imprints have on mam-malian development, foetal growth and behaviour, and inhuman disease, has led to the suggestion that the wholeprocess of genome-wide demethylation in PGCs reflects abyproduct of the necessity to reset locus-specific imprints.Indeed, as imprinted loci are resistant to demethylationduring zygotic reprogramming [82], they may have intrin-sic or epigenetic properties that preclude erasure exceptunder exceptional cellular conditions, such as exists in

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R1 R2

Removal of epigenetic barriers to totipotency

X-chromosome reactivation

Imprint erasure

Germline gene reactivation

Epimutation erasure

Retrotransposon reactivation

Setting for meiosis

~E7.75–9.0 ~E10.5–12.5

(a)

(b)

(c)

(d)

(e)

(f)

(g)

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Figure 4. Potential functions of epigenetic reprogramming in primordial germ cells (PGCs). The distinct roles of epigenetic reprogramming in PGCs and the time point at

which each occurs (rectangles). (a) Epigenetic reprogramming events during PGC migration (R1) and post-migration (R2) overcomes the layers of epigenetic modifications

acquired by epiblast cells, which form a barrier to acquisition of totipotency. (b) In female cells, reprogramming contributes to X-chromosome reactivation. (c) Erasure of

epigenetic marks in PGCs also prevents perpetuation of epimutations through the germline. (d) Global DNA demethylation at R2 triggers activation of stably silenced genes

necessary for germ cell development (e) A fundamental role of reprogramming is to erase parent-of-origin imprints, thereby enabling subsequent sex-specific methylation

marks to be established during gametogenesis (f) Erasure of methylated cytosine (5mC) may permit transient expression of retrotransposons, which are subsequently

targeted for stable transcriptional repression by small RNAs, thereby ensuring that potentially harmful genetic elements are strongly silenced in the germline. (g)

Reprogramming in PGCs contributes to establishing a chromatin and transcriptional environment that is primed for entry into meiosis. Abbreviation: E, embryonic day.


PGCs. Global DNA demethylation in PGCs has otherimportant functions, including the activation of stablysilenced genes that are required for germ cell development[32,83]. Genes such as deleted in azoospermia-like (Dazl)and synaptonemal complex protein 3 (Sycp3) are robustlysilenced by methylated CpG-dense promoters during post-implantation development, potentially to prevent ectopicactivation that may drive malignant tumour growth, butare activated by demethylation specifically in the germline[32,39,84,85]. Another key role of reprogramming in PGCsis to erase aberrant epigenetic information or epimuta-tions. Erasure of such modifications in the zygote andparticularly in PGCs prevents their inheritance and per-petuation through successive generations with potentiallydetrimental effects.

Global DNA demethylation may generate an epigeneticenvironment susceptible to expression of harmful trans-posable elements (TE), yet paradoxically it is possible that

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transient activation of TEs during reprogrammingenables silencing mechanisms to target repressive chro-matin, possibly directed by small RNAs derived fromexpressed TEs [86]. This putative mechanism would en-sure that harmful genetic elements that may have escapedrepressive mechanisms are rendered transcriptionallyquiescent throughout germ cell maturation. Notably,the transient erasure of the PGC epigenome may alsoprovide an opportunity for epigenetic writers to accesschromatin and establish new modifications that directevents that are crucial for meiosis. Indeed, the absenceof some chromatin-modifying proteins, including G9a,MLL2, SUV39H1, SUV39H2, LSH and PRDM9, onlymanifests in germ cells during meiosis [87–91]. One in-triguing possibility is that epigenetic erasure is necessaryto promote chiasmata formation at recombination hot-spots during meiosis. Here, reprogramming may enablethe lysine methylase PRDM9 access to target H3K4me3


marks specifically to 13-mer recognition sites, which act asbeacons for recombination [92–95]. Thus, it is probablethat functionally, epigenetic reprogramming in PGCs is aprotracted event (E7.75–E12.5) that contributes to myriadinterconnected processes that are collectively essential forgerm cell potency and development.

Concluding remarksConsiderable advances have been made in understandinghow new epigenetic information can be introduced, but lessis known about the mechanisms that can erase existingmodifications. Studies on PGCs provide an unprecedentedopportunity to unravel the role of key factors and theircombined roles in resetting the epigenome. Fundamentalknowledge gained from these studies may potentially findwider application. Somatic cells are prone to epimutationsthrough ageing and environmental factors, whereas thegerm line can reset the epigenome with the potential to‘rejuvenate’ adult cells. Insights from research on earlygerm cells may provide knowledge and tools for applica-tions in ageing-related diseases and generally improveability to manipulate cell fates for applications in repro-ductive and regenerative medicine.

AcknowledgementsWe would like to thank Roopsha Sengupta and Harry Leitch for criticalreading of the manuscript and to apologise to the authors whose workcould not be cited here owing to space constraints. JAH was funded by aWellcome Trust Grant and JJZ is the recipient of a Wellcome Trust PhDScholarship. MAS is supported by The Wellcome Trust (RG49135).

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parallel mechanisms of epigenetic reprogramming in the germline

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