ReviewParallel mechanisms of epigeneticreprogramming in the germlineJamie A. Hackett, Jan J. Zylicz and M. Azim SuraniWell...
Review                                                                              Trends in Genetics April 2012, Vol. 28...
Review                                                                                                               Trend...
Review                                                                              Trends in Genetics April 2012, Vol. 28...
Review                                                                                                                    ...
Review                                                                              Trends in Genetics April 2012, Vol. 28...
Review                                                                             Trends in Genetics April 2012, Vol. 28,...
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Parallel mechanisms of epigenetic reprogramming in the germline


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Germ cells possess the extraordinary and unique capacity to give rise to a new organism and create an enduring link between all generations. To acquire this property, primordial germ cells (PGCs) transit through an unprecedented programme of sequential epigenetic events that culminates in an epigenomic basal state that is the foundation of totipotency. This process is underpinned by genome-wide DNA demethylation, which may occur through several overlapping pathways, including conversion to 5-hydroxymethylcytosine. We propose that the epigenetic programme in PGCs operates through multiple parallel mechanisms to ensure robustness at the level of individual cells while also being flexible through functional redundancy to guarantee high fidelity of the process. Gaining a better understanding of the molecular mechanisms that direct epigenetic reprogramming in PGCs will enhance our ability to manipulate epigenetic memory, cell-fate decisions and applications in regenerative medicine.

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Parallel mechanisms of epigenetic reprogramming in the germline

  1. 1. ReviewParallel mechanisms of epigeneticreprogramming in the germlineJamie A. Hackett, Jan J. Zylicz and M. Azim SuraniWellcome Trust/Cancer Research UK Gurdon Institute and Department of Physiology, Development and Neuroscience,University of Cambridge, Cambridge, CB2 1QN, UKGerm cells possess the extraordinary and unique capac- [5,6]. This process is initiated in response to localised sig-ity to give rise to a new organism and create an enduring nals, including BMP4 and WNT3, which direct activation oflink between all generations. To acquire this property, the key transcriptional regulators B-lymphocyte-inducedprimordial germ cells (PGCs) transit through an unprec- maturation protein 1 (Blimp1) and PR domain containingedented programme of sequential epigenetic events that 14 (Prdm14) in competent epiblast cells [1,7–9]. Lineage-culminates in an epigenomic basal state that is the restricted PGCs then embark on an orchestrated sequencefoundation of totipotency. This process is underpinned of reprogramming that culminates in a basal epigeneticby genome-wide DNA demethylation, which may occur state. The number of early PGCs is highly restricted (ap-through several overlapping pathways, including con- proximately 40 by E7.25) so it is crucial that the complexversion to 5-hydroxymethylcytosine. We propose that series of epigenetic events be robust to ensure that most, ifthe epigenetic programme in PGCs operates through not all, cells efficiently transit through the process [10].multiple parallel mechanisms to ensure robustness at Reprogramming must also proceed rapidly because of strictthe level of individual cells while also being flexiblethrough functional redundancy to guarantee high fideli- Glossaryty of the process. Gaining a better understanding of the 5-hydroxymethylcytosine (5hmC): oxidation of methylated cytosines (5mC) bymolecular mechanisms that direct epigenetic repro- TET proteins generates 5hmC, which may be an intermediate during DNAgramming in PGCs will enhance our ability to manipu- demethylation and can be further converted to 5caC and 5fC. 5hmC is enrichedlate epigenetic memory, cell-fate decisions and in pluripotent and some neuronal cell types, but its precise functional consequences in the genome and its role in DNA demethylation are unclear.applications in regenerative medicine. Base excision repair (BER): a cellular mechanism for repair of nonhelix- distorting base mutations or lesions in the genome. BER is initiated by a DNAReprogramming PGCs towards totipotency glycosylase (e.g. TDG) that removes inappropriate bases and forms anDevelopment from the zygote to adulthood is characterised apurinic/apyrimidinic (AP) site, which is then cleaved by an AP endonuclease and repaired by specific lyases and polymerases. BER may function to removeby a progressive restriction of cellular potential that gives downstream derivatives of 5mC, such as 5caC, and mediate repair torise to all the differentiated somatic cell types. A unique unmodified C [60]. Basal epigenetic state: the unique epigenetic state of PGCs followingexception to this unidirectional process occurs in the germ- reprogramming. By E13.5, the PGC epigenome has undergone extensiveline, where an unprecedented reprogramming event in reorganisation of histone modifications and is stripped of genome-wide DNAPGCs (see Glossary) reverses epigenetic barriers to plas- methylation, rendering it at its most basal level during the mammalian life cycle.ticity and resets genomic potential. Reprogramming in Bisulfite sequencing: a technique used to determine the pattern of allelic DNAPGCs results in chromatin remodelling, erasure of genomic methylation (5mC) at specific genomic regions. Bisulfite sequencing cannotimprints and extensive DNA demethylation [1]. This pro- distinguish 5mC from 5hmC [33]. Additionally, 5caC is indistinguishable from unmodified C by bisulfite sequencing [60].cess represents the most comprehensive erasure of epige- DNA demethylation: the removal of a methyl group from position 5 of anetic information in the mammalian life cycle and cytosine base (5mC), which usually resides within a CpG genomic context, tounderpins the totipotent state. Therefore, unravelling generate an unmodified C. DNA demethylation may occur through either a ‘passive’ mechanism that relies on replication-dependent dilution or an ‘active’the mechanisms that drive reprogramming, particularly process driven by enzymatic replacement independently of DNA replication. AsDNA demethylation, in the unique context of PGCs is of DNA methylation is associated with transcriptional silencing, DNA demethyla-great interest. tion can generate a transcriptionally competent state. Epigenetic reprogramming: genome-wide reorganisation of epigenetic mod- In mice, PGCs are specified from a subset of posterior ifications that overcomes stable epigenetic barriers and enables acquisition ofproximal epiblast cells at approximately embryonic day (E) genomic potential. During the mammalian life cycle, epigenetic reprogram- ming occurs in PGCs and in early zygotic development.6.25, resulting in the establishment of a founder population Genomic imprints: genomic sequences that exhibit differences in CpG methyla-of PGCs at E7.25 [1,2]. These nascent PGCs subsequently tion according to the parent of origin. These differentially methylated regionsmigrate to the genital ridges by approximately E10.5 and, (DMRs) can influence the allele-specific expression of one or more genes. Primordial germ cell (PGC): the precursors of mature germ cells that arefrom E12.5 onwards, they undergo sex-specific development specified during post-implantation development. In vivo, PGCs are restrictedin the gonads [3,4]. Because mammalian PGCs are specified as a unipotent lineage and only give rise to gametes, which generate thefrom cells that are already primed towards a somatic fate, totipotent state upon fertilisation. Early PGCs also possess an underlying genomic plasticity, as evidenced through their capacity to form pluripotent EGnascent PGCs must both repress the ongoing somatic pro- cells upon in vitro culture.gramme and activate the germ cell transcriptional network Totipotency: the ability of a cell to give rise to all the cell types of the embryonic and extra-embryonic lineages. By contrast, pluripotency refers to the capacity of a cell to generate all the cell types of the embryo. Corresponding author: Surani, M.A. ( 0168-9525/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2012.01.005 Trends in Genetics, April 2012, Vol. 28, No. 4
  2. 2. Review Trends in Genetics April 2012, Vol. 28, No. 4temporal constraints imposed by the entry of male germ sperm or oocytes in vivo, during migration (E8.5–E11.5),cells into mitotic arrest and female germ cells into meiotic PGCs show multiple transcriptional and epigenetic simi-arrest at approximately E13.5. To overcome these con- larities to pluripotent ES cells. Indeed, E8.5–E11.5 PGCsstraints, epigenetic reprogramming in PGCs is probably can form pluripotent embryonic germ (EG) cells in vitro,directed by multiple parallel mechanisms that ensure the which resemble ES cells rather than the post-implantationfidelity, flexibility and efficiency of this fundamental pro- epiblast cells from which PGCs were originally specifiedcess. As such, events like genome-wide erasure of DNA [19,20]. It is possible that this epigenetic state of PGCs ismethylation in PGCs may occur through several intercon- an underlying requirement for the initiation of meiosisnected mechanisms, including both active and passive path- and the eventual acquisition of totipotency in the zygote.ways, that collectively confer redundancy and hence Interestingly, migrating PGCs additionally exhibit upre-robustness to PGC reprogramming and development. gulation of histone H2A/H4 arginine 3 symmetrical meth- Here, we discuss the epigenetic events in PGCs and ylation (H2A/H4R3me2 s), which is catalysed by proteinthe mechanisms that may operate to drive epigenetic arginine methyltransferase 5 (PRMT5) [21]. This modifi-reprogramming. We suggest that an integrated process cation may contribute to maintaining PGCs in a unipotentinvolving parallel systems is at play and consider the state in vivo and to repression of the somatic programmepotential pathways of DNA demethylation. We also high- [22].light some of the potential roles and developmental Changes in histone modifications occur in parallel withprocesses that epigenetic reprogramming contributes to a reported reduction in global levels of DNA methylationin PGCs. (5mC) in migrating PGCs from approximately E8.0 [11]. Any loss of 5mC might reflect the effects of BLIMP1 andEpigenetic events in PGCs PRDM14, which repress both DNA (cytosine-5)-methyl-At the point at which PGCs are specified from post-im- transferase 3a and 3b (Dnmt3a and Dnmt3b) and ubiqui-plantation epiblast cells, they are epigenetically indistin- tin-like, containing PHD and RING finger domains 1guishable at the global level from their neighbours, which (Uhrf1), which are essential components of the de novoare destined for a somatic fate [9,11]. Therefore, nascent and maintenance methylation machinery, respectivelyPGCs inherit stable epigenetic states, including DNA (Figure 1) [7,8,23–26]. Additionally, repression of GLPmethylation and X-inactivation, which constitute an epi- may directly affect DNA methylation through a parallelgenetic barrier against the eventual acquisition of totipo- mechanism that is both dependent and independent oftency [1,12]. It is thus an important early step in PGC H3K9me2 [27,28]. However, most analysed genomicdevelopment to initiate a process of reprogramming that regions, including transposable elements, imprinted locierases these stable epigenetic blocks. The first gross epi- and single-copy genes, apparently retain DNA methylationgenetic changes in PGCs entail a reciprocal loss of histone at CpG sites in PGCs until at least E10.5 [29–32], althoughH3 lysine 9 dimethylation (H3K9me2) from E7.75 and, in this does not exclude the possibility that there is conver-most PGCs, a global increase of H3 lysine 27 trimethyla- sion of 5mC to 5-hydroxymethylcytosine (5hmC) [33].tion (H3K27me3) by E9.5 (Figure 1) [11,13,14]. The ge- Therefore, it is unclear to what extent DNA demethylationnome-wide depletion of H3K9me2 is potentially a contributes to the early stages of epigenetic reprogram-consequence of the downregulation of GLP, a methyltrans- ming (from E8.0) in PGCs and whether conversion to 5hmCferase that forms a heteromeric complex with G9a (also plays a role. Conversely, it is unclear how 5mC (or 5hmC) isknown as EHMT2) that is required for deposition of H3K9 maintained at analysed genomic regions in migratingmono- and dimethylation, and the parallel upregulation of PGCs given that essential components of the de novospecific lysine demethylases ([15] and unpublished obser- and maintenance machinery, particularly UHRF1, arevations). By contrast, the mechanisms responsible for the absent [7]. One possibility is that UHRF2, which is con-increased H3K27me3 levels in PGCs remain unclear, al- served with UHRF1 at the sequence level and preferen-though notably enhancer of zeste homologue 1 (Ezh1), tially binds hemi-methylated DNA associated withwhich has H3K27me3 methyltransferase activity, is upre- H3K9me3, can compensate for UHRF1 to maintain DNAgulated in PGCs [16]. Because H3K9me2 and H3K27me3 methylation in PGCs, either globally or at specific loci [34].marks are both associated with transcriptional repression, In support of this, UHRF2 is specifically upregulatedit has been postulated that the loss of H3K9me2 is com- during PGC specification, at least in vitro [7,35]. Addition-plemented by the gain of H3K27me3 to maintain a repres- ally, unlike H3K9me2, global H3K9me3 levels are main-sive chromatin state in PGCs [17]. However, the precise tained in migrating PGCs [14]. The in vitro PGC-like cellsgenomic location and relationship between these epigenet- (PGCLC) generated in an elegant recent study may enableic changes remains to be determined. Nonetheless, the further mechanistic insights into epigenetic events in na-global enrichment of H3K27me3 and loss of H3K9me2 scent PGCs at a higher resolution [36].establishes a chromatin environment in PGCs that is The early stages of epigenomic reorganisation in PGCsgrossly similar to that in pluripotent embryonic stem are followed by, and are probably a prerequisite for, the(ES) cells and is coupled to upregulation of pluripotency dramatic genome-wide erasure of DNA methylation andgenes, such as Nanog and SRY Sox2 [1]. Additionally, extensive chromatin remodelling subsequent to entry intounlike global H3K27me3 levels, the inactive X-chromo- the genital ridges at approximately E10.5. Because grosssome (Xi) exhibits a protracted decline in H3K27me3 in DNA demethylation seems to occur rapidly at a distinctfemale PGCs, which is linked to initiation of X-reactivation time point, while most PGCs are in G2 phase, it is held that[18]. Thus, while PGCs are unipotent and only form either this process is an ‘active’ event occurring independently 165
  3. 3. Review Trends in Genetics April 2012, Vol. 28, No. 4 (a) Chromatin dynamics 5mC H3K27me3 Relative levels H3K9 me2 E5.5 E6.5 E8.0 E10.5 E11.5 (b) Specification and chromatin-modifying factors Blimp1 Relative expression Prdm14 G9a Glp E5.5 E6.5 E8.0 E10.5 E11.5 (c) DNA methylation factors Uhrf2 Relative expression Dnmt1 Dnmt3a Dnmt3b Uhrf1 E5.5 E6.5 E8.0 E10.5 E11.5 (d) Tet dioxygenases Relative expression Tet1 Tet2 Tet3 E5.5 E6.5 E8.0 E10.5 E11.5 Spec. R1 R2 TRENDS in GeneticsFigure 1. Chromatin and transcriptional changes in primordial germ cells (PGCs). Following specification (Spec.), PGCs transit through two sequential phases ofreprogramming during migration (R1) and subsequent to entry into the genital ridge (R2). (a) Epigenomic reorganisation in PGCs. At R1, PGCs exhibit global erasure ofhistone 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 furtherdramatic loss of DNA methylation, which includes erasure of imprints. This correlates with a transient reorganisation of H3K27me3, loss of heterochromatic chromocentresand remodelling of other chromatin modifications, including histone H2A/H4 arginine 3 symmetrical methylation (H2A/H4R3me2; not shown). (b–d) Transcriptional changesduring PGC development [13]. (b) Specification and chromatin-modifying factors. Upregulation of B-lymphocyte-induced maturation protein 1 (Blimp1) and PR domaincontaining 14 (Prdm14) is necessary for specification of PGC fate, whereas the parallel downregulation of glucagon-like peptide (Glp)/G9a and upregulation of lysinedemethylases (not shown) contribute to erasure of H3K9me2 at R1. (c) DNA methylation proteins. Downregulation of DNA (cytosine-5)-methyltransferase 3b (Dnmt3b) andubiquitin-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 tomaintain 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 is [29,32]. The process of DNA methylation erasure alsoyet to be elucidated [14]. Reprogramming in PGCs at this initiates a cascade of chromatin remodelling in PGCs atstage results in almost full erasure of DNA methylation by approximately E11.5. This includes a transient reorgani-E13.5 [37], with complete stripping of parental imprints sation of linker histone H1, H3K27me3 and H3K9me3 andand promoter CpG methylation at germline-specific genes stable remodelling of global H3K9ac and H2A/H4R3me2 s166
  4. 4. Review Trends in Genetics April 2012, Vol. 28, No. 4[14]. Some sequences, including intracisternal A particle replace 5mC during global DNA demethylation in PGCs(IAP) retrotransposons, partially evade DNA demethyla- [50].tion in PGCs, although the mechanism that protects these Mechanistically, conversion of 5mC to 5hmC could leadelements remains to be determined [31]. Nevertheless, the to unmodified cytosine through several routes in PGCs,erasure of DNA methylation and extensive chromatin including through providing a substrate for base excisionreorganisation at approximately E11.5 renders PGCs in repair (BER)-mediated active demethylation [40]. Severala basal epigenetic state that represents an epigenomic recent studies have linked BER components to demethyl-nadir during mammalian development. It is of note that ation during zygotic reprogramming and at specific loci inthe apparent biphasic nature of demethylation in PGCs (at somatic contexts [50–52]. As BER components are alsoapproximately E8.0 and approximately E11.5) may poten- upregulated in PGCs relative to somatic neighbours duringtially represent a continuous process of 5mC erasure. In epigenetic erasure, genome-wide demethylation in PGCsany case, reprogramming of the PGC genome as a whole is may also occur, at least partially through BER. Indeed, thedistinguished from reprogramming during zygotic devel- presence of chromatin-associated XRCC1 and active poly[-opment, where imprints, DNA methylation at multiple loci ADP-ribose] polymerase 1 (PARP1) in PGCs at approxi-and polycomb-based chromatin modifications remain; this mately E11.5, which signify single-strand DNA breaksimplies that reprogramming in PGCs represents a more associated with BER, further support this possibility [50].extensive process [32]. Although both reprogramming One potential involvement of BER-mediated DNA de-events probably share some mechanistic similarities, it methylation in PGCs is that TET-generated 5hmC is furtheris also likely that both employ unique systems to achieve processed by deamination to 5-hydroxymethyluridinedistinct levels of epigenomic resetting. (5hmU) by the activation-induced cytidine deaminase/apo- lipoprotein B mRNA-editing, enzyme-catalytic, polypeptideErasure of DNA methylation and cellular identity (AID/APOBEC) family of deaminases, and subsequentlyDNA methylation (5mC) within a CpG context is a highly excised by a glycosylase and repaired to unmodified C.heritable epigenetic mark that is associated with tran- Consistent with this, loss of AID impairs global demethyla-scriptional repression and that contributes to stable line- tion in PGCs by E13.5, indicating that 5mC erasure in PGCsage commitment [38–41]. In this respect, global erasure of depends, at least in part, on AID [37]. Indeed, AID alsoDNA methylation during PGC development is a fundamen- enhances locus-specific active demethylation mediated bytal event towards the acquisition of totipotency. The ca- TET1 in somatic cells and has been reported to interact withpacity of global DNA demethylation to alter cellular thymine DNA glycosylase (TDG), which can excise theidentity, for example in the course of derivation of induced deamination product 5hmU [52,53]. Moreover, AID haspluripotent stem cells (iPS) from somatic cells, makes been proposed to be required for DNA demethylation ofunravelling the mechanisms that mediate this process of octamer-binding transcription factor 4 (Oct4) and Nanoggreat importance [42,43]. Although extensive studies have during somatic cell reprogramming induced by heterokary-established how and where 5mC is and can be introduced, on formation with ES cells as well as in demethylation init remains enigmatic precisely how DNA methylation is zebrafish embryos [54,55]. However, murine PGCs stillremoved from the genome [44]. The global DNA demethyl- undergo extensive demethylation by E13.5 in the absenceation during PGC development represents a unique in vivo of AID; global 5mC is reduced to 22% and 20% in male andevent in that it results in near-complete stripping of CpG female mutant PGCs, respectively, as opposed to 16% andmethylation at almost all genomic loci, including imprints. 8% in wild-type PGCs and compared with approximatelyThe magnitude of DNA demethylation makes PGCs an 74% in E13.5 embryonic soma. Moreover, AID-mutant miceunparalleled system to understand the process of 5mC are both viable and fertile, as are APOBEC 1- and APOBECerasure in an in vivo context. 2/3-null mice [56–58]. It is also unclear whether AID is expressed to any significant degree in PGCs at the time ofActive demethylation in PGCs gross demethylation (approximately E11.5), as it has onlyThe recent identification of three 5mC-dioxygenases [ten- been detected from E12.5 in PGCs, suggesting that AID haseleven translocation gene 1, 2 and 3 (TET1, TET2 and a role in PGC demethylation either after global 5mC erasureTET3)], which can convert 5mC to 5-hydroxymethylcyto- (approximately E12.5) or perhaps at an earlier stage (ap-sine, presented a potential solution to the longstanding proximately E8.5), when the expression of AID is unknowndebate regarding the mechanism of DNA demethylation [50,59]. Likewise, TDG is not detectable in PGCs between[45,46]. Conversion of 5mC to 5hmC enables several alter- E10.5 and E13.5, as judged by immunofluorescence studiesnative but partially overlapping routes to generate an [50]. However, TDG-null PGCs accumulate biallelic CpGunmodified cytosine (C) residue independently of, or de- methylation by E11.0 at the insulin-like growth factor 2pendent on, DNA replication (Figure 2). In the zygote, the receptor (Igf2r)-imprinted differentially methylated regionrapid loss of 5mC from the male pronucleus correlates with (DMR), suggesting that TDG has a role in maintaininga concomitant gain in 5hmC, implying that 5mC is con- a methylation-free state at this locus, presumably by de-verted to 5hmC in this context and has a fundamental role methylation, although this event may occur prior to PGCin reprogramming [47,48]. Indeed, loss of TET3, which is specification [52]. Although further clarification of TDGthe only 5mC-dioxigenase significantly expressed in function and expression in PGCs is necessary, its putativezygotes, led to a failure to erase 5mC and neonatal lethality absence at E11.5 argues against a direct AID- and/or TDG-[49]. Although TET3 is not detectable in PGCs, TET1 and mediated active demethylation reaction in PGCs, as hasTET2 are present in these cells, suggesting that 5hmC may been reported in other contexts [52]. Therefore, alternative 167
  5. 5. Review Trends in Genetics April 2012, Vol. 28, No. 4 (a) C OBE 5mC / AP D AI TET1/2 C T BE 5hmC APO D/ TET1/2 AI 5hmU 5fC TET1/2 5caC ? BE ive R ss Pa (b) C 5mC Relative level Pa ss ive Ac ti ve 5hmC No conversion E5.5 E6.5 E8.0 E10.5 E11.5 Spec R1 R2 TRENDS in GeneticsFigure 2. Multiple parallel mechanisms of DNA demethylation. (a) Methylated cytosine (5mC) can be demethylated through several overlapping pathways, includingpassive and active mechanisms, which may occur in parallel. Passive demethylation (right) can occur through direct replication-dependent depletion of 5mC owing to anabsence or reduction of DNA methyltransferase activity. Alternatively, TET oncogene (TET) proteins can catalyse oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) orfurther conversion to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which may all lead to passive demethylation. Active erasure of 5mC (left) can occur throughdeamination of either 5mC or 5hmC to thymidine (T) or 5-hydroxymethyluridine (5hmU), respectively, which can act as a substrate for base excision repair (BER) tounmodified 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 aresult 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/oractive mechanisms. Abbreviations: AID/APOBEC, activation-induced cytidine deaminase/apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide; R1 and R2, the twosequential phases of reprogramming during migration (R1) and subsequent to entry into the genital ridge (R2); Spec. specification.168
  6. 6. Review Trends in Genetics April 2012, Vol. 28, No. 4deaminase–glycosylase complexes may operate down- 5hmU or further oxidation to 5fC and 5caC, all of which arestream of 5hmC (or 5mC) in PGCs. Investigation of potential BER substrates, ultimately leading to repair-driven de-candidates, such as other members of the APOBEC family, methylation.and methyl-CpG-binding domain protein 4 (MBD4), single-strand selective monofunctional uracil DNA glycosylase Passive demethylation in PGCs(SMUG1), nth endonuclease III-like 1 (NTH1) or nei endo- There is accumulating evidence that genome-wide DNAnuclease VIII-like 1 (NEIL1) glycosylases, may shed light on demethylation events include at least a partial ‘passive’the issue. component. Conversion of 5mC to 5hmC in the zygote has An alternative possibility is that TET-mediated hydro- recently been shown to lead to replication-dependent pas-xymethylation has a deamination-independent role in ac- sive demethylation, rather than to active removal of 5hmCtive demethylation in PGCs, as TET proteins can further or its derivatives [68]. Thus, chromosomes originating inoxidise 5hmC to 5-formylcytosine (5fC) and subsequently to the paternal pronucleus retain 5hmC through successive5-carboxylcytosine (5caC) [60,61]. Interestingly, these new cleavage divisions, whereas newly synthesised sister chro-C derivatives could also be targets for BER excision [60]. matids are devoid of 5mC and 5hmC, leading to a progres-Indeed, 5fC and 5caC are substrates for TDG, and poten- sive dilution of DNA modifications during development.tially other glycosylases [60,62]. 5caC could also theoreti- The passive loss of DNA modification is consistent with thecally be ‘actively’ removed from the genome by a BER- fact that the human maintenance DNA (cytosine-5)-independent pathway that would involve decarboxylation methyltransferase 1 (DNMT1) cannot recognise 5hmCby an as yet unknown enzyme. It will be important to and there are no other known 5hmC maintenance mecha-determine the prevalence of 5fC and 5caC in PGCs during nisms (although notably UHRF1 does efficiently recognisethe key stages of reprogramming to establish whether they 5hmC) [69,70]. Nevertheless, bisulfite sequencing has in-contribute to demethylation through any mechanism. A dicated there may be an additional active mechanism infurther possibility is that 5hmC is directly targeted for zygotes that is dependent on BER and operates prior toBER-mediated excision by 5hmC-specific glycosylases with- DNA replication, which may target specific genomic land-out the requirement for processing by deamination or fur- marks [51]. Additionally, the high sensitivity of the 5hmCther oxidation. Indeed, 5hmC glycosylase activity has been antibody may mask partial or locus-specific active erasurereported in calf thymus extract [63]. Taken together, a of 5hmC on the paternally derived pronuclear chromatidsmechanism based on BER may play at least a partial role [71]. However, it seems probable that the bulk of paternalin PGC demethylation, although it is currently unclear pronuclear demethylation occurs passively followingwhich protein complexes direct the process or whether there 5hmC conversion. It remains unclear how the maternalis a degree of redundancy. Likewise, it is not clear which pronucleus, which does not undergo 5mC to 5hmC conver-5hmC or 5mC derivative substrate [5hmC, 5hmU, 5fC, 5caC sion, undergoes concomitant passive demethylation, asor thymidine (T)] might be targeted by the BER machinery DNMT1 is present and sufficient to maintain maternalfor active DNA demethylation in PGCs. imprints [68]. Nonetheless, these studies indicate that It is also possible that alternative mechanisms, indepen- conversion of 5mC to 5hmC coupled with passive demeth-dent of 5hmC, contribute to, or drive, active demethylation ylation is feasible and could operate in PGCs. In Arabidopsis thaliana, demethylation occurs via In murine PGCs, although there is an overall loss ofdirect excision of 5mC by the specific bifunctional DNA DNA methylation over a relatively short period at approx-glycosylase/lyases, DEMETER (DME) and REPRESSOR imately E11.5, it is important to reconsider whether era-OF SILENCING 1 (ROS1), followed by recruitment of sure of imprinted genes can occur over a protracted periodBER machinery [64,65]. Although no mammalian orthologs of 2 days [30], which would not be compatible with a singleof DME–ROS1 or 5mC-specific glycosylases have been de- genome-wide wave of ‘active’ demethylation model [14].scribed, it remains a formal possibility that 5mC is directly Indeed, erasure of DNA methylation from imprinted lociexcised from the genome in PGCs. Another possibility is that and repeat elements in porcine PGCs occurs over an ex-5mC is deaminated directly to T followed by excision by a tended period of up to 20 days, which suggests that ‘active’glycosylase that recognises the T–G mismatch, such as TDG DNA demethylation does not apply in all mammalianor MBD4, and repaired to unmodified cytosine by BER. It is PGCs, possibly including those in mice [72]. Similarly toadditionally possible that a demethylation route based on zygotes, conversion of 5mC to 5hmC (or 5fC and 5caC) inradical sterile alpha motif (SAM) enzymes operates, al- PGCs would facilitate passive replication-dependent de-though experimental evidence for this is lacking in PGCs methylation (Figure 2). Such a conversion of 5mC to 5hmC[66]. Similarly, active 5mC erasure has been reported to be would account for the dramatic loss of 5mC staining ob-linked to the nucleotide excision repair (NER) pathway, and served in PGCs at E11.5 and the protracted nature ofthis could contribute to DNA demethylation during PGC imprint erasure, given that bisulfite conversion wouldmigration (at approximately E8.5–E10.5) [67]. However, it identify hydroxymethylated loci as methylated but 5mCremains unclear whether such a mechanism could operate antibodies would not [14,30,33]. Interestingly, conversionin PGCs at approximately E11.5 owing to the absence of of 5mC to 5hmC may also skew bisulfite sequencingseveral key NER components between E10.5 and E12.5 [50]. results, as polymerases appear to amplify unmethylatedThus, the consensus of available data points towards a alleles preferentially over 5hmC-modified alleles after bi-putative active DNA demethylation mechanism in PGCs sulfite treatment, which may falsely imply demethylationat approximately E11.5 being based on initial TET-mediat- has occurred rather than conversion to 5hmC [73]. Addi-ed hydroxylation of 5mC and either deamination towards tionally, TET-mediated conversion to 5hmC may proceed 169
  7. 7. Review Trends in Genetics April 2012, Vol. 28, No. 4to 5caC in PGCs, which is read as unmethylated by bisul- [49]. This suggests that rapid epigenetic reprogramming isfite sequencing, erroneously indicating the occurrence of involved in diminishing developmental barriers that someactive demethylation [60]. Although functional conse- cells can nevertheless still overcome in a stochastic man-quences of 5hmC, 5caC or other C derivatives are as yet ner. Similarly, Tet1-null mice sire reduced litter sizes andunclear, the inherent biases and lack of derivative speci- mutant pups have a smaller body size at birth, potentiallyficity of bisulfite sequencing mean that observing an owing to a delay in overcoming epigenetically imposedunmethylated allele via this technique does not necessarily barriers [77]. The presence of multiple means of 5mCsignify the presence of unmodified C or the implied active erasure could compensate for the loss of function of Tet1demethylation. As such, conversion to 5hmC or down- through the use of alternative routes in a stochastic man-stream C derivatives could at least partially account for ner. For example, given that key maintenance-methylationthe apparent active ‘replication-independent’ demethyla- enzymes, including UHRF1, are absent in PGCs, it istion that has been reported in PGCs. possible that direct passive demethylation of 5mC can An alternative to passive erasure through 5hmC con- partly offset the absence of any putative 5hmC-mediatedversion is that PGCs undergo direct passive dilution of mechanisms, a possibility that might also affect interpre-5mC, based on an absence or exclusion of maintenance tation of the demethylation that still occurs in AID-defi-components, such as DNMT1 or UHRF1. As the doubling cient PGCs by E13.5 [7,37].time of PGCs at this stage is approximately 16 hr [13,74], In addition to contributing to a robust system, paralleldirect replication-dependent dilution could account for a mechanisms of 5mC erasure could function to target spe-significant reduction in global 5mC in PGCs between E10.5 cific genomic loci in PGCs. For example, passive demeth-and E11.5. Because the 5mC antibody (33D3) exhibits ylation could account for the bulk of demethylation,relatively weak avidity (e.g. as compared to the 5hmC analogously to the zygote, whereas erasure of imprintedantibody [71]) and 5mC levels at E10.5 may already be regions may be targeted by specific active mechanisms thatpartially depleted relative to somatic neighbours, this are absent in the zygote, perhaps based on BER. Interest-passive reduction of 5mC may appear as a largely complete ingly, targeting specific loci with distinct demethylationand, therefore, active erasure of global DNA methylation, mechanisms may also lead to particular functional out-while there would also be a significant loss of methylation comes, perhaps based on the specific C derivative (5hmC,by bisulfite sequencing. Although it is not clear precisely 5fC, 5caC, etc.) that directed demethylation. This couldhow a passive demethylation mechanism might be trig- have an important functional role during meiosis, similarlygered, the existence of such a system would circumvent the to the role of inherited histone modifications at develop-potential genetic damage that may result from genome- mental loci in the zygote [81]. Active DNA demethylationwide BER. mechanisms could also operate in parallel to a passive system to initiate DNA repair-driven chromatin remodel-Parallel mechanisms of epigenetic reprogramming ling [14]. Notably, other phases of epigenetic reprogram-Although cumulative evidence indicates that several mo- ming in PGCs also utilise parallel systems. For example,lecular pathways of DNA demethylation may operate, erasure of H3K9me2 in early PGCs may occur throughthe precise mechanism(s) that mediate the comprehen- both downregulation of the methyltransferase GLP andsive methylation erasure in PGCs remain to be clarified upregulation of specific lysine demethylases at approxi-[44,75,76]. Indeed, DNA demethylation and chromatin mately E8.5, which potentially contribute to erasure of thisremodelling in PGCs may occur through several comple- modification in a redundant manner. Similarly, the histonementary parallel pathways, including active and passive replacement that is associated with reorganisation of glob-systems, which would provide a degree of redundancy al H3K27me3 and H3K9me3 marks in PGCs at approxi-and confer robustness and flexibility to the programme mately E11.5 also occurs in parallel with upregulation of(Figure 3). This seems necessary because of the funda- specific lysine demethylases, which may contribute to themental importance of epigenetic reprogramming to germ efficient erasure. Indeed, both Tet1 and Tet2 are alsocell development. Furthermore, there are very limited upregulated at this stage, indicating a possible parallelnumbers of PGCs and most, if not all, of them must role [50]. Thus, we propose that the unique importance oftransit through the process efficiently and prior to the reprogramming in PGCs necessitates a system of multipleentry of female germ cells into meiosis shortly after epigenetic erasure mechanisms to confer efficiency, fidelityreprogramming. and robustness to the process. A robust system based on multiple parallel mechanismsand inbuilt redundancy may account for the observation Potential roles of epigenetic reprogramming in PGCsthat Tet1-null and Tet2-null mice are viable and fertile, The fundamental role of epigenetic reprogramming indespite a probable role for 5hmC in demethylation in PGCs PGCs is to overcome multiple epigenomic barriers to the[77–80]. Although functional redundancy coupled with eventual acquisition of totipotency acquired by epiblastgenetic background effects may explain this observation, cells during early development. This is necessary becauseit is also possible that impeded DNA demethylation in mammals utilise an inductive mechanism of germ cellTET-deficient PGCs would not cause complete loss of the specification (they specify the germline from cells primedcells but rather a reduction in their numbers. In support of towards a somatic fate) rather than acquire germ cell fatethis, impeded demethylation of the paternal genome in through an inherited germplasm. This essential role ofTET3-deficient zygotes is only associated with a severe reprogramming in mammals is evident in mice lacking thedevelopmental phenotype in a subset of mutant embryos transcriptional regulator PRDM14. Nascent PGCs in170
  8. 8. Review Trends in Genetics April 2012, Vol. 28, No. 4 t 3 a / b , U h r f 1 d o wn r e Dnm gu l a t io n T ET 1 / 2 a c t i v i t y Deamination 5mC 5mC 5mC Me e Pa s M sive Ac BE R Ac e Me M Reprogramming DNA demethyla M e Ac Primed state tion H M T/ Histo HDM HA ne e /HD Td xch AC ow an nr up eg ge reg ul ati ula on on ti His ton em ark r eprog M Ac ramming e Progress ion toward Basal sta s te TRENDS in GeneticsFigure 3. Parallel routes towards the basal epigenetic state. Post-implantation epiblast cells acquire epigenetic modifications that constitute a barrier against their reversionto 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 ofreprogramming that overcomes the epigenetic barrier and establishes a basal epigenetic environment that underpins totipotency. Multiple parallel mechanisms contributeto epigenetic reprogramming in PGCs to ensure robustness and redundancy to the process. Global erasure of DNA methylation (upper routes) can occur through passive oractive mechanisms directed through several interconnected processes. Likewise, reorganisation of chromatin architecture (lower routes) can occur through the parallelupregulation 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 reprogramming RNA Xist necessary to complete the process [18,82]. Theof chromatin and DNA methylation or to activate the germ wave of global DNA demethylation in PGCs at approxi-cell genetic programme and consequently exhibit severely mately E11.5 seems to be a trigger for several otherimpaired PGC development and are sterile [8]. Evidence important processes, including the erasure of parentalshows that PGC specification and epigenetic reprogram- imprints to enable establishment of new methylationming are intimately linked. Furthermore, within the broad marks according to the sex of the embryo [17,29]. Theremit of establishing a state of ‘underlying totipotency’, significant impact that aberrant imprints have on mam-epigenomic reprogramming has many distinct roles that malian development, foetal growth and behaviour, and incollectively contribute to a continuous process of PGC human disease, has led to the suggestion that the wholedevelopment (Figure 4). process of genome-wide demethylation in PGCs reflects a An important event driven by reprogramming is reacti- byproduct of the necessity to reset locus-specific imprints.vation of Xi in female PGCs. This requires the integration Indeed, as imprinted loci are resistant to demethylationof multiple genetic and epigenetic systems; with early during zygotic reprogramming [82], they may have intrin-chromatin reorganisation initiating X-reactivation and sic or epigenetic properties that preclude erasure exceptDNA demethylation and repression of the long noncoding under exceptional cellular conditions, such as exists in 171
  9. 9. Review Trends in Genetics April 2012, Vol. 28, No. 4 (a) Removal of epigenetic barriers to totipotency (b) X-chromosome reactivation (c) Epimutation erasure (d) Germline gene reactivation (e) Imprint erasure (f) Retrotransposon reactivation (g) Setting for meiosis R1 R2 ~E7.75–9.0 ~E10.5–12.5 TRENDS in GeneticsFigure 4. Potential functions of epigenetic reprogramming in primordial germ cells (PGCs). The distinct roles of epigenetic reprogramming in PGCs and the time point atwhich each occurs (rectangles). (a) Epigenetic reprogramming events during PGC migration (R1) and post-migration (R2) overcomes the layers of epigenetic modificationsacquired by epiblast cells, which form a barrier to acquisition of totipotency. (b) In female cells, reprogramming contributes to X-chromosome reactivation. (c) Erasure ofepigenetic marks in PGCs also prevents perpetuation of epimutations through the germline. (d) Global DNA demethylation at R2 triggers activation of stably silenced genesnecessary for germ cell development (e) A fundamental role of reprogramming is to erase parent-of-origin imprints, thereby enabling subsequent sex-specific methylationmarks to be established during gametogenesis (f) Erasure of methylated cytosine (5mC) may permit transient expression of retrotransposons, which are subsequentlytargeted 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 other transient activation of TEs during reprogrammingimportant functions, including the activation of stably enables silencing mechanisms to target repressive chro-silenced genes that are required for germ cell development matin, possibly directed by small RNAs derived from[32,83]. Genes such as deleted in azoospermia-like (Dazl) expressed TEs [86]. This putative mechanism would en-and synaptonemal complex protein 3 (Sycp3) are robustly sure that harmful genetic elements that may have escapedsilenced by methylated CpG-dense promoters during post- repressive mechanisms are rendered transcriptionallyimplantation development, potentially to prevent ectopic quiescent throughout germ cell maturation. Notably,activation that may drive malignant tumour growth, but the transient erasure of the PGC epigenome may alsoare activated by demethylation specifically in the germline provide an opportunity for epigenetic writers to access[32,39,84,85]. Another key role of reprogramming in PGCs chromatin and establish new modifications that directis to erase aberrant epigenetic information or epimuta- events that are crucial for meiosis. Indeed, the absencetions. Erasure of such modifications in the zygote and of some chromatin-modifying proteins, including G9a,particularly in PGCs prevents their inheritance and per- MLL2, SUV39H1, SUV39H2, LSH and PRDM9, onlypetuation through successive generations with potentially manifests in germ cells during meiosis [87–91]. One in-detrimental effects. triguing possibility is that epigenetic erasure is necessary Global DNA demethylation may generate an epigenetic to promote chiasmata formation at recombination hot-environment susceptible to expression of harmful trans- spots during meiosis. Here, reprogramming may enableposable elements (TE), yet paradoxically it is possible that the lysine methylase PRDM9 access to target H3K4me3172
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