562 RESEARCH ARTICLE

Development 140, 562-572 (2013) doi:10.1242/dev.089268 © 2013. Published by The Company of Biologists Ltd Plasticity in Dnmt3L-dependent and -independent modes of de novo methylation in the developing mouse embryo Mounia Guenatri*, Rachel Duffié*, Julian Iranzo, Patricia Fauque and Déborah Bourc’his‡

SUMMARY A stimulatory DNA methyltransferase co-factor, Dnmt3L, has evolved in mammals to assist the process of de novo methylation, as genetically demonstrated in the germline. The function of Dnmt3L in the early embryo remains unresolved. By combining developmental and genetic approaches, we find that mouse embryos begin development with a maternal store of Dnmt3L, which is rapidly degraded and does not participate in embryonic de novo methylation. A zygotic-specific promoter of Dnmt3l is activated following gametic methylation loss and the potential recruitment of pluripotency factors just before implantation. Importantly, we find that zygotic Dnmt3L deficiency slows down the rate of de novo methylation in the embryo by affecting methylation density at some, but not all, genomic sequences. Dnmt3L is not strictly required, however, as methylation patterns are eventually established in its absence, in the context of increased Dnmt3A protein availability. This study proves that the postimplantation embryo is more plastic than the germline in terms of DNA methylation mechanistic choices and, importantly, that de novo methylation can be achieved in vivo without Dnmt3L.

KEY WORDS: DNA methylation, Gametes, Early mouse embryo

INTRODUCTION not interchangeable. Dnmt3A is strictly required for gametic Cytosine methylation profoundly impacts genome expression and methylation but is globally dispensable for de novo methylation in stability. In mammals, its occurrence at CpG-rich promoters is the early embryo. Conversely, gametic methylation can occur correlated with transcriptional repression of retrotransposons and without Dnmt3B, whereas embryonic methylation cannot (Okano various single-copy sequences, such as pluripotency and germline- et al., 1999; Kaneda et al., 2004; Borgel et al., 2010; Kaneda et al., expressed , as well as genes subject to genomic imprinting 2010). Conditional deletions of the Dnmt3a further highlight and X- inactivation. DNA methylation is essential for its involvement in methylation processes that take place later, in mammalian development and reproduction, as shown by the early postnatal cell lineages (Nguyen et al., 2007; Challen et al., 2012). embryonic lethality and sterility phenotypes of mouse models of The reasons for this stage specificity in Dnmt3A and Dnmt3B global DNA methylation deficiency (Li et al., 1992; Okano et al., function are unknown. 1999; Bourc’his et al., 2001; Kaneda et al., 2004; Bostick et al., Whereas Dnmt3a and Dnmt3b orthologs are found widely 2007). Abnormal methylation patterns are also associated with among distant eukaryotic species, a third Dnmt3 member, Dnmt3- pathological states in humans, including cancer (Rakyan et al., like (Dnmt3l), has evolved uniquely in mammals (Yokomine et al., 2011). DNA methylation patterns are set genome-wide during two 2006). This gene encodes a catalytically inert protein that is unable main developmental stages: gametogenesis and early to methylate DNA substrates alone but serves as a regulator of the embryogenesis (Morgan et al., 2005; Borgel et al., 2010). These de novo methylation machinery. In biochemical assays performed establishment phases follow a genome-wide clearance of in vitro and in cellular systems, human DNMT3L increases the methylation, which occurs concomitantly with the expression of efficiency of the methylation reaction by stimulating DNMT3A and pluripotency factors, in primordial germ cells and preimplantation DNMT3B catalytic activity (Chédin et al., 2002; Suetake et al., embryos (Rougier et al., 1998; Smallwood et al., 2011; Guibert et 2004; Moarefi and Chédin, 2011) and the deposition of more al., 2012; Smith et al., 2012). uniform methylation patterns (Wienholz et al., 2010). DNMT3L is DNA methylation patterns are driven de novo by two DNA moreover involved in the organization of tetrameric complexes methyltransferase enzymes, Dnmt3A and Dnmt3B, which can with DNMT3A (Jia et al., 2007), leading to the formation of target cytosines in both CG and non-CG contexts (Chédin, 2011). DNMT3L-DNMT3A filaments that polymerize along the DNA Expression of these proteins peaks at times of major de novo molecule in vitro (Jurkowska et al., 2008). Dnmt3L-dependent methylation in developing germ cells, early embryos and stimulation of Dnmt3 enzyme activity has been linked to increased embryonic stem (ES) cells (Watanabe et al., 2002; La Salle et al., affinity for the methyl donor S-adenosyl-L-methionine (SAM), 2004; Sakai et al., 2004; Hirasawa et al., 2008). Although these longer residency on DNA, and improved processivity, none of enzymes may show some degree of functional redundancy, they are these mechanisms being mutually exclusive (Kareta et al., 2006; Holz-Schietinger and Reich, 2010). In vivo, Dnmt3l is highly expressed during gametogenesis under the control of sex-specific promoters (Bourc’his et al., 2001; INSERM U934/UMR3215, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France. Shovlin et al., 2007). The canonical transcript is produced in prospermatogonia, whereas in oocytes a longer transcript originates *These authors contributed equally to this work. from an upstream promoter embedded in an intron of the ‡Author for correspondence ([email protected]) neighboring Aire gene. Although different in length, these

Accepted 12 November 2012 transcripts produce a protein of similar size (O’Doherty et al., DEVELOPMENT De novo methylation without Dnmt3L RESEARCH ARTICLE 563

2011). Dnmt3L activity is strictly required for gametic methylation (Dnmt3l+/– ϫ Dnmt3l+/–) crosses, whereas Dnmt3lm– (Dnmt3l–/+) and and fertility. Dnmt3l–/– males do not produce mature sperm, in Dnmt3lm–z– (Dnmt3l–/–) embryos were obtained from (Dnmt3l–/– ϫ association with retrotransposon hypomethylation and reactivation Dnmt3l+/–) crosses (Bourc’his et al., 2001). At E6.5, epiblasts were during spermatogenesis (Bourc’his and Bestor, 2004). Dnmt3l–/– separated from the extra-embryonic ectoderm (Nagy et al., 2003). At E9.5, females produce methylation-deficient oocytes and their Dnmt3l–/+ only embryos were kept for analysis. Dnmt3l and sex genotyping were performed by PCR on extra-embryonic tissues for E6.5 and E9.5 progeny systematically die at mid-gestation, in association with the conceptuses. dysregulation of imprinted (Bourc’his et al., 2001; Smallwood et al., 2011; Kobayashi et al., 2012). Constitutive Quantitative RT-PCR (RT-qPCR) Dnmt3l mutant phenotypes mimic a conditional knockout of For oocytes and preimplantation embryos, ~300 diploid genomes were Dnmt3a in germ cells (Kaneda et al., 2004). collected per stage, total RNA was obtained by three freeze/thaw cycles as Gametic de novo methylation therefore occurs under the control previously described (Jeong et al., 2005) and lysis extracts were processed of a developmentally regulated Dnmt3L-dependent pathway. for cDNA conversion (Superscript III, Invitrogen). For E6.5 embryos, ten epiblasts of sex-matched wt and Dnmt3lz– genotype were separately However, whether embryonic de novo methylation occurs in a pooled, extracted using the RNA Picopure kit (Applied Biosystems) and Dnmt3L-dependent manner is still controversial. Dnmt3L is treated with DNase (TURBO DNase, Ambion) prior to cDNA conversion. abundantly expressed in ES cells from the same promoter used in Quantitative amplification of cDNA was performed using the PCR primers prospermatogonia (Bourc’his et al., 2001; Shovlin et al., 2007). listed in supplementary material Table S1 on an Mx4000 QPCR system Dnmt3L protein is found in postimplantation embryos from E5.5 according to manufacturer instructions (Stratagene). to E8.5 (Hu et al., 2008), in a pattern mirroring embryonic methylation acquisition. On the one hand, Dnmt3l deficiency in ES Bisulfite analysis Bisulfite genomic treatment on postimplantation embryos was cells impairs both de novo methylation of newly integrated proviral performed using the Epitect kit (Qiagen). For E6.5, seven Dnmt3lwt and DNA and maintenance of genomic methylation patterns upon five Dnmt3lz– epiblasts were pooled for DNA extraction. For E9.5, DNA multiple passages in culture (Ooi et al., 2010; Arand et al., 2012). from two independent embryos was treated separately for each On the other hand, Dnmt3L deficiency in living organisms does not genotype. For oocytes (300) and blastocysts (50), DNA was extracted as seem to compromise somatic methylation. Indeed, although described (Smith et al., 2009). For oocytes, bisulfite conversion was Dnmt3l–/– mice are sterile they are perfectly viable with no overt performed on agarose-embedded DNA. For blastocysts, bisulfite phenotype and present normal methylation patterns in somatic conversion was carried out with the Epitect kit. Single, nested or semi- tissues in pre- and postnatal life (Bourc’his et al., 2001; Schulz et nested PCR was conducted using the primers listed in supplementary al., 2010). By contrast, Dnmt3b–/– embryos die at mid-gestation, material Table S2. Amplification products were cloned using the TOPO- –/– TA vector system (Invitrogen) and individually sequenced, or directly and Dnmt3a animals develop to term but die within one month processed by mass array technology (Sequenom) as described previously (Okano et al., 1999). Contrary to the data obtained in cellular (Popp et al., 2010). Lack of somatic contamination in oocyte samples systems, this in vivo observation strongly argues against an was assessed by profiling the methylation of the paternally and essential role of Dnmt3L in embryonic methylation. Alternatively, maternally imprinted loci H19 DMD and Kv DMR, respectively (data the presence of maternally inherited Dnmt3L has been proposed as not shown). Quma Analyzer software was used for sequence alignments a potential source of Dnmt3L to support the formation of normal (quma.cdb.riken.jp) and clones with identical patterns of conversion methylation patterns in Dnmt3l–/– embryos. were removed from the final Pileup. Sequence logos were created using Whether de novo methylation can occur with or without Dnmt3L WebLogo (weblogo.berkeley.edu). depending on the developmental context is an important issue that Immunofluorescence on whole-mount embryos will shed light on the mechanisms governing methylation patterns Whole-mount immunostaining was performed essentially as previously in normal and pathological states. Here we present a detailed in described (Santos et al., 2005), with some minor modifications. Briefly, vivo analysis of the regulation and requirement of Dnmt3L in following ZP removal, embryos were fixed in 3% paraformaldehyde for 15 gametogenesis and early embryogenesis. Our work illustrates that, minutes at room temperature, and then permeabilized with 0.5% Triton X- contrary to gametic methylation, embryonic methylation can be 100 in PBS at 4°C from 3 to 20 minutes (oocyte to E4.5). After blocking achieved without Dnmt3L, potentially through the ability of the in 5% BSA and 0.1% Triton X-100 in PBS, embryos were incubated with embryo to express and assemble various combinations of the de primary antibodies at 4°C overnight: mouse anti-mouse Dnmt3A (Abcam ab13888; 1:200), mouse anti-mouse Dnmt3B (Abcam ab13604; 1:100), novo methylation complex. rabbit anti-mouse Dnmt3L [from S. Tajima (Sakai et al., 2004); 1:200], mouse anti-mouse Cdx2 (Biogenex AM392; 1:50), and mouse anti-mouse MATERIALS AND METHODS Nanog (BD Biosciences 560259; 1:100). Two-dimensional images were Embryo collection obtained using an epifluorescence photomicroscope (Digital Module R, All procedures using animals were reviewed and approved by the Institut Leica). Confocal sections were obtained at 0.2 µm intervals on an Curie Animal Care and Use Committee. Oocytes and preimplantation UltraVIEW spinning disk laser-scanning confocal multifocal monophoton embryos were recovered from ~5-week-old superovulated B6/CBA F1 Mus (PerkinElmer) using 60ϫ and 40ϫ objectives for E2.5 and E3.5-4.5 musculus females. Trophectoderm (TE) cells were dissected as follows. embryos, respectively. E3.5 embryos were placed individually in 5 l M2 medium under oil, held in place by gentle suction using a holding micropipette (Humagen, Origio, Western blot France) and rotated until the inner cell mass (ICM) was at the 3 o’clock For western blotting, 30 E6.5 embryos were collected from wt and position. A large opening in the zona pellucida (ZP) was created by laser Dnmt3lz– genotypes and homogenized in 100 l ice-cold buffer comprising force (MTM Medical Technology, Montreux SA, Switzerland), from which 10 mM NaH2PO4, 150 mM NaCl, 6 mM CaCl2, 0.5% NP40 and protease the ICM was aspirated with a biopsy micropipette (Humagen). The ZP was inhibitors (Roche). Protein extracts were run on 4/16% SDS-PAGE removed by gentle suction and the TE cells were aspirated with the holding polyacrylamide gradient gels. Blocking was performed in 5% milk, 0.1% micropipette and dissected from the blastocyst using four laser pulses of 3- Tween 20 in PBS. Anti-Dnmt3A (Abcam ab13888; 1:500) and anti-tubulin msecond duration. TE cells were directly frozen at –80°C. ICM cells were (Oncogene CP06; 1:1000) antibodies were incubated overnight at 4°C and isolated by immunosurgery (Nagy et al., 2003). Postimplantation Dnmt3lwt detected by HRP-conjugated secondary antibodies (GE Healthcare). The +/+ z– –/–

(Dnmt3l ) and Dnmt3l (Dnmt3l ) embryos were obtained from ECL system (Amersham) was used to detect the signal. DEVELOPMENT 564 RESEARCH ARTICLE Development 140 (3)

RESULTS regained Dnmt3L protein, with cell-to-cell variation in intensity but Dnmt3L expression profile during mouse no obvious delineation between the inner cell mass (ICM) and the preimplantation development trophectoderm (TE). At E4.5, cells positive for Dnmt3L were Little is known about how the specificity of Dnmt3l transcripts is clearly restricted to a discrete area of the expanded blastocyst. Co- regulated in gametes or which forms are found in early embryos staining for the TE marker caudal type homeobox 2 (Cdx2) before implantation. We determined the kinetics of Dnmt3L confirmed that the Dnmt3L protein progressively disappears from expression during preimplantation development from the ovulated the mural and the polar TE from which the placenta and other oocyte (E0) to the late blastocyst (E4.5) so as to address the extra-embryonic membranes originate, and is instead specific to the transition between maternally inherited and autonomous zygotic ICM, which will give rise to the embryo proper (Fig. 1B). The expression. For protein assessment, we performed whole-mount Dnmt3L territory was larger than that of the pluripotency factor immunostaining with an antibody raised against the C-terminal Nanog, suggesting that Dnmt3L expression might extend to the region of mouse Dnmt3L (Sakai et al., 2004), the specificity of primitive endoderm (supplementary material Fig. S2A). which we confirmed on fertilized Dnmt3l–/– oocytes In RT-qPCR assays, the total amount of Dnmt3l mRNA followed (supplementary material Fig. S1). the same developmental kinetics as observed at the protein level. In the wild-type (wt) ovulated oocyte, Dnmt3L protein We observed a sharp decrease from the oocyte to the 2-cell stage accumulated on meiotic (Fig. 1A). In the 1-cell embryo, which is a common trait for maternally inherited zygote, the maternal protein was distributed equally on the transcripts, followed by reinitiation of expression between E2.5 and maternal and paternal pronuclei (supplementary material Fig. S1), E3.5, and a dramatic increase from E3.5 to E4.5 (Fig. 1C). with enrichment in DAPI-dense ring structures around nucleolar Moreover, there was negligible Dnmt3l transcript in the TE as early bodies, a staining reminiscent of pericentromeric heterochromatin. as E3.5, suggesting that transcript disappearance precedes Dnmt3L The protein persisted only briefly in the nucleus until the 2-cell protein loss in these cells (supplementary material Fig. S2B). Using stage, and was mostly absent from the 4-cell to compacted morula primers specific to different Dnmt3l transcript isoforms, we (E2.5) stages. At E3.5, all the blastomeres of the blastocyst confirmed that the protein present at the 1-cell stage is the

Fig. 1. Dnmt3L protein and mRNA levels throughout mouse preimplantation development. (A) Representative images of indirect immunostaining of Dnmt3L (green) from ovulated oocytes to E4.5 embryos. (B) Dnmt3L protein becomes gradually excluded from Cdx2-positive trophectoderm (TE) cells (red) from E2.5 to E4.5. (C) Dnmt3l transcription can initiate at three alternative cell-specific promoters. The round spermatid promoter (blue) produces a transcript with no coding capacity (Shovlin et al., 2007). RT-qPCR assays indicate a switch from the oocyte- specific transcript Dnmt3loo (red) to the isoform previously described in prospermatogonia and ES cells, named here Dnmt3lzygo (green). Total Dnmt3l mRNA levels (black) are determined with primers that recognize both isoforms. Values are expressed relative to Hprt1 mRNA levels and are calibrated to the stage with the highest expression level for each transcript (100%). Error bars indicate s.d. from two technical replicates of pooled

oocytes and embryos. DEVELOPMENT De novo methylation without Dnmt3L RESEARCH ARTICLE 565 translation product of the oocyte transcript (Dnmt3loo), and that the methylation (Weber et al., 2007), we tested whether differential zygotic transcript activated after E2.5 is the same as that expressed DNA methylation acts as a regulator of promoter switching and in prospermatogonia and ES cells (Dnmt3lzygo) (Fig. 1C). lineage restriction of Dnmt3l transcription in the peri-conception The kinetics of Dnmt3lzygo expression is reminiscent of window. We analyzed the DNA methylation pattern of the pluripotency-dependent transcriptional regulation. Examination of Dnmt3loo and Dnmt3lzygo promoters in gametes, E3.5 publicly available ChIP-Seq data from ES cells revealed a preimplantation and E6.5 postimplantation embryos using a significant enrichment of pluripotency factor [Oct4 (Pou5f1), Sox2 bisulfite cloning approach. We investigated the regions spanning and Nanog] binding just upstream from Dnmt3lzygo (P<10–9), but the transcription start site (TSS) of each isoform, with Dnmt3loo not in the vicinity of the Dnmt3loo promoter (supplementary containing eight CpGs and an observed/expected CpG ratio of 0.6, material Fig. S2C) (Marson et al., 2008). Pluripotency factor and Dnmt3lzygo with 16 CpGs and a CpG ratio of 0.45. availability is therefore likely to underlie the specificity of Both promoters showed sex-specific methylation in gametes, but Dnmt3lzygo recruitment in pluripotent blastomeres of the with an opposite pattern: Dnmt3loo is specifically methylated in preimplantation embryo. sperm, whereas Dnmt3lzygo is methylated in the oocyte (Fig. 2A). This pattern qualifies these regions as germline differentially Dnmt3l promoter methylation in gametes and methylated regions (gDMRs) of paternal and maternal origin, peri-implantation embryos respectively. For the Dnmt3lzygo promoter, the region of oocyte- In terms of CpG enrichment, the Dnmt3loo and Dnmt3lzygo specific methylation extended at least 1 kb upstream from the TSS promoters belong to an intermediate class between those with high (Fig. 2A), but was previously shown to exclude the Aire promoter CpG content (CpG islands) and low CpG content. As the activity located 6 kb upstream (O’Doherty et al., 2011). As shown for other of these intermediate promoters is inversely correlated with DNA oocyte-methylated regions, abundant non-CG methylation was

Fig. 2. Gametic methylation patterns of Dnmt3l promoters. (A) The Dnmt3loo and Dnmt3lzygo promoters are methylated in a sex- specific manner in gametes, showing opposite patterns in oocyte and sperm, as determined by bisulfite sequencing. Nucleotide numbers are expressed relative to the TSS (+1) at each promoter. Black circle: methylated CG, white circle: unmethylated CG. (B) Cytosine methylation occurs at CG and non-CG contexts at the Dnmt3lzygo promoter (–186/+338 depicted 5Ј to 3Ј on the x-axis) in the oocyte. The percentage of methylation was determined at each cytosine position from 43 independent clones by bisulfite sequencing (A). (C) Sequence logos of non-CG methylation at the Dnmt3lzygo promoter in the oocyte. (D) Dnmt3lzygo methylation occurs in a Dnmt3L-dependent manner in the oocyte. Dnmt3l–/– oocytes show a reduction in CG methylation compared with the wild-type (wt) oocyte pattern (A). (E) Both CG and non-CG methylation are reduced in Dnmt3l–/– oocytes. Methylation percentage was determined from 43 and 27 clones for wt and Dnmt3l–/– oocytes, respectively. DEVELOPMENT 566 RESEARCH ARTICLE Development 140 (3) found at the Dnmt3lzygo promoter, mostly in the CA and CT context (Fig. 2B,C) (Tomizawa et al., 2011; Kobayashi et al., 2012). The dramatic reduction in methylation observed in Dnmt3l- deficient oocytes further confirmed that Dnmt3lzygo promoter methylation during oogenesis is controlled by the Dnmt3L protein itself, both in CG and non-CG contexts (Fig. 2D,E). The gDMRs of the Dnmt3l locus were, however, lost during preimplantation, as demonstrated by the hypomethylated state of both the Dnmt3loo and Dnmt3lzygo promoters in E3.5 blastocysts (Fig. 3A). Loss of parent-of-origin methylation was associated with equal expression of the paternal and maternal alleles of the Dnmt3l transcript in hybrid E3.5 blastocysts derived from crosses between C57BL/6J and CASTEi/J mouse strains (Fig. 3B), and further confirmed in hybrid ES cells. Interestingly, although Dnmt3lzygo transcription was repressed in the TE as early as E3.5, no distinction in promoter methylation was found between the ICM and the TE at this stage (supplementary material Fig. S2D). After implantation, in E6.5 epiblasts, methylation is gained at both the Dnmt3loo and Dnmt3lzygo promoters (~75% methylated), as differentiation occurs and pluripotency is lost (Fig. 3A).

Zygotic Dnmt3L is involved in, but not strictly required for, the initiation of embryonic de novo methylation Zygotic Dnmt3L expression is concomitant with the wave of de novo methylation that occurs around the time of implantation. The timing of methylation initiation has not been precisely determined, but most data converge toward a window from E4.5 to E6.5, with locus-to-locus heterogeneity (Dean et al., 2001; Borgel et al., 2010). To functionally assess the role of Dnmt3L in this process, Fig. 3. Lack of parental specificity in DNA methylation and we analyzed the kinetics of methylation acquisition in expression of the Dnmt3l locus in the early mouse embryo. postimplantation embryos lacking zygotic Dnmt3l (Dnmt3l–/–, (A) Dnmt3loo and Dnmt3lzygo promoters are unmethylated before referred to here as Dnmt3lz–), as generated from heterozygous implantation in E3.5 blastocysts and do not show evidence of allelic crosses (Dnmt3l+/– ϫ Dnmt3l+/–), compared with their wt differences. They both globally regain methylation after implantation in E6.5 epiblasts. (B) Dnmt3l transcription occurs equally from both littermates. Various genomic sequences that are known to acquire parental alleles in hybrid E3.5 blastocysts derived from C57BL/6J ϫ methylation around the time of implantation were analyzed after CASTEi/J crosses. The yellow box highlights the detection of the two bisulfite conversion in dissected epiblasts at E6.5 for the beginning parental single-nucleotide polymorphisms (G+A) by Sanger sequencing. of de novo methylation and in E9.5 embryos for a readout at the end of the process; these comprised promoters of germline-specific and pluripotency genes, secondary or somatic DMRs of imprinted of alleles had at least one methylated CpG but with a much lower loci, and repetitive sequences such as LINE-1 elements and major average of 3.8 methylated CpGs per allele. satellite DNA. We included intracisternal A-particle (IAP) elements A significantly lower methylation density in E6.5 Dnmt3lz– as a control of maintenance methylation, as the methylation of embryos was also observed for three secondary, somatically these sequences has been reported to be globally unaltered acquired imprinted DMRs associated with the promoters of the throughout development (Lane et al., 2003; Guibert et al., 2012). Cdkn1c, Igf2r and Igf2 genes (P<10–5) (Fig. 4A; supplementary We found that the germline-expressed Scp3 (Sycp3 – Mouse material Fig. S3A). At E9.5, methylation levels were close to the Genome Informatics) and Dazl genes acquired complete expected 50% (in agreement with only one of the parental alleles methylation as early as E6.5 in wt epiblasts, confirming previous being methylated) both in wt and mutant embryos, except for Igf2 data (Borgel et al., 2010). CpG methylation levels of 90% were DMR1, which remained significantly hypomethylated in mutants observed at E6.5 for both promoters and were maintained until (36% versus 19.9%; P=0.0005). For genes associated with E9.5 (Fig. 4A). A dramatic reduction in methylation was observed pluripotency, the Oct4 promoter exhibited a methylation level in Dnmt3lz– embryos at E6.5, but significant methylation below 10% at both stages in wt, with increased methylation in E9.5 differences disappeared by E9.5. For Scp3, values of 89.1% and Dnmt3lz– embryos (8.4% versus 23.2%; P<10–4). The Dnmt3lzygo 20% CpG methylation (P<10–90, Fisher’s exact test) were observed and Dnmt3loo promoters were equally methylated in the presence at E6.5 and values of 87.9% and 75.3% (P=1.00) at E9.5 in wt and or absence of Dnmt3L, at E6.5 and E9.5. The embryonic context Dnmt3lz– embryos, respectively (Fig. 4A). Examination of therefore differs from that of the oocyte, where Dnmt3lzygo individual alleles at E6.5 indicated that Dnmt3L deficiency leads methylation was strictly dependent upon Dnmt3L. Finally, to a reduction in the number of alleles with methylated CpGs, but, methylation of repetitive sequences was globally unaltered in even more dramatically, in the number of methylated CpGs per Dnmt3lz– embryos, and this was observed both for clustered allele (Fig. 4B). In wt embryos, 100% of Scp3 alleles had at least centromeric repeats (major satellite DNA) and IAP elements. one methylated CpG, with an average of 16.9 methylated CpGs per However, for transposons of the LINE-1 family, some z–

allele (out of 19 analyzed CpGs). In the Dnmt3l background, 80% discrepancies were observed among subclasses of active elements, DEVELOPMENT De novo methylation without Dnmt3L RESEARCH ARTICLE 567

Fig. 4. Methylation and expression patterns of genes/ sequences targeted by embryonic de novo methylation in Dnmt3lz– embryos. (A) Average CG methylation of germline genes, somatic imprinted DMRs, pluripotency genes and repetitive sequences in wt and Dnmt3lz– mouse embryos at E6.5 and E9.5. Percentages were calculated after bisulfite sequencing of pools of embryos on 15-30 individual alleles for single-copy sequences, and by bisulfite followed by Sequenom for repetitive sequences. Although some sequences are hypomethylated in Dnmt3lz– embryos at E6.5, normal methylation levels are restored at E9.5, in comparison to wt embryos. *P<10–5, ** P<10–30, Fisher’s exact test. (B) Details of Scp3 promoter methylation in E6.5 wt and Dnmt3lz– embryos. Global methylation levels are reported (percentages as in A) as well as P-values between Dnmt3lz– and wt embryos. (C) Lower DNA methylation levels are not associated with increased transcription. Expression levels of the Scp3 gene and LINE-1 Tf elements are similar in E6.5 wt and Dnmt3lz– embryos. RT-qPCR values are expressed relative to Ppia mRNA. Error bars indicate s.d. from two technical replicates of pools of ten sex-matched embryos of each genotype.

which can be identified by specific monomeric repeats in their 5Ј methylation levels by E9.5, continue development, are born in UTR. The type A subclass was equally densely methylated in wt the expected Mendelian ratios (supplementary material Table and Dnmt3lz– embryos at both stages, whereas the Tf subclass S3), and progress throughout adulthood with normal somatic exhibited a delay in acquiring methylation in E6.5 mutant embryos, methylation patterns and no overt phenotype aside from sterility but gained normal methylation levels by E9.5. (Bourc’his et al., 2001). In summary, we provide evidence that the absence of Dnmt3L slows the establishment of embryonic methylation patterns. To The maternal store of Dnmt3L is not involved in rule out a developmental delay as a potential cause of reduced embryonic de novo methylation methylation, we confirmed that Dnmt3lz– embryos are Next, we investigated the potential developmental compensation of morphologically indistinguishable and present the same growth zygotic Dnmt3L deficiency, which would allow Dnmt3lz– embryos rate as their wt counterparts at E6.5 (supplementary material Fig. to successfully acquire methylation at some genomic sequences at S4), implying a direct effect of Dnmt3L on de novo methylation E6.5 and to fully recover normal methylation patterns at E9.5. In efficiency. Functionally, the temporary hypomethylation of the the homozygous state the Dnmt3l mutation prevents the production germline Scp3 gene and the LINE-1 Tf retrotransposons does not of a functional protein, from both the zygotic and oocyte promoters lead to increased transcription of these sequences in E6.5 (Shovlin et al., 2007). However, it has been argued that the Dnmt3lz– embryos (Fig. 4C; supplementary material Fig. S3B). maternal store of Dnmt3L could be maintained throughout This was an important point to address because reactivation of preimplantation development and could participate in de novo these sequences has been previously linked to genomic methylation, even in minute amounts. To functionally assess the instability (Bourc’his and Bestor, 2004; Borgel et al., 2010). contribution of maternal Dnmt3L to embryonic methylation, we Finally, the hypomethylation phenotype is largely compensated generated embryos deficient for both maternal and zygotic Dnmt3l z– m–z– –/–

a few days later, so that Dnmt3l embryos attain normal expression (Dnmt3l ) by crossing Dnmt3l females with DEVELOPMENT 568 RESEARCH ARTICLE Development 140 (3)

Fig. 5. Combined depletion of maternal and zygotic Dnmt3L does not affect the completion of embryonic methylation at E9.5. (A) Breeding scheme for the generation of maternally deficient (Dnmt3lm–z+) and maternally plus zygotically deficient (Dnmt3lm–z–) mouse embryos. Phenotypes are indistinguishable between Dnmt3lm–z+ and Dnmt3lm–z– embryos at E9.5, showing typical signs of maternal Dnmt3L deficiency (abnormal head morphology and enlarged pericardium). (B) Methylation analysis of Scp3, Igf2r DMR1 and Dnmt3lzygo sequences at E9.5. Dnmt3lm–, Dnmt3lz– and Dnmt3lm–z– do not show significant methylation changes compared with wt embryos. (C) Full methylation of three classes of repetitive sequences in each category of embryos at E9.5, as determined by Southern blotting. The same membrane was consequently hybridized with radiolabeled probes specific to LINE-1 type A, IAP and major satellite elements. Resistance of high molecular weight DNA to HpaII (H) digestion is indicative of DNA methylation. The MspI (M) isoschizomer is used as a control for full digestion equivalent to unmethylated states.

Dnmt3l+/– males (Fig. 5A). Embryos derived from Dnmt3l–/– transcribed in the embryo as early as E2.5, while Dnmt3lzygo females (Dnmt3lm–) die at E10.5 (Bourc’his et al., 2001). We found transcription was not yet activated. At E3.5 to E4.5, the Dnmt3B that Dnmt3lm–z– embryos did not progress past this stage and were protein is highly expressed. By contrast, Dnmt3A expression only phenotypically similar to their Dnmt3lm–z+ littermates (Dnmt3lm–), becomes obvious at E4.5 in the ICM of the late blastocyst, with a confirming the minor to zero impact of the zygotic mutation at this timing of activation more similar to that of Dnmt3lzygo stage of development, as compared with the severe effect of the (Fig. 6A,B). maternal Dnmt3l mutation. Interestingly, upregulation in Dnmt3a expression was observed We then analyzed the methylation status of sequences that we in response to zygotic Dnmt3l deficiency at E6.5, as demonstrated had previously found to be properly methylated at E9.5 in the by a twofold increase in transcription in Dnmt3lz– epiblasts absence of zygotic Dnmt3l. Using bisulfite sequencing, we compared with wt littermates (P<0.01) detected by RT-qPCR assessed the Scp3 promoter, Igf2r DMR1 and the Dnmt3lzygo (Fig. 6C). Dnmt3b transcript levels were comparatively unaffected. promoter, and a methylation-sensitive Southern blot approach was The specific upregulation of Dnmt3a could not be explained by a used to analyze three classes of repetitive sequences (LINE-1 type loss of promoter methylation in the Dnmt3lz– context: the two A, IAP and major satellite DNA). None of the analyzed loci alternative CpG-rich promoters of this locus – a 5Ј promoter that differed significantly in methylation in the absence of maternal or gives rise to Dnmt3a and an intragenic promoter that gives rise to zygotic Dnmt3L, nor, most importantly, in the absence of both the Dnmt3a2 isoform – are constitutively unmethylated at E3.5, maternal and zygotic Dnmt3L, as compared with wt embryos at E6.5 and E9.5 in wt embryos, as suggested from publicly available E9.5 (Fig. 5B,C). This convincingly illustrates that the maternal methylated DNA immunoprecipitation (MeDIP)-chip and reduced form of Dnmt3L inherited from the oocyte does not play a role in representation bisulfite sequencing (RRBS) datasets (Borgel et al., setting up methylation patterns around the time of implantation. 2010; Smith et al., 2012) (supplementary material Fig. S5). These results provide ultimate proof that, in striking contrast to the Importantly, increased steady-state levels of Dnmt3a transcripts situation in germ cells, de novo methylation can be achieved in the resulted in higher levels of Dnmt3A protein in Dnmt3lz– epiblasts. embryo without any source of Dnmt3L. Notably, Dnmt3A2, which is the most active form of Dnmt3A and abundant in ES cells and early postimplantation embryos (Nimura Dnmt3L deficiency is associated with higher levels et al., 2006), was clearly overexpressed in E6.5 Dnmt3lz– embryos, of Dnmt3A protein as revealed by western blotting (Fig. 6D). Although the details of Finally, we assessed which of the de novo methylation enzymes, the compensatory feedback of Dnmt3L deficiency on Dnmt3A Dnmt3A and Dnmt3B, is available at the time of methylation expression are not known, a greater availability of Dnmt3A protein pattern formation in the early embryo, in the presence or absence provides a likely mechanism for the normal acquisition of of Dnmt3L. By RT-qPCR and immunofluorescence, we confirmed embryonic methylation patterns in the absence of a Dnmt3L

that Dnmt3B is the first active DNA methyltransferase to be stimulatory effect. DEVELOPMENT De novo methylation without Dnmt3L RESEARCH ARTICLE 569

Fig. 6. Interplay between Dnmt3L and the Dnmt3 de novo DNA methyltransferases in the early mouse embryo. (A) Expression kinetics of Dnmt3l, Dnmt3a and Dnmt3b transcripts from ovulated oocytes to E4.5 embryos as determined by RT-qPCR. The three transcripts are maternally inherited by the embryo, then display variable timing of zygotic activation. Values are expressed relative to the Hprt1 mRNA level and are calibrated to expression in the oocyte, which represents the highest expression level for all transcripts (100%). Error bars indicate s.d. from two technical replicates of pooled oocytes and embryos. (B) Representative images of immunostaining showing Dnmt3B as the first Dnmt3 member to be expressed in the early embryo, accumulating at E3.5. Dnmt3A protein only becomes visible at E4.5. (C) Relative quantities of Dnmt3a and Dnmt3b transcripts in E6.5 Dnmt3lz– embryos compared with their wt littermates. Note the significant upregulation of Dnmt3a transcripts with primers detecting both isoforms (*P<0.01) and similar levels of Dnmt3b transcripts in Dnmt3lz– versus wt embryos, relative to - actin mRNA. Error bars indicate s.d. from two biological replicates. (D) Western blot analysis confirms the upregulation of Dnmt3A at the protein level in E6.5 Dnmt3lz– embryos. The arrow indicates the short, most active Dnmt3A protein isoform Dnmt3A2, which is the most abundant isoform in ES cells (left). Anti-tubulin antibody provides a loading control.

DISCUSSION DNA methylation has previously been shown to regulate Although Dnmt3L is strictly required for de novo methylation in Dnmt3l transcriptional output and promoter usage in the context germ cells, clear evidence was still lacking as to whether its of ES cells and adult tissues, as well as in vitro transfection stimulatory function is necessary in the developing embryo. studies (Aapola et al., 2004; Hu et al., 2008; O’Doherty et al., Our in vivo study demonstrates that a promoter switch underlies 2011). Here we confirm the mutually exclusive relationship the autonomous and time-staged expression of Dnmt3L in the between expression and promoter methylation in the oocyte, embryo, which appears to have an innate role in accelerating where the Dnmt3loo promoter is active and unmethylated and the de novo methylation process. However, we found that the the Dnmt3lzygo promoter is inactive and methylated. This is also absence of Dnmt3L can be compensated for by other factors. true for the postimplantation embryo, where the two promoters Compared with the germline, the early embryo is more plastic in are silenced and methylated. However, we identified two terms of the mechanisms by which DNA methylation patterns situations in which transcription control is achieved without can be acquired. DNA methylation: (1) in the E3.5 blastocyst, where both Dnmt3l Although the existence of alternative Dnmt3l promoters was promoters are unmethylated but only the Dnmt3lzygo transcript known (Shovlin et al., 2007), we reveal here that these promoters is found; and (2) in the TE, where the Dnmt3lzygo promoter is are transmitted to the embryo with opposite allelic states of DNA unmethylated but the corresponding transcript is not produced. methylation acquired in the parental gametes: Dnmt3lzygo is If transcriptional regulation is involved, it occurs in a DNA methylated on the maternal allele whereas Dnmt3loo is methylated methylation-independent manner and/or additional factors are on the paternal allele. Recent genome-wide methylation studies required to activate hypomethylated promoters in these contexts. have highlighted certain genomic features that are conducive to In support of this hypothesis, the Dnmt3lzygo promoter is oocyte methylation: high CpG density and intragenic location physically bound by pluripotency factors in ES cells (Marson et (Chotalia et al., 2009; Smallwood et al., 2011; Kobayashi et al., al., 2008), which fits with our in vivo observation of Dnmt3L 2012). The maternal Dnmt3lzygo gDMR fulfills these two criteria, restriction to the pluripotent part of the embryo. Our study as it is located within the transcription unit defined by the 5Ј highlights a two-step regulatory model whereby clearance of Dnmt3loo promoter, which is specifically active in the oocyte. parent-inherited methylation could potentiate promoter activity, Although subject to parent-specific methylation at fertilization, the then embryonic pluripotency factors further specify this activity. two promoters of Dnmt3l are not imprinted, as methylation is not This double security system might prevent spurious activation of maintained during embryonic cleavage. Accordingly, we found no key factors, such as the DNA methylation effectors, to provide evidence of binding sites at these loci for Zfp57, which is likely to efficient spatiotemporal control of genome activity and lineage be an obligate factor for the protection of gamete-inherited decision. This type of regulation was formerly reported for the methylation (Quenneville et al., 2011; Messerschmidt et al., 2012; Nanog gene (Farthing et al., 2008). Our study suggests that it

Proudhon et al., 2012). could apply more generally to targets of pluripotency factors. DEVELOPMENT 570 RESEARCH ARTICLE Development 140 (3)

The intricate regulation of Dnmt3l expression may attest to its zygotic in origin. This situation is at odds with the obligate important function during embryonic development. Our work requirement of Dnmt3L for full methylation in gametes (Bourc’his formally demonstrates that Dnmt3L is indeed involved in de novo et al., 2001). This unique property of the embryo occurs in the methylation at implantation. The germline-expressed Scp3 and context of higher levels of the Dnmt3A enzyme, and in particular Dazl genes, some somatic imprinted DMRs and LINE-1 Tf of the most active Dnmt3A2 isoform. By contrast, Dnmt3L elements are significantly, although not completely, deficiency has no effect on Dnmt3 enzyme levels in hypomethylated in Dnmt3lz– embryos at E6.5, within the early prospermatogonia (Niles et al., 2011), and, although it leads to steps of methylation acquisition. The most potent effect we upregulation of Dnmt3b transcripts in oocytes the amount of observed is on methylation density per DNA strand, thus providing Dnmt3B protein is unchanged (Lucifero et al., 2007). Increased developmental evidence to support the proposal that Dnmt3L Dnmt3A availability might result from transcriptional and/or post- enhances the deposition of long methylation tracts (Wienholz et al., transcriptional mechanisms. Because both Dnmt3a promoters are 2010), potentially by stimulating the processivity and/or the constitutively methylation free in wt peri-implantation embryos, catalytic rate of Dnmt3 enzymes once they are loaded on DNA methylation changes in Dnmt3lz– embryos should not affect (Kareta et al., 2006; Holz-Schietinger and Reich, 2010; Chédin, Dnmt3a expression levels directly (Borgel et al., 2010; Smith et al., 2011). However, Dnmt3L-dependent stimulation is not universal, 2012). Of note, several microRNA families have been shown to as other sequences are properly methylated at E6.5 in the absence modulate Dnmt3a and Dnmt3b expression. Downregulation of of Dnmt3L. Moreover, Dnmt3L-dependent effects appear to be miR-29 could cause higher levels of Dnmt3A in the Dnmt3l mutant time specific, as full methylation patterns are globally recovered in background (Takada et al., 2009). Alternatively, increased Dnmt3A Dnmt3lz– embryos at E9.5. Taken altogether, our work suggests that levels could be the indirect consequence of upregulation of the Dnmt3L facilitates the formation of methylation patterns in the miR-290 species, which target the Dnmt repressor Rbl2 (Benetti et embryo in its initiation phase. Only by undertaking a al., 2008; Sinkkonen et al., 2008). developmental analysis of ongoing de novo methylation at Although we cannot conclude that increased Dnmt3A individual loci was it possible to reveal such subtle spatiotemporal availability is responsible for the acquisition of normal methylation details of the influence of Dnmt3L on embryonic de novo patterns, it could mechanistically counterbalance Dnmt3L methylation. depletion. In biochemical assays, maximal Dnmt3L-mediated Differential Dnmt3L-dependent stimulation might reflect stimulation is achieved at a 1:1 molar stoichiometry of Dnmt3L and differential Dnmt3A or Dnmt3B target specificity. On the one hand, Dnmt3A, but increasing Dnmt3A protein alone can also lead to expression and locus-specific analyses have identified Dnmt3B as increased recruitment of methyl group donors (SAM). Moreover, the main enzyme responsible for de novo methylation in the although Dnmt3L interacts with Dnmt3A2 to form tetrameric embryo, although some rare sequences require Dnmt3A (Okano et structures, Dnmt3A2 is capable of auto-assembling into large al., 1999; Watanabe et al., 2002; Borgel et al., 2010). On the other polymers of up to 30 monomers in solution and likely also in vivo hand, Dnmt3L can potentiate both Dnmt3A and Dnmt3B activity (Kareta et al., 2006; Holz-Schietinger and Reich, 2010). Our work in vitro, but Dnmt3A appears to be the favored partner in vivo and therefore illustrates that the embryo may be flexible in its ability to in particular in ES cells (Nimura et al., 2006). Of direct relevance use various combinations of Dnmt3A, Dnmt3B and Dnmt3L to our results, de novo methylation of Dazl and Dnmt3lzygo homo- and heterocomplexes, depending on relative partner promoters was demonstrated to be strictly Dnmt3B dependent (Hu availability. By contrast, the assembly of Dnmt3L-associated et al., 2008; Borgel et al., 2010). The fact that these sequences are z– complexes is strictly required for proper methylation in the either hypomethylated or normally methylated in E6.5 Dnmt3l germline (Bourc’his et al., 2001; Bourc’his and Bestor, 2004; embryos does not support preferential Dnmt3L stimulation of Kaneda et al., 2004; Kaneda et al., 2010). Interestingly, DNA Dnmt3A or Dnmt3B. Differential methylation kinetics provides replication might play an enhancing role in de novo methylation another potential explanation for the observed difference in and alleviate the need for Dnmt3L stimulation: indeed, whereas Dnmt3L requirement. In this regard, X-linked CpG islands that embryonic methylation occurs in rapidly cycling cells, gametic acquire methylation slowly upon X-chromosome inactivation were methylation occurs in non-dividing cell types during male and shown to require the activity of the Smchd1 chromatin protein, female germ cell development (Bourc’his et al., 2001; Bourc’his whereas fast-acquiring sequences do not (Gendrel et al., 2012). and Bestor, 2004). Such a relationship might not apply to Dnmt3L, however. For To conclude, whereas abnormal methylation patterns are usually example, although the Scp3 promoter and Cdkn1c DMR are both z– associated with dramatic outcomes, the slight delay in acquiring hypomethylated in E6.5 Dnmt3l embryos, Scp3 is a fast-acquiring full methylation has no somatic impact on Dnmt3lz– individuals, sequence that is already completely methylated in E6.5 wt underscoring the efficiency and precision of Dnmt3L-independent embryos, whereas the Cdkn1c DMR is a slow-acquiring sequence, methylation in the developing embryo. attaining full methylation only by E9.5. More generally, the process of de novo methylation has been shown to be influenced by local Acknowledgements CpG content, gene density, transcription and histone H3K4 We thank Anne-Valérie Gendrel for assistance with Sequenom analysis; Renata methylation states (Ooi et al., 2007; Weber et al., 2007; Borgel et Kozyraki for help with protein analysis in early embryos; Shoji Tajima for providing the anti-Dnmt3L antibody; Mickael Weber for sharing the MeDIP- al., 2010; Lienert et al., 2011; Gendrel et al., 2012). Our study chip data on Dnmt3a promoters; the PICT IBiSA Genetics and Developmental represents an indispensable proof of principle for further Biology Imaging Facility for help with microscopy; and Isabelle Grandjean and investigation of the sequence elements and/or chromatin contexts Colin Jouanneau for excellent mouse husbandry. genome-wide that require Dnmt3L for a fast rate of DNA methylation acquisition in the early developing embryo. Funding This work was supported by a grant from the Schlumberger Foundation and The most important finding of our work is the formal by a European Young Investigator (EURYI) Award from the European Union to demonstration that embryos can attain normal genomic methylation D.B. R.D. is the recipient of an Institut Curie PhD fellowship. M.G. is the

levels in the absence of any source of Dnmt3L, either maternal or recipient of a DIM-Stem Pole post-doctoral fellowship. DEVELOPMENT De novo methylation without Dnmt3L RESEARCH ARTICLE 571

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