REPRODUCTIONREVIEW

Genomic imprinting in germ cells: imprints are under control

Philippe Arnaud Institut de Ge´ne´tique Mole´culaire, CNRS UMR-5535 and Universite´s de Montpellier, 1919, route de Mende, 34090 Montpellier, France Correspondence should be addressed to P Arnaud; Email: [email protected]

Abstract

The cis-acting regulatory sequences of imprinted loci, called imprinting control regions (ICRs), acquire specific imprint marks in germ cells, including DNA methylation. These epigenetic imprints ensure that imprinted are expressed exclusively from either the paternal or the maternal allele in offspring. The last few years have witnessed a rapid increase in studies on how and when ICRs become marked by and subsequently maintain such epigenetic modifications. These novel findings are summarised in this review, which focuses on the germline acquisition of DNA methylation imprints and particularly on the combined role of primary sequence specificity, configuration, non-histone and transcriptional events. Reproduction (2010) 140 411–423

Introduction their imprinted status in several mammalian species (if known) can be found elsewhere (see ‘Websites of In the mid-1980s, elegant embryological studies in the interest’ section). Briefly, many imprinted genes have mouse demonstrated the functional non-equivalence of roles in the regulation of foetal and/or placental growth the maternal and paternal genomes. Specifically, con- and development. Others play key roles in neurological ceptuses derived from zygotes containing either two sets pathways and in behaviour (Smith et al. 2006, Wilkinson of the maternal or two sets of the paternal et al. 2007). As a consequence, perturbations in imprinted chromosomes failed to develop beyond mid-gestation gene expression are an important cause of several growth (Barton et al. 1984, McGrath & Solter 1984). These and behavioural syndromes in humans including the findings demonstrated that normal embryonic develop- Silver–Russell, Beckwith–Wiedemann, Prader–Willi and ment requires both a maternal and a paternal genome Angelman syndromes (Hirasawa & Feil 2010). and suggested the existence of genes whose expression The allele-specific expression of imprinted genes (and depends on whether they are inherited from the mother more broadly of imprinted loci) is regulated by or from the father. This specific regulation of gene epigenetic modifications that differentially mark the expression was named ‘genomic imprinting’, and the parental alleles as either active or repressed at discrete genes involved was named as ‘imprinted genes’. Since cis-acting elements known as imprinting control regions then, more than 100 of imprinted genes have been (ICRs). These regions exhibit patterns of parental allele- identified in the mouse, and the imprinting of many of specific DNA methylation, which are essential for them, though not of all, is conserved in human as well. orchestrating mono-allelic gene expression. The last About half of the imprinted genes are expressed from the few years have witnessed a rapid increase in studies on maternal allele and the other half from the paternal how and when ICRs become marked by epigenetic allele. Imprinted genes are found throughout the modifications. These recent findings are described in this genome. Some of them appear to be isolated, but the review with emphasis on the mechanisms that are majority of them reside in domains that cover hundreds involved in the acquisition of imprints in the germlines. to a thousand of kilobases. These imprinted clusters comprise both maternally and paternally expressed genes as well as non-imprinted genes, and usually, Differentially methylated regions control imprinting they contain at least one non-coding RNA (Fig. 1). Virtually all examined imprinted loci contain a differen- Genomic imprinting is conserved among viviparous tially methylated region (DMR) of up to several kilobases mammals, especially eutherians, and, to a lesser extent, in size that is characterised by DNA methylation marks in marsupials. Imprinting has not been detected in egg- inherited from the male or the female gamete (the laying monotremes (Renfree et al. 2009). Up-to-date germline DMRs). To date, 21 of such germline DMRs repertoires of imprinted genes, their diverse functions and have been identified in the mouse (Fig. 1), and indirect

q 2010 Society for Reproduction and Fertility DOI: 10.1530/REP-10-0173 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 06:42:56PM via free access 412 P Arnaud

Imprinted Chrom Localisation of Parental origin ICR ? Associated imprinted genes and non-coding RNA References Locus Germline of methylation (germline DMR/ICR) DMR Zdbf2 1 10 kb upstream Zdbf2 Paternal ND Kobayashi et al. (2009) Exon1 Mcts2 2 Mcts2 Promoter/Exon1 Maternal ND H13 Wood et al. (2007) GnasXL/Nespas promoter Coombes et al. (2003), and Gnas 1A promoter ICR Liu et al. (2000), and Gnas 2 Maternal (GnasXL / Gnasxl; Nesp; Nespas; Gnas exon1A (domain containing two NespasDMR) Williamson et al. germline DMRs) (2004, 2006) Peg10 6 Peg10/Sgce promoter Maternal ND Sgce; Ppp1r9a; Pon1; Pon2; Pon3; Asb4, Calcr, TpfI2 Ono et al. (2003) Mest (Peg1) 6 Mest promoter/exon1 Maternal ND Klf14; Copg2; Copg2as; Mirn335 Lucifero et al. (2004) Nap1L5 6 Nap1L5 promoter/exon1 Maternal ND Woods et al. (2007) Peg3 7 Peg3 promoter/exon1 Maternal ND Zim1; Zim2; Zim3; Usp29; Zfp264 Kim et al. (2003) Snrpn 7 Snrpn promoter/exon1 Maternal ICR Atp10a; Ube3a; Snurf; Ndn; Magel2; Mkrn3; Pec2; Shemer et al. (1997) Pec3; Peg12;Ube3a-as; Zfp127as; SnoRNas and Bielinska et al. (2000) Inpp5f-V2 Inpp5f 7 promoter/exon1 Maternal ND Inpp5f-V2; Inpp5f-V3 Wood et al. (2007) 2 kb upstream of H19 et al. Igf2 7 Paternal ICR H19 ; Ins2 Tremblay (1997) and (H19DMR) Thorvaldsen et al. (1998) Kcnq1-ot 1 promoter Th; Ascl2; Tspan32; Cd81; Tssc4; Kcnq1-ot1; Cdknlc; Fitzpatrick et al. (2002) and Kcnq1 7 Maternal ICR (Kvdmr1) Slc22a18; Phlda2; Nap1l4; Osbpl5; Yastsuki et al. (2002) 30 kb upstream of Shibata et al. (1998) and Rasgrf1 9 Paternal ICR A19; Mir184 Rasgrf1 exon1 Yoon et al. (2002) Plagl1 (Zac1) 10 Plagl1 promoter/exon1 Maternal ND Smith et al. (2002) Grb10 CpG island2 - Arnaud et al. (2003) and Grb10 11 Maternal ICR Ddc; Cobl; Grb10as Brain-specific promoter Shiura et al. (2009) U2af1-rs1 U2af1-rs1 11 Maternal ND Commd1 Wood et al. (2007) (Zrsr1) promoter/exon1 13 kb upstream of Gtl2 Lin et al. (2003) and Dlk1 12 Paternal ICR Begain; Dio3; Rtl1; Gtl2; Rian; C/D SnoRNAs; Mirg; miRNAs (IG-DMR) Takada et al. (2002) KcnK9 15 Peg13 promoter Maternal ND Peg13 Ruf et al. (2007) Slc38aA 15 Slc38a4 promoter Maternal ND Chotalia et al. (2009) Stoger et al. (1993) and Igf2r 17 Airn promoter Maternal ICR Airn ; Slc22a2: Slc22a3 Wutz et al. (2001) Impact 18 Impact promoter/exon1 Maternal ND Okamura et al. (2000)

Figure 1 List of germline DMRs identified in the mouse genome. Imprinted loci are named from the -coding gene closest to the germline DMR. Only imprinted loci with a characterised germline DMR are cited. For further information, please refer to the links provided in the ‘Websites of interest’ section. evidence suggests that there is an additional one at the and can be found in other comprehensive review texts Nnat promoter (Schulz et al. 2009). It is clear that there is (Ideraabdullah et al. 2008, Bartolomei 2009). Schema- an imbalance in the parental origin of DNA methylation, tically, differential DNA methylation of germline as only four of the known germline DMRs are methylated DMRs/ICRs is thought to initiate a sequence of events on the paternal allele (Fig. 1). Targeted deletion that ultimately gives rise to coordinated allele-specific experiments performed to assess the role of DMRs have expression for clusters of imprinted genes that are up to demonstrated that all but one of the nine germline DMRs one megabase in size (Fig. 1). The simplest scenario is assessed so far coincide with ICRs (Fig. 1). The exception when the ICR is also a promoter, thus leading to direct comes from Gnas 1A DMR that controls imprinted silencing of only the methylated allele. In the case of expression of a single transcript in the Gnas domain large imprinted domains, associated ‘long distance’ (Williamson et al. 2004); the whole domain being regulatory mechanisms, mainly through chromatin controlled by a second maternally methylated germline insulators and non-coding RNAs, are also required. DMR (Williamson et al. 2006; Fig. 1). It is thus likely that Importantly, for many imprinted genes, these ‘reading’ the germline DMRs identified at other imprinted loci are mechanisms and the ensuing imprinted gene expression ICRs as well. This idea is also supported by the are limited to specific tissues or developmental stages, demonstration that DNA methylation is a major despite the constitutive presence of the allelic DNA component in the control of imprinting (Li et al. 1993, methylation imprint. Methylation at ICRs can thus be Bourc’his et al. 2001, Hata et al. 2002, Kaneda et al. considered as the first, necessary level of control that acts 2004). In the remainder of this text, therefore, both upstream of diverse tissue- and developmental-specific ‘germline DMR’ and ‘ICR’ will be used to describe the 21 regulatory mechanisms. regions listed in Fig. 1. Once acquired in the germ cells, DNA methylation imprints are maintained in all the somatic lineages Reprogramming of imprints between generations throughout the development (Fig. 2), where the marks Imprints need to be reset at each new generation. First, the are ‘read’ in different ways to ensure appropriate parental existing imprints at ICRs are erased, and then, a new set of allele-specific expression. A detailed description of the imprints according to the sex of the developing germ cells reading mechanisms is outside the scope of this review, is acquired. Subsequently, these imprint marks are

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Maintenance

Blastocyst

Zygote Embryo Adult

Erasure

Oocyte

Germline-specific establishment

Sperm Primordial germ cells (female or male)

Figure 2 Imprint life cycle. This schematic description of the imprint life cycle is based on the acquisition of DNA methylation (black lollipop), a consistent hallmark of imprints (see text). Two hypothetical germline DMRs, one with paternally and the other with maternally derived DNA methylation, are depicted (as grey barrels) on a . The maternally inherited chromosome is shown in red, and the paternal chromosome is shown in blue. Methylation marks are established in the male and the female germline respectively. After fertilisation, these opposite imprints are somatically maintained throughout the development. In the new germline, the methylation imprints are erased in early germ cells. They are then re-established in later-stage germ cells according to the sex of embryo, to be transmitted to the next generation. maintained from the zygote to all somatic tissues (Fig. 2). precise mechanism is not fully elucidated, recent studies Because of the fundamental role of DNA methylation in show that the activation-induced cytidine deaminase conferring a functional imprint (Obata & Kono 2002), the (AID) contributes to this second demethylation step ‘imprint life cycle’ can be recapitulated by the methylation (Popp et al. 2010), and that histone replacement might dynamics at germline DMRs. Primordial germ cells (PGCs) also play an important role in this active process (Hajkova originate from 7 days post coitum (dpc)-old proximal et al.2008). epiblast and, following migration, colonise the developing New methylation imprints are acquired at later gonads at around 10.5 dpc to differentiate into gametes. At stages of mouse germ cell development, with a different 13.5 dpc, female germ cells enter meiosis, whereas male timing in the two sexes (Fig. 3). In the male germline, germ cells undergo mitotic arrest. During this develop- paternal imprints are acquired in mitotically arrested mental window, germ cells of both sexes undergo a major germ cells. Imprint establishment starts before birth, in epigenetic reprogramming believed to be necessary to pro-spermatogonia (at around 14.5 dpc), is completed restore totipotency. This reprogramming occurs in two in peri-natal pro-spermatogonia, and is maintained steps. First, a (possibly passive) decrease in DNA through the meiotic and haploid stages (Davis et al. methylation, which is associated with reprogramming of 2000, Kato et al. 2007). histone modifications, initiates in migrating PGCs at Maternal imprints, in contrast, are acquired after birth around 8 dpc (Seki et al.2005, Hajkova et al. 2008). only, during oocyte growth (Fig. 3). This happens in cells After they have reached the gonads (at around 10.5 dpc), that have already initiated meiotic recombination (with PGCs undergo a second (possibly active) genome-wide 4n DNA content), and is dependent on the size of the DNA demethylation process. Importantly,all sequences in oocyte (Lucifero et al. 2004, Hiura et al. 2006). Maternal the genome do not seem to be affected equally by these methylation marks are acquired asynchronously at two demethylation steps. Particularly, erasure of genomic different loci: some germline DMRs, such as Snrprn imprints occurs only during the second (active) step, since and Peg3, are methylated earlier, while others, like the ICRs retain their methylation marks up to 11.5 dpc before Mest DMR, become methylated at later stages of oocyte undergoing rapid demethylation that is complete by growth. Nonetheless, maternal methylation marks are 13.5 dpc (Hajkova et al.2002; Fig. 3). Although the fully acquired by the metaphase II stage. www.reproduction-online.org Reproduction (2010) 140 411–423

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(Male gonads)

Meiotic Mitotic divisions recombination Colonizing Round Post-migratory Prospermatogonia Prospermatogonia Sperm Male PGCs spermatid PGCs germline Methylation acquisition DNMT3A/3L DNMT3B? Migrating PGCs Erasure DNMT3A/3L KDM1B; ZFP57 Transcription-through CpG spacing ? Methylation acquisition Colonizing Quiescent Growing Post-migratory Fully growth MII Female PGCs oocyte oocyte PGCs Meiotic oocyte oocyte germline recombination

(Female gonads) Birth

Figure 3 Dynamic of methylation imprints in the developing male and female germline. This diagram is based on information obtained in the mouse. Factors known to be involved in methylation imprint acquisition are shown. See text and Fig. 4 for details. Not all developmental stages are represented. PGC, primordial germ cells; MII oocyte, oocyte in metaphase II of meiosis.

Interestingly, the timing of methylation acquisition the genome-wide reprogramming processes that charac- could be different in the two alleles of DMRs. Davis et al. terise the pre- and post-implantation stages (Morgan (2000) reported that in male germ cells, DNA methyl- et al. 2005; see below). In conclusion, the developmental ation at the H19 DMR is first acquired on the paternally life cycle of imprinting appears to be a complex and inherited allele (i.e. the previously methylated allele). tightly regulated process that is likely to be mediated by Similar observations were made in the female germ line several other factors in addition to the DNA methylation for the Snrpn, Mest and Plagl1 DMRs, which first acquire machinery. DNA methylation on the maternally-inherited allele in growing oocytes (Lucifero et al. 2004, Hiura et al. 2006). These observations suggest that both in male and in Factors that mediate imprint establishment female germ cells, a ‘signal’, possibly involving other The methylation machinery involved in imprint acqui- epigenetic modifications, could allow parental alleles to sition in the female germline is well characterised. A key remember their origin and influence the process of factor is DNMT3L, a protein that belongs to the DNMT3 reacquisition of methylation. de novo methyltransferase family even though it lacks a Timing of imprint establishment in humans is less well functional methyltransferase domain. In DNMT3LK/K documented. The H19 ICR (putative) is unmethylated mice, germline DMRs (as observed for Snrpn, Igf2r and in foetal male germ cells. Methylation starts to be acquired Peg3) failed to be methylated in oocytes (Bourc’his et al. in adult spermatogonia only, before meiotic division 2001, Lucifero et al.2007), and consequently, (Kerjean et al.2000). In the female germ line, only a Dnmt3LK/K females generate embryos, in which limited number of studies are available. In one detailed virtually all ‘maternal’ germline DMRs lack methylation, analysis, methylation at the KvDMR1 was shown to be whilst, importantly, methylation of the rest of the genome acquired progressively during oocyte maturation and to be is apparently unaffected (Fig. 4). Because of the completed by the metaphase II stage (Khoueiry et al. deregulated expression of associated imprinted genes 2008). Similarly, the SNRPN DMR was found to be fully (aberrant bi-allelic expression or complete repression), methylated in mature oocytes (Geuns et al.2003), these conceptuses arrest development before embryonic suggesting a timing similar to that observed in the day 10.5 (Bourc’his et al. 2001, Hata et al. 2002). mouse. However, in another study, the SNRPN DMR was Biochemical and genetic studies demonstrated that reported to be unmethylated in mature oocytes, suggesting DNMT3L is an essential factor of maternal imprint that this methylation mark is acquired only upon, or after, establishment through its interaction with de novo fertilisation in humans (El-Maarri et al.2001). methyltransferase DNMT3A (Kaneda et al.2004, Once acquired in the germline, and following Suetake et al. 2004). fertilisation, methylation imprints persist throughout the Although there are some discrepancies, studies in the development and adult life. This occurs in the context of male germline support the idea that the acquisition of

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Germline DMR(s) Name of Germline DMR(s) known to known not to be Comments Type of factor References factors be affected by its deletion affected by its (see text for details) deletion - Depletion effect is less severe in male Bourc'his et al. (2001) All germline DMRs tested so far than in female germline Hata et al. (2002) Non-catalytic co- (i.e: KvDMR1, Grb10, U2af1-rs1, Webster et al. (2005) Arnaud et al. (2006) DNMT3L factor of Dnmt3a/b Plagl1, Snrpn, Gnasx1, Gnas1A, - DNMT3A and DNMT3B are thought Kato et al. (2007) Nap1l5; Peg1; Peg3; Igf2r; Peg10, to act redundantly on Rasgrf1 DMR Mcts2; H19; Rasgrf1; IG-DMR) Monk et al. (2008) Wood et al. (2008) and Henckel et al. (2009)

DNA All germline DMRs tested so far Hata et al. (2002) methyltransferase DNMT3A (i.e: Snrpn, Peg1; Peg3; Igf2r, H19; Rasgrf1? Kaneda et al. (2004) and with de novo IG-DMR) Kato et al. (2007) activity KvDMR1; Igf2r; H3K4me2-histone Almost exclusively expressed in KDM1B Mest; Grb10; Plagl1, Impact Snrpn; Rasgrf1; Ciccone et al. (2009) growing oocytes H19 Transcription factor ZFP57 of the KRAB zinc- Ig-DMR; Peg1; Factor (maternal+zygotic) also involved Snrpn Li et al. (2008) (maternal) finger protein Peg3; Igf2r; Nnat in maintenance. See Table 3 family

- Mutation found associated with PEG3, SNRPN, KVDMR1, GNAS1A familial mole and likely to affect most Members of the (Also found associated with abnormal Murdoch et al. (2006) NLRP7 (if not all) ICRs CATERPILLER gain of methylation on H19 DMR E1-Maari et al. (2003) (human) - Mutation has partial penetrance protein family maternal allele) and Kou et al. (2008) involved in - Possible role in post-zygotic inflammation and maintenance also apoptosis NLRP2 SNRPN and KVDMR1; PEG1 Meyer et al. (2009) (human) PLAGL1DMRs Transcription- Germline DMRs at Gnas domain Also detected at : through Chotalia et al. (2009) (GnasX1 and 1A DMRs) Igf2r; Grb10; Plagl1, Impact and Kvdmr1 events

Figure 4 Factors known to be involved in germline establishment of imprint in mouse and human (when indicated). Maternally methylated germline DMRs are in red, and paternally methylated DMRs are in blue.

‘paternal’ methylation imprints also relies on DNMT3L line-specific manner is not known. Recent studies (Fig. 3). Specifically, the DNMT3A–DNMT3L complex suggest that several components could contribute to contributes to methylation acquisition at the H19 and this process, including primary sequence specificity, IG-DMR DMRs (Bourc’his & Bestor 2004, Webster et al. chromatin configuration, non-histone proteins and 2005, Kato et al. 2007). On the other hand, DNA transcriptional events. methylation at the Rasgrf1 DMR is only slightly affected in Dnmt3aK/K or Dnmt3bK/K male gem cells, Imprinting acquisition: sequence specificities suggesting that both enzymes are redundantly involved in association with DNMT3L (Kato et al. 2007; Fig. 4). It Both in mice and humans, the genomic features of should be noted, however, that unlike the female paternal and maternal germline DMRs are clearly germline, where absence of DNMT3A or DNMT3L different. Maternal germline DMRs are enriched in leads to complete failure of methylation imprint CpG dinucleotides, most of them fulfilling the criteria acquisition, ‘paternal’ germline DMRs are only partially of a CpG island, and they are always located in a unmethylated in Dnmt3aK/K or Dnmt3lK/K male promoter region. By contrast, all the four known germ cells. This suggests that DNMT3A and DNMT3B paternally methylated germline DMRs reside in inter- are functionally redundant in the male germline, and/or genic regions, and are CpG poor. It is somehow that other yet to be characterised factors are involved. In intriguing that most of the maternal germline DMRs are contrast to the female germ line, DNMT3L shows a CpG islands as this class of sequences is normally broader action in the male germline as it is also unmethylated in the genome, suggesting a sequence- responsible for the de novo methylation and transcrip- specific capacity of ‘imprinted’ CpG islands to attract tional silencing of dispersed repeated sequences in DNA methylation. It was postulated that arrays of spermatogonia (Bourc’his & Bestor 2004, Webster tandem repeat motifs (Neumann et al. 1995), which et al. 2005, Kato et al. 2007). are frequently found in or close to germline DMRs In summary, altogether these findings indicate that the (Neumann et al. 1995, Hutter et al. 2006), could have a DNMT3A/DNMT3L complex is the core of the DNA role in imprint acquisition. Such repeats, however, have methylation machinery involved in imprint acquisition been convincingly shown to be necessary for correct in both female and male germline (Fig. 3). How, differential methylation only at the Rasgrf1 locus, precisely, this complex is targeted to ICRs in a germ whereas they are dispensable for imprint acquisition at www.reproduction-online.org Reproduction (2010) 140 411–423

Downloaded from Bioscientifica.com at 10/02/2021 06:42:56PM via free access 416 P Arnaud the H19 and the KvDMR1 DMRs (Reed et al. 2001, Yoon model, in which repressive histone marks are a et al. 2002, Mancini-Dinardo et al. 2006). The CpG prerequisite for DNA methylation imprints. Consistently, spacing at the targeted sequences might be more the emerging, alternative model does not implicate relevant for imprint acquisition. Structural analyses repressive histone marks in the maternal imprint showed that DNMT3A and DNMT3L form a tetrameric acquisition, but rather, involves the specific erasure of complex that preferentially methylates pairs of CpGs that a permissive histone mark. In agreement with such a are 8–10 bp apart (Jia et al. 2007). Such CpG spacing has scenario, biochemical approaches have shown that been observed at all maternally methylated germline DNMT3L interacts with histone H3 only when it is DMRs but not at the paternally methylated germline unmethylated on lysine 4, suggesting that H3K4 DMRs so far. The distribution of CpGs could help methylation (permissive mark) prevents the recruitment explaining why the absence of DNMT3L, though of DNMT3A/DNMT3L to germline DMRs (Ooi et al. affecting both germlines, has a more drastic effect on 2007). The proof that H3K4 methylation needs to be DMRs that are methylated in oocytes than on those removed to allow DNA methylation to occur is brought methylated in sperms (see above). Nevertheless, CpG about by a recent functional study on the histone spacing is probably not specific to germline DMRs as it is demethylase KDM1B (Ciccone et al. 2009). This enzyme found in many other CpG islands in the is an H3K4 demethylase with specificity towards as well (Ferguson-Smith & Greally 2007). This finding H3K4me2. Its targeted disruption has no effect on suggests that CpG spacing, if involved in imprint somatic development; however, embryos derived from acquisition, does not act on its own, but synergistically KDM1BK/K oocytes show gross developmental with others components. defects, and do not survive beyond 10.5 dpc. This phenotype is reminiscent of the developmental defects observed in the progeny of DNMT3LK/K females. Imprint acquisition: a chromatin connection Consistently, the authors demonstrate that in the absence Concerning other possible components involved in of KDM1B, a subset of germline DMRs fails to acquire imprint acquisition, it is interesting to note that in their methylation imprint in oocytes, leading to aberrant addition to DNA methylation, germline DMRs are also expression of the associated genes in the derived marked by allelic histone modifications in somatic cells. embryos. This defect is probably specific to these regions Several studies, initially based on locus-specific analysis as methylation at several classes of repetitive elements of histone modifications and further supported by was unaffected. KDM1B appears thus to be a new key genome-wide analyses, revealed the existence of a player specifically involved in imprint acquisition germline DMR-specific chromatin signature. Speci- (Ciccone et al.2009; Fig. 3). However, unlike fically, at both maternal and paternal germline DMRs, DNMT3L, its action is limited to a subset of maternal the DNA unmethylated allele is associated with H3/H4 germline DMRs (Fig. 4). acetylation and dimethylation of lysine 4 of histone H3 A similar mechanism could also be present in the (H3K4me2), hallmarks of permissive chromatin. By paternal germline, but it would be independent of contrast, the DNA methylated allele is consistently KDM1B, which is expressed almost exclusively in associated with histone marks that are characteristic growing oocytes. Alternatively, indirect evidence of repressive chromatin: tri-methylation on lysine 9 of suggests an implication of repressive marks in paternal histone H3 (H3K9me3), trimethylation on lysine 20 of imprint acquisition. Experiments conducted in Xenopus histone H4 (H4K20me3) and symmetrical dimethylation laevis oocytes demonstrated that to fully acquire its on arginine 3 of histones H4/H2A (H4/H2AR3me2s; methylation imprint, the mouse H19 DMR required, in Henckel et al. 2009). Based on observations in other addition to plasmids encoding the methylation organisms, particularly in the fungus Neurospora crassa, machinery (DNMT3A/DNMT3B/DNMT3L) and the where it has been clearly established that H3K9me3 is a cofactor CTCF-like (CTCFL; see below) also the arginine prerequisite for DNA methylation (Tamaru et al. 2003), it methyltransferase PRMT7, suggesting a role for histone is tempting to speculate that this (or part of) specific arginine methylation in this process (Jelinic et al. 2006). chromatin signature could be involved in recruiting the Whether this mark is involved in paternal imprint DNMT3A/DNMT3L complex. However, due to tech- acquisition at the endogenous locus remains however nical limitations, it is currently unknown whether these to be established. On the other hand, H3K9me3 and specific histone marks are also present in the germ cells, H4K20me3, two repressive marks constitutively associ- particularly in growing oocytes. Of concern, none- ated with the methylated allele in somatic cells, can theless, is the recent observation that mouse embryos probably be discarded for a role in imprint acquisition. derived from Dnmt3lK/K oocytes (i.e. that failed to Indeed, at a late spermatogenesis stage (i.e. following acquire their DNA methylation imprint) are devoid of methylation imprint acquisition), the H19 and IG-DMR repressive histone marks at the maternal germline DMRs DMRs do not show enrichment for these marks, (Henckel et al. 2009). This finding suggests an upstream suggesting that they are acquired after DNA methylation role for DNA methylation and challenges the classical (Delaval et al. 2007).

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Imprint acquisition: a role for non-histone proteins some cases of FHM present abnormal gains of methylation at the H19 DMR (El-Maarri et al. 2003). In addition to the components of the methylation Combined, these observations indicate that mutations in machinery, several recent studies underline the role of NLRP7 lead to a complex and heterogeneous pattern of other factors in imprint acquisition. These include the abnormal methylation imprints, and suggest a role in both KRAB zinc finger protein ZFP57. Following both zygotic germline acquisition and post-zygotic maintenance. and oocyte-specific depletion of Zfp57 in the mouse, Li Finally, the CTCFL protein, also known as BORIS, is a et al. (2008) demonstrated that besides maintaining DNA factor, which might be involved in imprint acquisition at methylation at several germline DMRs (see below, the paternally methylated H19 DMR. CTCFL is a testis- Fig. 5), ZFP57 is also required for acquisition of specific paralogue of CTCF, an insulator protein that methylation in oocytes at the Snrpn ICR (Fig. 4). Two binds to the unmethylated H19 DMR. Immunoprecipita- members of the nucleotide-binding oligomerisation tions conducted on chromatin purified from embryonic domain, leucine-rich repeat and pyrin domain (NLRP) day 15.5 testes have suggested that CTCFL is physically family also are involved in the acquisition of methylation bound to the H19 ICR in male germ cells. In addition, imprints in humans. Specifically, in a recent study CTCFL interacts with PRMT7, and the experiments conducted on a familial case of the growth disorder conducted in Xenopus oocytes further support the role Beckwith–Wiedemann, a mutation in the NLRP2 gene of these two factors in establishing the methylation was found to be associated with absence of methylation imprint at the H19 DMR (Jelinic et al. 2006). However, it at the KVDMR1 and PEG1 DMRs (Meyer et al. 2009). remains to be formally demonstrated that CTCFL and Furthermore, a subset of familial hydatidiform moles PRMT7 are indeed involved in imprint acquisition in the (FHMs) that are abnormal forms of pregnancy, which are mammalian male germ line, particularly in view of the thought to result from a genetic defect which prevents fact that functional CTCF-binding sites are dispensable the establishment of maternal germline imprints ( Judson for methylation imprint acquisition at the H19 DMR et al. 2002, Djuric et al. 2006, Murdoch et al. 2006, Kou during spermatogenesis (Engel et al. 2006). et al. 2008), has been associated with a mutation in NALP7/NLRP7 (Murdoch et al. 2006, Kou et al. 2008). How mutation in NLRP genes leads to methylation Imprint acquisition: a role for transcriptional events imprint defects is unclear. Of interest is the observation The role of small RNAs in triggering de novo that NLRP7 mutations can be associated with partial DNA methylation is well documented, especially in disease penetrance; some women that carried an NLRP7 the plant kingdom (Matzke et al. 2009). In mammals, mutation nevertheless had a normal pregnancy de novo DNA methylation of repetitive elements (IAP (Moglabey et al. 1999, Murdoch et al. 2006), and and Line-1) during spermatogenesis appears to be

Factors known to Germline DMR(s) be involved in Germline DMR(s) known to Comments Type of factor known not to be References methylation be affected by its deletion (see text for details) affected by its deletion maintenance Transcription In mouse: Snrpn, Peg1; Peg3, Nnat; Protect the methylated allele ZFP57 factor from KRAB IG-DMR In mouse: Igf2r Li et al. (2008) and against the pre-implantation (maternal+zygotic) Zinc-finger In human: PLAGL1; GRB10 and (partial effect?) Mackay et al. (2008) DNA demethylation wave protein family PEG3 Maternal factor PGC7/Stella essential for early Peg1; Peg3; Peg10, H19; Rasgrf-1 Snrpn,Nnat; IG-DMR Nakamura et al. (2007) development Snrpn Methyl CpG Methylation is reduced MBD3 H19 indirect evidences for Peg3 Reese et al. (2007) binding protein 3 but not abolished and IG-DMR RBBP1 and RB binding Snrpn Wu et al. (2006) RBBP1-like 1 proteins Methylation is reduced Linker-histone H1 H19 and IG-DMR Fan et al. (2005) but not abolished Factors known to Germline DMR(s) be involved in Germline DMR(s) known to Type of factor known to be Comments References protecting against be affected by its deletion affected by its deletion methylation

Protect unmethylated allele Engel et al. (2003) and CTCF Insulator protein H19 during post-implantation Schoenherr et al. (2003) de novo methylation wave

Figure 5 Factors known to be involved in imprint maintenance in mouse and human (when indicated). Maternally methylated germline DMRs are in red, and paternally methylated germline DMRs are in blue. www.reproduction-online.org Reproduction (2010) 140 411–423

Downloaded from Bioscientifica.com at 10/02/2021 06:42:56PM via free access 418 P Arnaud regulated by MILI and MIWI2, two Piwi-interacting interest are Snrpn and Peg3 ICRs, which were found to be small RNA (piRNA)-binding proteins (Kuramochi-Miyagawa fully maternally methylated in respectively 30 and 14% et al. 2008). Although these pathways might provide an of the progeny of Dnmt3lK/K females (Arnaud et al. attractive mechanism for imprint establishment, their 2006, Henckel et al. 2009). Since bisulphite analyses in involvement in this process has not been demonstrated Dnmt3lK/K oocytes failed to detect methylation at so far (see Kacem & Feil (2009) and ref inside). Similarly, these loci (Bourc’his et al. 2001, Lucifero et al. 2007), the it is well documented that non-coding RNAs are methylation imprints observed in the progeny (in both involved in imprinting (Ideraabdullah et al.2008). 9.5 dpc embryos and trophoblast cells) must have been However, the described imprinted non-coding tran- acquired at the implantation stage (Henckel et al. 2009). scripts are controlled by the DNA methylation imprints, Similarly, failure to establish the Snrpn ICR methylation and their function so far seems limited to ‘reading’ the imprint in Zfp57K/K eggs could be partially rescued by imprints rather than in their establishment. the paternally derived ZFP57 at post-implantation stages Evidence for a transcription-based event in imprint (Li et al. 2008). These observations, especially at the acquisition comes from the Gnas locus. This complex Snrpn ICR, are consistent with studies that report that locus comprises a series of imprinted overlapping coding methylation at the SNRPN ICR in humans might occur and non-coding transcripts, and is characterised by the after fertilisation (El-Maarri et al. 2001, Kaufman et al. presence of two maternally methylated germline DMRs 2009; note, however, that Geuns et al. (2003) found that (Fig. 1). An original study (Chotalia et al. 2009) the human SNRPN ICR is methylated already in oocytes). demonstrated that truncation of the Nesp transcript, Combined, these analyses suggest that (some) ICRs which initiates furthest upstream in the locus and covers carry a germline-derived (DNA methylation independent) the entire domain, disrupts the acquisition of methyl- signature that can mediate DNA methylation acquisition ation at the two germline DMRs in oocytes. This on the right parental allele at implantation during the observation is somehow reminiscent of an earlier study genome-wide de novo methylation. The nature of such in humans, in which absence of methylation at the signature and whether the ‘embryonic’ establishment GNASXL and 1A DMRs was associated with deletion of recapitulates the mechanism that normally occurs in the the NESP promoter region (Bastepe et al. 2005). germline remains to be discovered. However, it is Combined, these observations suggest that transcription tempting to speculate that a germline-derived chromatin across the germline DMRs of the Gnas locus is required signature and/or transcription through ICR at implantation for the establishment of their DNA methylation imprint will be recognised by the somatic DNMT3A/DNMT3L in oocytes. It is postulated that transcription could serve complex and by relevant other non-histone proteins. The to ‘open’ the chromatin and facilitate the recruitment of post-fertilisation establishment of DNA methylation the DNA methylation machinery. The observation that imprints could constitute a safeguard system to sense the five other maternal germline DMRs tested are also and correct imprint methylation defects that could traversed by protein-coding transcripts (some of which otherwise lead to serious developmental defects. are oocyte specific) in growing oocytes suggests that this newly discovered mechanism might occur at other imprinted domains as well (Chotalia et al. 2009; Factors that mediate the maintenance of imprints Fig. 4). Conversely, it is not known whether such a Following their establishment, methylation imprints have mechanism might also be relevant for the establishment to be maintained and faithfully transmitted to all somatic of intergenic paternally methylated germline DMRs. lineages. Remarkably, this occurs despite genome-wide reprogramming events. Indeed, the first issue is to understand how the paternal imprints resist the active Imprinting in the absence of germline DNA methylation demethylation of the paternal genome that occurs acquisition after fertilisation (Morgan et al. 2005). One exciting The canonical starting point of the imprint cycle is the hypothesis comes from the observation that in the human acquisition of DNA methylation in the germline (Figs 2 sperm, ICRs and some developmentally regulated genes and 3). However, several studies suggest that this key are apparently not subject to histone-to-protamine step can be by-passed, and that methylation imprints can exchange (Hammoud et al. 2009). This could be be acquired later in development. For instance, when the consistent with the involvement of a specific chromatin H19 DMR is inserted at ectopic positions in the genome, structure in the transmission of the paternal imprint from paternal-specific DNA methylation at this DMR can be spermatozoa to the zygote. Following fertilisation and up established partially, or even completely, after fertilisa- to implantation, the methylated alleles of all germline tion, during early development (Park et al. 2004, DMRs are protected from the global wave of demethyl- Tanimoto et al. 2005). Furthermore, methylation ation. Continuous expression of maternal and then imprints at some ICRs can be present in a stochastic zygotic DNMT1 in pre-implantation embryos are manner in some embryos derived from DNMT3L- required for this process (Biniszkiewicz et al. 2002, deficient oocytes (Arnaud et al. 2006). Of particular Hirasawa et al. 2008).

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Besides, the methylation machinery, other protein evidence for a role of repressive histone marks in imprint factors are involved in maintaining parental-specific maintenance has not been documented. Of relevance imprints. One of these is the KRAB zinc finger protein could be the observation that DNA methylation is ZFP57. In addition to being involved in the acquisition of reduced at several ICRs regions in ES cells that lack the methylation imprint at the Snrpn ICR, ZFP57 is also the G9a H3K9me2 HMT (Dong et al. 2008, Tachibana required to maintain DNA methylation at several et al. 2008). However, this is more likely due to the paternal and maternal imprinted regions (Li et al. 2008; absence of G9a itself rather than to the loss of Fig. 5). The role of ZFP57 in maintaining methylation H3K9me2 (Dong et al. 2008, Tachibana et al. 2008). imprints is conserved between mice and humans as Furthermore, this observation was not confirmed in suggested by a study in patients with transient neonatal 9.5 dpc G9aK/K embryos (Tachibana et al. 2008). diabetes mellitus (TNDM; Mackay et al. 2008). In these Absence of H4K20me3 (another chromatin repressive patients, mutations in ZFP57 were associated with hypo- mark) also failed to affect methylation imprint mainten- methylation at the (putative) ICR of PLAGL1, a defect ance in vivo (Wu et al. 2006, Pannetier et al. 2008), thus causally involved in TNDM. Interestingly, these patients questioning the role of this histone modification at ICRs. often present hypo-methylation at others ICRs as well Indirect evidence indicates a role for chromatin in (Mackay et al. 2008). How ZFP57 mediates its action is imprint maintenance. In ES cells depleted of the three unknown. However, this class of transcription factors is subtypes of the linker histone H1, hypo-methylation and thought to act as transcriptional repressors by recruiting aberrant expression of the associated genes were the KAP-1/TIF1B repressive complex (Abrink et al. observed at the H19 and IG-DMR ICRs, while most of 2001). This complex includes histone methyltransferases the genome remained unaffected (Fan et al. 2005). and deacetylases potentially involved in the mainten- Similarly, RBBP1 and RBBP1-like1, two RB-binding ance of DNA methylation imprints. Another relevant proteins, are required for maintenance of both DNA protein is PGC7/Stella, a maternal factor that is required and histone (H4K20me3 and H3K9me3) methylation for embryonic development and partially protects marks, at the mouse Snrpn ICR (Wu et al. 2006). against loss of DNA methylation at specific paternal and maternal ICRs (Fig. 5; Nakamura et al. 2007). Interestingly, ZFP57 and Stella have only partially Outlooks overlapping effects (Fig. 5). The methyl CpG-binding In recent years, major advances have been made in our domain protein 3 (MBD3) was also found to be involved understanding of the mechanisms that govern the in imprint maintenance, though its role appears limited establishment of genomic imprints. The emerging to the paternally methylated H19 ICR (Reese et al. 2007). picture suggests that CpG spacing, removal of H3K4me This was unexpected, since MBD3 itself does not bind and transcriptional read-through are likely to act in a directly to methylcytosines indicating that maintenance concerted and not exclusive manner to recruit the could rely on MBD3-containing protein complexes such DNMT3A/DNMT3L complex on ICRs in growing as NuRD. oocytes (Fig. 3). With the notable exception of CpG The last step of the early development reprogramming periodicity, these factors could also play a role in (after the wave of demethylation) is characterised by a paternal imprint acquisition. Furthermore, recent studies wave of genome-wide de novo methylation that occurs emphasise that imprint acquisition relies on components soon after implantation. The specific association of the that could differ not only between the two germlines, but unmethylated alleles of germline DMRs with H3K4me2, also between ICRs in the same germline. This could a mark linked with chromatin that is permissive for probably account for the temporally asynchronous transcription, probably contributes to protect DMRs from acquisition of imprints at different DMRs in the same aberrant acquisition of methylation (Ooi et al. 2007, germline. It is tempting to speculate that besides the Ciccone et al. 2009). Besides chromatin structure, the ‘universal’ imprinting regulator DNMT3L, imprint acqui- CTCF zinc finger domain protein is also implicated in sition relies on a common ‘core’ mechanism that is itself protection against DNA methylation. Indeed, in absence controlled by and/or associated with factors acting in an of CTCF binding, the mouse H19 ICR is susceptible to ICR(s)-specific manner. For instance, structural studies acquire methylation at the normally unmethylated on the DNMT3A/DNMT3L complex (Ooi et al. 2007) parental allele during post-implantation de novo methyl- and the observation that the presence of the permissive ation (Schoenherr et al. 2003, Engel et al. 2006; Fig. 5). mark H3K4me2 is inversely correlated with DNA The interplay between histone and DNA methylation is methylation in mammalian cells supports the idea that thought to be important for the somatic maintenance of erasure of H3K4me could be generally used for imprint imprints. The recent observation that absence of methy- acquisition. Nevertheless, the specificity of KDM1B for lation imprints acquisition drastically affects the chroma- only a few of the maternal DMRs (Ciccone et al. 2009) tin signature at ICRs is consistent with the existence of a suggests that other H3K4 could be mechanistic link between both types of methylation involved at the other ICRs. Similarly, if transcription marks (Henckel et al.2009). However, functional through ICRs turns out to be generally used in imprint www.reproduction-online.org Reproduction (2010) 140 411–423

Downloaded from Bioscientifica.com at 10/02/2021 06:42:56PM via free access 420 P Arnaud acquisition, one may expect that the control of such The insights provided by these new studies, combined transcripts might rely on a variety of transcription factors. with the development of sensitive techniques to analyse Further understanding of the mechanisms that regulate germ cells, pave the way for an exciting time during imprint acquisition necessarily requires defining the which the mechanisms leading to imprint establishment temporal relationship between transcriptional events, might finally be unravelled. H3K4me erasure and de novo methylation. This will be obviously facilitated by the recent development of sensitive ‘scaled-down’ techniques dedicated to study Websites of interest epigenetic modifications in limited numbers of cells, For up-to-date information on imprinted genes, their including at the genome-wide level (Dahl & Collas species distribution and their functions: The Medical 2009, Popp et al. 2010). Such approaches will also help Research Council, Harwell (UK) Mouse Imprinting to determine whether, besides the absence of H3K4me, Website: http://www.har.mrc.ac.uk/research/genomic_ other chromatin signatures are involved in this process. imprinting/. The University of Otago (New Zealand) Another clue about a putative chromatin structure could Imprinting Website: http://igc.otago.ac.nz/home.html. arise through biochemical analysis of KDM1B. Several The King’s College, London (UK), WAMIDEX website: studies indeed showed that histone demethylases can be a Web Atlas of Murine Genomic Imprinting and associated with specific HMTs in the same complex Differential Expression: https://atlas.genetics.kcl.ac.uk/. (Agger et al. 2008). For example, UTX, a demethylase for the repressive mark H3K27me3, is associated with MLL2, the H3K4-specific HMT. This supports a dynamic Declaration of interest model, in which the removal of a specific histone tag is The author declares that there is no conflict of interest associated with the addition of another histone methyl- that could be perceived as prejudicing the impartiality of the ation mark. Identification of the HMT that associates work reported. specifically with KDM1B should help to unravel the chromatin structure that preclude, and eventually control, methylation imprint acquisition. Funding Further characterisation of HMTs involved in ICR This work was supported by the Association Pour la Recherche histone modification in somatic cells is also of sur le Cancer (grant ‘subvention fixe’ no. 4980; 2010) and importance. Except for SUV4-20H, known to regulate LA LIGUE contre le Cancer (grant ‘subvention comite´ H4K20me3 at ICRs (Pannetier et al. 2008), the HMTs Herault’; 2010). involved in the deposition of other repressive marks at ICRs remain to be determined. Their identification would permit to properly establish the nature of the relationship Acknowledgements between histone and DNA methylation at ICRs. The observation that DNA methylation imprints are faithfully I thank Ryutaro Hirasawa, Satya Kota, Lionel Sanz and Robert maintained in MEF cells deficient for SUV4-20H Feil for advice and critical reading of the manuscript. I also thank the two anonymous reviewers whose comments helped (Pannetier et al. 2008) suggests a redundant role for the to improve this manuscript. panel of histone marks detected at the methylated allele of ICRs. It would thus be important to test, individually and concomitantly, the impact of HMT depletion in germline acquisition and somatic maintenance of DNA References methylation imprints. Abrink M, Ortiz JA, Mark C, Sanchez C, Looman C, Hellman L, Chambon P Mechanisms that protect ICRs against DNA methyl- & Losson R 2001 Conserved interaction between distinct Kruppel- ation in the opposite germline have also to be taken into associated box domains and the transcriptional intermediary factor 1b. PNAS 98 1422–1426. consideration. For instance, high level of H3K4me is Agger K, Christensen J, Cloos PA & Helin K 2008 The emerging functions of detected at maternal ICRs in early spermatogonia cells histone demethylases. 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