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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.
INTRODUCTION
Cytosine methylation profoundly impacts genome expression and
stability. In mammals, its occurrence at CpG-rich promoters is
correlated with transcriptional repression of retrotransposons and
various single-copy sequences, such as pluripotency and germlineexpressed genes, as well as genes subject to genomic imprinting
and X-chromosome inactivation. DNA methylation is essential for
mammalian development and reproduction, as shown by the early
embryonic lethality and sterility phenotypes of mouse models of
global DNA methylation deficiency (Li et al., 1992; Okano et al.,
1999; Bourc’his et al., 2001; Kaneda et al., 2004; Bostick et al.,
2007). Abnormal methylation patterns are also associated with
pathological states in humans, including cancer (Rakyan et al.,
2011). DNA methylation patterns are set genome-wide during two
main developmental stages: gametogenesis and early
embryogenesis (Morgan et al., 2005; Borgel et al., 2010). These
establishment phases follow a genome-wide clearance of
methylation, which occurs concomitantly with the expression of
pluripotency factors, in primordial germ cells and preimplantation
embryos (Rougier et al., 1998; Smallwood et al., 2011; Guibert et
al., 2012; Smith et al., 2012).
DNA methylation patterns are driven de novo by two DNA
methyltransferase enzymes, Dnmt3A and Dnmt3B, which can
target cytosines in both CG and non-CG contexts (Chédin, 2011).
Expression of these proteins peaks at times of major de novo
methylation in developing germ cells, early embryos and
embryonic stem (ES) cells (Watanabe et al., 2002; La Salle et al.,
2004; Sakai et al., 2004; Hirasawa et al., 2008). Although these
enzymes may show some degree of functional redundancy, they are
INSERM U934/UMR3215, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05,
France.
*These authors contributed equally to this work.
‡
Author for correspondence ([email protected])
Accepted 12 November 2012
not interchangeable. Dnmt3A is strictly required for gametic
methylation but is globally dispensable for de novo methylation in
the early embryo. Conversely, gametic methylation can occur
without Dnmt3B, whereas embryonic methylation cannot (Okano
et al., 1999; Kaneda et al., 2004; Borgel et al., 2010; Kaneda et al.,
2010). Conditional deletions of the Dnmt3a gene further highlight
its involvement in methylation processes that take place later, in
postnatal cell lineages (Nguyen et al., 2007; Challen et al., 2012).
The reasons for this stage specificity in Dnmt3A and Dnmt3B
function are unknown.
Whereas Dnmt3a and Dnmt3b orthologs are found widely
among distant eukaryotic species, a third Dnmt3 member, Dnmt3like (Dnmt3l), has evolved uniquely in mammals (Yokomine et al.,
2006). This gene encodes a catalytically inert protein that is unable
to methylate DNA substrates alone but serves as a regulator of the
de novo methylation machinery. In biochemical assays performed
in vitro and in cellular systems, human DNMT3L increases the
efficiency of the methylation reaction by stimulating DNMT3A and
DNMT3B catalytic activity (Chédin et al., 2002; Suetake et al.,
2004; Moarefi and Chédin, 2011) and the deposition of more
uniform methylation patterns (Wienholz et al., 2010). DNMT3L is
moreover involved in the organization of tetrameric complexes
with DNMT3A (Jia et al., 2007), leading to the formation of
DNMT3L-DNMT3A filaments that polymerize along the DNA
molecule in vitro (Jurkowska et al., 2008). Dnmt3L-dependent
stimulation of Dnmt3 enzyme activity has been linked to increased
affinity for the methyl donor S-adenosyl-L-methionine (SAM),
longer residency on DNA, and improved processivity, none of
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;
Shovlin et al., 2007). The canonical transcript is produced in
prospermatogonia, whereas in oocytes a longer transcript originates
from an upstream promoter embedded in an intron of the
neighboring Aire gene. Although different in length, these
transcripts produce a protein of similar size (O’Doherty et al.,
DEVELOPMENT
KEY WORDS: DNA methylation, Gametes, Early mouse embryo
De novo methylation without Dnmt3L
MATERIALS AND METHODS
Embryo collection
All procedures using animals were reviewed and approved by the Institut
Curie Animal Care and Use Committee. Oocytes and preimplantation
embryos were recovered from ~5-week-old superovulated B6/CBA F1 Mus
musculus females. Trophectoderm (TE) cells were dissected as follows.
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,
France) and rotated until the inner cell mass (ICM) was at the 3 o’clock
position. A large opening in the zona pellucida (ZP) was created by laser
force (MTM Medical Technology, Montreux SA, Switzerland), from which
the ICM was aspirated with a biopsy micropipette (Humagen). The ZP was
removed by gentle suction and the TE cells were aspirated with the holding
micropipette and dissected from the blastocyst using four laser pulses of 3msecond duration. TE cells were directly frozen at –80°C. ICM cells were
isolated by immunosurgery (Nagy et al., 2003). Postimplantation Dnmt3lwt
(Dnmt3l+/+) and Dnmt3lz– (Dnmt3l–/–) embryos were obtained from
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(Dnmt3l+/– ⫻ Dnmt3l+/–) crosses, whereas Dnmt3lm– (Dnmt3l–/+) and
Dnmt3lm–z– (Dnmt3l–/–) embryos were obtained from (Dnmt3l–/– ⫻
Dnmt3l+/–) crosses (Bourc’his et al., 2001). At E6.5, epiblasts were
separated from the extra-embryonic ectoderm (Nagy et al., 2003). At E9.5,
only embryos were kept for analysis. Dnmt3l and sex genotyping were
performed by PCR on extra-embryonic tissues for E6.5 and E9.5
conceptuses.
Quantitative RT-PCR (RT-qPCR)
For oocytes and preimplantation embryos, ~300 diploid genomes were
collected per stage, total RNA was obtained by three freeze/thaw cycles as
previously described (Jeong et al., 2005) and lysis extracts were processed
for cDNA conversion (Superscript III, Invitrogen). For E6.5 embryos, ten
epiblasts of sex-matched wt and Dnmt3lz– genotype were separately
pooled, extracted using the RNA Picopure kit (Applied Biosystems) and
treated with DNase (TURBO DNase, Ambion) prior to cDNA conversion.
Quantitative amplification of cDNA was performed using the PCR primers
listed in supplementary material Table S1 on an Mx4000 QPCR system
according to manufacturer instructions (Stratagene).
Bisulfite analysis
Bisulfite genomic treatment on postimplantation embryos was
performed using the Epitect kit (Qiagen). For E6.5, seven Dnmt3lwt and
five Dnmt3lz– epiblasts were pooled for DNA extraction. For E9.5, DNA
from two independent embryos was treated separately for each
genotype. For oocytes (300) and blastocysts (50), DNA was extracted as
described (Smith et al., 2009). For oocytes, bisulfite conversion was
performed on agarose-embedded DNA. For blastocysts, bisulfite
conversion was carried out with the Epitect kit. Single, nested or seminested PCR was conducted using the primers listed in supplementary
material Table S2. Amplification products were cloned using the TOPOTA vector system (Invitrogen) and individually sequenced, or directly
processed by mass array technology (Sequenom) as described previously
(Popp et al., 2010). Lack of somatic contamination in oocyte samples
was assessed by profiling the methylation of the paternally and
maternally imprinted loci H19 DMD and Kv DMR, respectively (data
not shown). Quma Analyzer software was used for sequence alignments
(quma.cdb.riken.jp) and clones with identical patterns of conversion
were removed from the final Pileup. Sequence logos were created using
WebLogo (weblogo.berkeley.edu).
Immunofluorescence on whole-mount embryos
Whole-mount immunostaining was performed essentially as previously
described (Santos et al., 2005), with some minor modifications. Briefly,
following ZP removal, embryos were fixed in 3% paraformaldehyde for 15
minutes at room temperature, and then permeabilized with 0.5% Triton X100 in PBS at 4°C from 3 to 20 minutes (oocyte to E4.5). After blocking
in 5% BSA and 0.1% Triton X-100 in PBS, embryos were incubated with
primary antibodies at 4°C overnight: mouse anti-mouse Dnmt3A (Abcam
ab13888; 1:200), mouse anti-mouse Dnmt3B (Abcam ab13604; 1:100),
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
Nanog (BD Biosciences 560259; 1:100). Two-dimensional images were
obtained using an epifluorescence photomicroscope (Digital Module R,
Leica). Confocal sections were obtained at 0.2 µm intervals on an
UltraVIEW spinning disk laser-scanning confocal multifocal monophoton
(PerkinElmer) using 60⫻ and 40⫻ objectives for E2.5 and E3.5-4.5
embryos, respectively.
Western blot
For western blotting, 30 E6.5 embryos were collected from wt and
Dnmt3lz– genotypes and homogenized in 100 l ice-cold buffer comprising
10 mM NaH2PO4, 150 mM NaCl, 6 mM CaCl2, 0.5% NP40 and protease
inhibitors (Roche). Protein extracts were run on 4/16% SDS-PAGE
polyacrylamide gradient gels. Blocking was performed in 5% milk, 0.1%
Tween 20 in PBS. Anti-Dnmt3A (Abcam ab13888; 1:500) and anti-tubulin
(Oncogene CP06; 1:1000) antibodies were incubated overnight at 4°C and
detected by HRP-conjugated secondary antibodies (GE Healthcare). The
ECL system (Amersham) was used to detect the signal.
DEVELOPMENT
2011). Dnmt3L activity is strictly required for gametic methylation
and fertility. Dnmt3l–/– males do not produce mature sperm, in
association with retrotransposon hypomethylation and reactivation
during spermatogenesis (Bourc’his and Bestor, 2004). Dnmt3l–/–
females produce methylation-deficient oocytes and their Dnmt3l–/+
progeny systematically die at mid-gestation, in association with the
dysregulation of imprinted gene expression (Bourc’his et al., 2001;
Smallwood et al., 2011; Kobayashi et al., 2012). Constitutive
Dnmt3l mutant phenotypes mimic a conditional knockout of
Dnmt3a in germ cells (Kaneda et al., 2004).
Gametic de novo methylation therefore occurs under the control
of a developmentally regulated Dnmt3L-dependent pathway.
However, whether embryonic de novo methylation occurs in a
Dnmt3L-dependent manner is still controversial. Dnmt3L is
abundantly expressed in ES cells from the same promoter used in
prospermatogonia (Bourc’his et al., 2001; Shovlin et al., 2007).
Dnmt3L protein is found in postimplantation embryos from E5.5
to E8.5 (Hu et al., 2008), in a pattern mirroring embryonic
methylation acquisition. On the one hand, Dnmt3l deficiency in ES
cells impairs both de novo methylation of newly integrated proviral
DNA and maintenance of genomic methylation patterns upon
multiple passages in culture (Ooi et al., 2010; Arand et al., 2012).
On the other hand, Dnmt3L deficiency in living organisms does not
seem to compromise somatic methylation. Indeed, although
Dnmt3l–/– mice are sterile they are perfectly viable with no overt
phenotype and present normal methylation patterns in somatic
tissues in pre- and postnatal life (Bourc’his et al., 2001; Schulz et
al., 2010). By contrast, Dnmt3b–/– embryos die at mid-gestation,
and Dnmt3a–/– animals develop to term but die within one month
(Okano et al., 1999). Contrary to the data obtained in cellular
systems, this in vivo observation strongly argues against an
essential role of Dnmt3L in embryonic methylation. Alternatively,
the presence of maternally inherited Dnmt3L has been proposed as
a potential source of Dnmt3L to support the formation of normal
methylation patterns in Dnmt3l–/– embryos.
Whether de novo methylation can occur with or without Dnmt3L
depending on the developmental context is an important issue that
will shed light on the mechanisms governing methylation patterns
in normal and pathological states. Here we present a detailed in
vivo analysis of the regulation and requirement of Dnmt3L in
gametogenesis and early embryogenesis. Our work illustrates that,
contrary to gametic methylation, embryonic methylation can be
achieved without Dnmt3L, potentially through the ability of the
embryo to express and assemble various combinations of the de
novo methylation complex.
RESEARCH ARTICLE
RESEARCH ARTICLE
RESULTS
Dnmt3L expression profile during mouse
preimplantation development
Little is known about how the specificity of Dnmt3l transcripts is
regulated in gametes or which forms are found in early embryos
before implantation. We determined the kinetics of Dnmt3L
expression during preimplantation development from the ovulated
oocyte (E0) to the late blastocyst (E4.5) so as to address the
transition between maternally inherited and autonomous zygotic
expression. For protein assessment, we performed whole-mount
immunostaining with an antibody raised against the C-terminal
region of mouse Dnmt3L (Sakai et al., 2004), the specificity of
which we confirmed on fertilized Dnmt3l–/– oocytes
(supplementary material Fig. S1).
In the wild-type (wt) ovulated oocyte, Dnmt3L protein
accumulated on meiotic chromosomes (Fig. 1A). In the 1-cell
zygote, the maternal protein was distributed equally on the
maternal and paternal pronuclei (supplementary material Fig. S1),
with enrichment in DAPI-dense ring structures around nucleolar
bodies, a staining reminiscent of pericentromeric heterochromatin.
The protein persisted only briefly in the nucleus until the 2-cell
stage, and was mostly absent from the 4-cell to compacted morula
(E2.5) stages. At E3.5, all the blastomeres of the blastocyst
Development 140 (3)
regained Dnmt3L protein, with cell-to-cell variation in intensity but
no obvious delineation between the inner cell mass (ICM) and the
trophectoderm (TE). At E4.5, cells positive for Dnmt3L were
clearly restricted to a discrete area of the expanded blastocyst. Costaining for the TE marker caudal type homeobox 2 (Cdx2)
confirmed that the Dnmt3L protein progressively disappears from
the mural and the polar TE from which the placenta and other
extra-embryonic membranes originate, and is instead specific to the
ICM, which will give rise to the embryo proper (Fig. 1B). The
Dnmt3L territory was larger than that of the pluripotency factor
Nanog, suggesting that Dnmt3L expression might extend to the
primitive endoderm (supplementary material Fig. S2A).
In RT-qPCR assays, the total amount of Dnmt3l mRNA followed
the same developmental kinetics as observed at the protein level.
We observed a sharp decrease from the oocyte to the 2-cell stage
embryo, which is a common trait for maternally inherited
transcripts, followed by reinitiation of expression between E2.5 and
E3.5, and a dramatic increase from E3.5 to E4.5 (Fig. 1C).
Moreover, there was negligible Dnmt3l transcript in the TE as early
as E3.5, suggesting that transcript disappearance precedes Dnmt3L
protein loss in these cells (supplementary material Fig. S2B). Using
primers specific to different Dnmt3l transcript isoforms, we
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 oocytespecific 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
564
translation product of the oocyte transcript (Dnmt3loo), and that the
zygotic transcript activated after E2.5 is the same as that expressed
in prospermatogonia and ES cells (Dnmt3lzygo) (Fig. 1C).
The kinetics of Dnmt3lzygo expression is reminiscent of
pluripotency-dependent transcriptional regulation. Examination of
publicly available ChIP-Seq data from ES cells revealed a
significant enrichment of pluripotency factor [Oct4 (Pou5f1), Sox2
and Nanog] binding just upstream from Dnmt3lzygo (P<10–9), but
not in the vicinity of the Dnmt3loo promoter (supplementary
material Fig. S2C) (Marson et al., 2008). Pluripotency factor
availability is therefore likely to underlie the specificity of
Dnmt3lzygo recruitment in pluripotent blastomeres of the
preimplantation embryo.
Dnmt3l promoter methylation in gametes and
peri-implantation embryos
In terms of CpG enrichment, the Dnmt3loo and Dnmt3lzygo
promoters belong to an intermediate class between those with high
CpG content (CpG islands) and low CpG content. As the activity
of these intermediate promoters is inversely correlated with DNA
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565
methylation (Weber et al., 2007), we tested whether differential
DNA methylation acts as a regulator of promoter switching and
lineage restriction of Dnmt3l transcription in the peri-conception
window. We analyzed the DNA methylation pattern of the
Dnmt3loo and Dnmt3lzygo promoters in gametes, E3.5
preimplantation and E6.5 postimplantation embryos using a
bisulfite cloning approach. We investigated the regions spanning
the transcription start site (TSS) of each isoform, with Dnmt3loo
containing eight CpGs and an observed/expected CpG ratio of 0.6,
and Dnmt3lzygo with 16 CpGs and a CpG ratio of 0.45.
Both promoters showed sex-specific methylation in gametes, but
with an opposite pattern: Dnmt3loo is specifically methylated in
sperm, whereas Dnmt3lzygo is methylated in the oocyte (Fig. 2A).
This pattern qualifies these regions as germline differentially
methylated regions (gDMRs) of paternal and maternal origin,
respectively. For the Dnmt3lzygo promoter, the region of oocytespecific methylation extended at least 1 kb upstream from the TSS
(Fig. 2A), but was previously shown to exclude the Aire promoter
located 6 kb upstream (O’Doherty et al., 2011). As shown for other
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 sexspecific 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 5⬘ to 3⬘ 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
De novo methylation without Dnmt3L
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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 Dnmt3ldeficient 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).
Fig. 3. Lack of parental specificity in DNA methylation and
expression of the Dnmt3l locus in the early mouse embryo.
(A) Dnmt3loo and Dnmt3lzygo promoters are unmethylated before
implantation in E3.5 blastocysts and do not show evidence of allelic
differences. They both globally regain methylation after implantation in
E6.5 epiblasts. (B) Dnmt3l transcription occurs equally from both
parental alleles in hybrid E3.5 blastocysts derived from C57BL/6J ⫻
CASTEi/J crosses. The yellow box highlights the detection of the two
parental single-nucleotide polymorphisms (G+A) by Sanger sequencing.
of alleles had at least one methylated CpG but with a much lower
average of 3.8 methylated CpGs per allele.
A significantly lower methylation density in E6.5 Dnmt3lz–
embryos was also observed for three secondary, somatically
acquired imprinted DMRs associated with the promoters of the
Cdkn1c, Igf2r and Igf2 genes (P<10–5) (Fig. 4A; supplementary
material Fig. S3A). At E9.5, methylation levels were close to the
expected 50% (in agreement with only one of the parental alleles
being methylated) both in wt and mutant embryos, except for Igf2
DMR1, which remained significantly hypomethylated in mutants
(36% versus 19.9%; P=0.0005). For genes associated with
pluripotency, the Oct4 promoter exhibited a methylation level
below 10% at both stages in wt, with increased methylation in E9.5
Dnmt3lz– embryos (8.4% versus 23.2%; P<10–4). The Dnmt3lzygo
and Dnmt3loo promoters were equally methylated in the presence
or absence of Dnmt3L, at E6.5 and E9.5. The embryonic context
therefore differs from that of the oocyte, where Dnmt3lzygo
methylation was strictly dependent upon Dnmt3L. Finally,
methylation of repetitive sequences was globally unaltered in
Dnmt3lz– embryos, and this was observed both for clustered
centromeric repeats (major satellite DNA) and IAP elements.
However, for transposons of the LINE-1 family, some
discrepancies were observed among subclasses of active elements,
DEVELOPMENT
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,
we analyzed the kinetics of methylation acquisition in
postimplantation embryos lacking zygotic Dnmt3l (Dnmt3l–/–,
referred to here as Dnmt3lz–), as generated from heterozygous
crosses (Dnmt3l+/– ⫻ Dnmt3l+/–), compared with their wt
littermates. Various genomic sequences that are known to acquire
methylation around the time of implantation were analyzed after
bisulfite conversion in dissected epiblasts at E6.5 for the beginning
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
loci, and repetitive sequences such as LINE-1 elements and major
satellite DNA. We included intracisternal A-particle (IAP) elements
as a control of maintenance methylation, as the methylation of
these sequences has been reported to be globally unaltered
throughout development (Lane et al., 2003; Guibert et al., 2012).
We found that the germline-expressed Scp3 (Sycp3 – Mouse
Genome Informatics) and Dazl genes acquired complete
methylation as early as E6.5 in wt epiblasts, confirming previous
data (Borgel et al., 2010). CpG methylation levels of 90% were
observed at E6.5 for both promoters and were maintained until
E9.5 (Fig. 4A). A dramatic reduction in methylation was observed
in Dnmt3lz– embryos at E6.5, but significant methylation
differences disappeared by E9.5. For Scp3, values of 89.1% and
20% CpG methylation (P<10–90, Fisher’s exact test) were observed
at E6.5 and values of 87.9% and 75.3% (P=1.00) at E9.5 in wt and
Dnmt3lz– embryos, respectively (Fig. 4A). Examination of
individual alleles at E6.5 indicated that Dnmt3L deficiency leads
to a reduction in the number of alleles with methylated CpGs, but,
even more dramatically, in the number of methylated CpGs per
allele (Fig. 4B). In wt embryos, 100% of Scp3 alleles had at least
one methylated CpG, with an average of 16.9 methylated CpGs per
allele (out of 19 analyzed CpGs). In the Dnmt3lz– background, 80%
De novo methylation without Dnmt3L
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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.
methylation levels by E9.5, continue development, are born in
the expected Mendelian ratios (supplementary material Table
S3), and progress throughout adulthood with normal somatic
methylation patterns and no overt phenotype aside from sterility
(Bourc’his et al., 2001).
The maternal store of Dnmt3L is not involved in
embryonic de novo methylation
Next, we investigated the potential developmental compensation of
zygotic Dnmt3L deficiency, which would allow Dnmt3lz– embryos
to successfully acquire methylation at some genomic sequences at
E6.5 and to fully recover normal methylation patterns at E9.5. In
the homozygous state the Dnmt3l mutation prevents the production
of a functional protein, from both the zygotic and oocyte promoters
(Shovlin et al., 2007). However, it has been argued that the
maternal store of Dnmt3L could be maintained throughout
preimplantation development and could participate in de novo
methylation, even in minute amounts. To functionally assess the
contribution of maternal Dnmt3L to embryonic methylation, we
generated embryos deficient for both maternal and zygotic Dnmt3l
expression (Dnmt3lm–z–) by crossing Dnmt3l–/– females with
DEVELOPMENT
which can be identified by specific monomeric repeats in their 5⬘
UTR. The type A subclass was equally densely methylated in wt
and Dnmt3lz– embryos at both stages, whereas the Tf subclass
exhibited a delay in acquiring methylation in E6.5 mutant embryos,
but gained normal methylation levels by E9.5.
In summary, we provide evidence that the absence of Dnmt3L
slows the establishment of embryonic methylation patterns. To
rule out a developmental delay as a potential cause of reduced
methylation, we confirmed that Dnmt3lz– embryos are
morphologically indistinguishable and present the same growth
rate as their wt counterparts at E6.5 (supplementary material Fig.
S4), implying a direct effect of Dnmt3L on de novo methylation
efficiency. Functionally, the temporary hypomethylation of the
germline Scp3 gene and the LINE-1 Tf retrotransposons does not
lead to increased transcription of these sequences in E6.5
Dnmt3lz– embryos (Fig. 4C; supplementary material Fig. S3B).
This was an important point to address because reactivation of
these sequences has been previously linked to genomic
instability (Bourc’his and Bestor, 2004; Borgel et al., 2010).
Finally, the hypomethylation phenotype is largely compensated
a few days later, so that Dnmt3lz– embryos attain normal
568
RESEARCH ARTICLE
Development 140 (3)
Dnmt3l+/– males (Fig. 5A). Embryos derived from Dnmt3l–/–
females (Dnmt3lm–) die at E10.5 (Bourc’his et al., 2001). We found
that Dnmt3lm–z– embryos did not progress past this stage and were
phenotypically similar to their Dnmt3lm–z+ littermates (Dnmt3lm–),
confirming the minor to zero impact of the zygotic mutation at this
stage of development, as compared with the severe effect of the
maternal Dnmt3l mutation.
We then analyzed the methylation status of sequences that we
had previously found to be properly methylated at E9.5 in the
absence of zygotic Dnmt3l. Using bisulfite sequencing, we
assessed the Scp3 promoter, Igf2r DMR1 and the Dnmt3lzygo
promoter, and a methylation-sensitive Southern blot approach was
used to analyze three classes of repetitive sequences (LINE-1 type
A, IAP and major satellite DNA). None of the analyzed loci
differed significantly in methylation in the absence of maternal or
zygotic Dnmt3L, nor, most importantly, in the absence of both
maternal and zygotic Dnmt3L, as compared with wt embryos at
E9.5 (Fig. 5B,C). This convincingly illustrates that the maternal
form of Dnmt3L inherited from the oocyte does not play a role in
setting up methylation patterns around the time of implantation.
These results provide ultimate proof that, in striking contrast to the
situation in germ cells, de novo methylation can be achieved in the
embryo without any source of Dnmt3L.
Dnmt3L deficiency is associated with higher levels
of Dnmt3A protein
Finally, we assessed which of the de novo methylation enzymes,
Dnmt3A and Dnmt3B, is available at the time of methylation
pattern formation in the early embryo, in the presence or absence
of Dnmt3L. By RT-qPCR and immunofluorescence, we confirmed
that Dnmt3B is the first active DNA methyltransferase to be
transcribed in the embryo as early as E2.5, while Dnmt3lzygo
transcription was not yet activated. At E3.5 to E4.5, the Dnmt3B
protein is highly expressed. By contrast, Dnmt3A expression only
becomes obvious at E4.5 in the ICM of the late blastocyst, with a
timing of activation more similar to that of Dnmt3lzygo
(Fig. 6A,B).
Interestingly, upregulation in Dnmt3a expression was observed
in response to zygotic Dnmt3l deficiency at E6.5, as demonstrated
by a twofold increase in transcription in Dnmt3lz– epiblasts
compared with wt littermates (P<0.01) detected by RT-qPCR
(Fig. 6C). Dnmt3b transcript levels were comparatively unaffected.
The specific upregulation of Dnmt3a could not be explained by a
loss of promoter methylation in the Dnmt3lz– context: the two
alternative CpG-rich promoters of this locus – a 5⬘ promoter that
gives rise to Dnmt3a and an intragenic promoter that gives rise to
the Dnmt3a2 isoform – are constitutively unmethylated at E3.5,
E6.5 and E9.5 in wt embryos, as suggested from publicly available
methylated DNA immunoprecipitation (MeDIP)-chip and reduced
representation bisulfite sequencing (RRBS) datasets (Borgel et al.,
2010; Smith et al., 2012) (supplementary material Fig. S5).
Importantly, increased steady-state levels of Dnmt3a transcripts
resulted in higher levels of Dnmt3A protein in Dnmt3lz– epiblasts.
Notably, Dnmt3A2, which is the most active form of Dnmt3A and
abundant in ES cells and early postimplantation embryos (Nimura
et al., 2006), was clearly overexpressed in E6.5 Dnmt3lz– embryos,
as revealed by western blotting (Fig. 6D). Although the details of
the compensatory feedback of Dnmt3L deficiency on Dnmt3A
expression are not known, a greater availability of Dnmt3A protein
provides a likely mechanism for the normal acquisition of
embryonic methylation patterns in the absence of a Dnmt3L
stimulatory effect.
DEVELOPMENT
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.
De novo methylation without Dnmt3L
RESEARCH ARTICLE
569
DISCUSSION
Although Dnmt3L is strictly required for de novo methylation in
germ cells, clear evidence was still lacking as to whether its
stimulatory function is necessary in the developing embryo.
Our in vivo study demonstrates that a promoter switch underlies
the autonomous and time-staged expression of Dnmt3L in the
embryo, which appears to have an innate role in accelerating
the de novo methylation process. However, we found that the
absence of Dnmt3L can be compensated for by other factors.
Compared with the germline, the early embryo is more plastic in
terms of the mechanisms by which DNA methylation patterns
can be acquired.
Although the existence of alternative Dnmt3l promoters was
known (Shovlin et al., 2007), we reveal here that these promoters
are transmitted to the embryo with opposite allelic states of DNA
methylation acquired in the parental gametes: Dnmt3lzygo is
methylated on the maternal allele whereas Dnmt3loo is methylated
on the paternal allele. Recent genome-wide methylation studies
have highlighted certain genomic features that are conducive to
oocyte methylation: high CpG density and intragenic location
(Chotalia et al., 2009; Smallwood et al., 2011; Kobayashi et al.,
2012). The maternal Dnmt3lzygo gDMR fulfills these two criteria,
as it is located within the transcription unit defined by the 5⬘
Dnmt3loo promoter, which is specifically active in the oocyte.
Although subject to parent-specific methylation at fertilization, the
two promoters of Dnmt3l are not imprinted, as methylation is not
maintained during embryonic cleavage. Accordingly, we found no
evidence of binding sites at these loci for Zfp57, which is likely to
be an obligate factor for the protection of gamete-inherited
methylation (Quenneville et al., 2011; Messerschmidt et al., 2012;
Proudhon et al., 2012).
DNA methylation has previously been shown to regulate
Dnmt3l transcriptional output and promoter usage in the context
of ES cells and adult tissues, as well as in vitro transfection
studies (Aapola et al., 2004; Hu et al., 2008; O’Doherty et al.,
2011). Here we confirm the mutually exclusive relationship
between expression and promoter methylation in the oocyte,
where the Dnmt3loo promoter is active and unmethylated and
the Dnmt3lzygo promoter is inactive and methylated. This is also
true for the postimplantation embryo, where the two promoters
are silenced and methylated. However, we identified two
situations in which transcription control is achieved without
DNA methylation: (1) in the E3.5 blastocyst, where both Dnmt3l
promoters are unmethylated but only the Dnmt3lzygo transcript
is found; and (2) in the TE, where the Dnmt3lzygo promoter is
unmethylated but the corresponding transcript is not produced.
If transcriptional regulation is involved, it occurs in a DNA
methylation-independent manner and/or additional factors are
required to activate hypomethylated promoters in these contexts.
In support of this hypothesis, the Dnmt3lzygo promoter is
physically bound by pluripotency factors in ES cells (Marson et
al., 2008), which fits with our in vivo observation of Dnmt3L
restriction to the pluripotent part of the embryo. Our study
highlights a two-step regulatory model whereby clearance of
parent-inherited methylation could potentiate promoter activity,
then embryonic pluripotency factors further specify this activity.
This double security system might prevent spurious activation of
key factors, such as the DNA methylation effectors, to provide
efficient spatiotemporal control of genome activity and lineage
decision. This type of regulation was formerly reported for the
Nanog gene (Farthing et al., 2008). Our study suggests that it
could apply more generally to targets of pluripotency factors.
DEVELOPMENT
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.
RESEARCH ARTICLE
The intricate regulation of Dnmt3l expression may attest to its
important function during embryonic development. Our work
formally demonstrates that Dnmt3L is indeed involved in de novo
methylation at implantation. The germline-expressed Scp3 and
Dazl genes, some somatic imprinted DMRs and LINE-1 Tf
elements are significantly, although not completely,
hypomethylated in Dnmt3lz– embryos at E6.5, within the early
steps of methylation acquisition. The most potent effect we
observed is on methylation density per DNA strand, thus providing
developmental evidence to support the proposal that Dnmt3L
enhances the deposition of long methylation tracts (Wienholz et al.,
2010), potentially by stimulating the processivity and/or the
catalytic rate of Dnmt3 enzymes once they are loaded on DNA
(Kareta et al., 2006; Holz-Schietinger and Reich, 2010; Chédin,
2011). However, Dnmt3L-dependent stimulation is not universal,
as other sequences are properly methylated at E6.5 in the absence
of Dnmt3L. Moreover, Dnmt3L-dependent effects appear to be
time specific, as full methylation patterns are globally recovered in
Dnmt3lz– embryos at E9.5. Taken altogether, our work suggests that
Dnmt3L facilitates the formation of methylation patterns in the
embryo in its initiation phase. Only by undertaking a
developmental analysis of ongoing de novo methylation at
individual loci was it possible to reveal such subtle spatiotemporal
details of the influence of Dnmt3L on embryonic de novo
methylation.
Differential Dnmt3L-dependent stimulation might reflect
differential Dnmt3A or Dnmt3B target specificity. On the one hand,
expression and locus-specific analyses have identified Dnmt3B as
the main enzyme responsible for de novo methylation in the
embryo, although some rare sequences require Dnmt3A (Okano et
al., 1999; Watanabe et al., 2002; Borgel et al., 2010). On the other
hand, Dnmt3L can potentiate both Dnmt3A and Dnmt3B activity
in vitro, but Dnmt3A appears to be the favored partner in vivo and
in particular in ES cells (Nimura et al., 2006). Of direct relevance
to our results, de novo methylation of Dazl and Dnmt3lzygo
promoters was demonstrated to be strictly Dnmt3B dependent (Hu
et al., 2008; Borgel et al., 2010). The fact that these sequences are
either hypomethylated or normally methylated in E6.5 Dnmt3lz–
embryos does not support preferential Dnmt3L stimulation of
Dnmt3A or Dnmt3B. Differential methylation kinetics provides
another potential explanation for the observed difference in
Dnmt3L requirement. In this regard, X-linked CpG islands that
acquire methylation slowly upon X-chromosome inactivation were
shown to require the activity of the Smchd1 chromatin protein,
whereas fast-acquiring sequences do not (Gendrel et al., 2012).
Such a relationship might not apply to Dnmt3L, however. For
example, although the Scp3 promoter and Cdkn1c DMR are both
hypomethylated in E6.5 Dnmt3lz– embryos, Scp3 is a fast-acquiring
sequence that is already completely methylated in E6.5 wt
embryos, whereas the Cdkn1c DMR is a slow-acquiring sequence,
attaining full methylation only by E9.5. More generally, the process
of de novo methylation has been shown to be influenced by local
CpG content, gene density, transcription and histone H3K4
methylation states (Ooi et al., 2007; Weber et al., 2007; Borgel et
al., 2010; Lienert et al., 2011; Gendrel et al., 2012). Our study
represents an indispensable proof of principle for further
investigation of the sequence elements and/or chromatin contexts
genome-wide that require Dnmt3L for a fast rate of DNA
methylation acquisition in the early developing embryo.
The most important finding of our work is the formal
demonstration that embryos can attain normal genomic methylation
levels in the absence of any source of Dnmt3L, either maternal or
Development 140 (3)
zygotic in origin. This situation is at odds with the obligate
requirement of Dnmt3L for full methylation in gametes (Bourc’his
et al., 2001). This unique property of the embryo occurs in the
context of higher levels of the Dnmt3A enzyme, and in particular
of the most active Dnmt3A2 isoform. By contrast, Dnmt3L
deficiency has no effect on Dnmt3 enzyme levels in
prospermatogonia (Niles et al., 2011), and, although it leads to
upregulation of Dnmt3b transcripts in oocytes the amount of
Dnmt3B protein is unchanged (Lucifero et al., 2007). Increased
Dnmt3A availability might result from transcriptional and/or posttranscriptional mechanisms. Because both Dnmt3a promoters are
constitutively methylation free in wt peri-implantation embryos,
methylation changes in Dnmt3lz– embryos should not affect
Dnmt3a expression levels directly (Borgel et al., 2010; Smith et al.,
2012). Of note, several microRNA families have been shown to
modulate Dnmt3a and Dnmt3b expression. Downregulation of
miR-29 could cause higher levels of Dnmt3A in the Dnmt3l mutant
background (Takada et al., 2009). Alternatively, increased Dnmt3A
levels could be the indirect consequence of upregulation of the
miR-290 species, which target the Dnmt repressor Rbl2 (Benetti et
al., 2008; Sinkkonen et al., 2008).
Although we cannot conclude that increased Dnmt3A
availability is responsible for the acquisition of normal methylation
patterns, it could mechanistically counterbalance Dnmt3L
depletion. In biochemical assays, maximal Dnmt3L-mediated
stimulation is achieved at a 1:1 molar stoichiometry of Dnmt3L and
Dnmt3A, but increasing Dnmt3A protein alone can also lead to
increased recruitment of methyl group donors (SAM). Moreover,
although Dnmt3L interacts with Dnmt3A2 to form tetrameric
structures, Dnmt3A2 is capable of auto-assembling into large
polymers of up to 30 monomers in solution and likely also in vivo
(Kareta et al., 2006; Holz-Schietinger and Reich, 2010). Our work
therefore illustrates that the embryo may be flexible in its ability to
use various combinations of Dnmt3A, Dnmt3B and Dnmt3L
homo- and heterocomplexes, depending on relative partner
availability. By contrast, the assembly of Dnmt3L-associated
complexes is strictly required for proper methylation in the
germline (Bourc’his et al., 2001; Bourc’his and Bestor, 2004;
Kaneda et al., 2004; Kaneda et al., 2010). Interestingly, DNA
replication might play an enhancing role in de novo methylation
and alleviate the need for Dnmt3L stimulation: indeed, whereas
embryonic methylation occurs in rapidly cycling cells, gametic
methylation occurs in non-dividing cell types during male and
female germ cell development (Bourc’his et al., 2001; Bourc’his
and Bestor, 2004).
To conclude, whereas abnormal methylation patterns are usually
associated with dramatic outcomes, the slight delay in acquiring
full methylation has no somatic impact on Dnmt3lz– individuals,
underscoring the efficiency and precision of Dnmt3L-independent
methylation in the developing embryo.
Acknowledgements
We thank Anne-Valérie Gendrel for assistance with Sequenom analysis; Renata
Kozyraki for help with protein analysis in early embryos; Shoji Tajima for
providing the anti-Dnmt3L antibody; Mickael Weber for sharing the MeDIPchip data on Dnmt3a promoters; the PICT IBiSA Genetics and Developmental
Biology Imaging Facility for help with microscopy; and Isabelle Grandjean and
Colin Jouanneau for excellent mouse husbandry.
Funding
This work was supported by a grant from the Schlumberger Foundation and
by a European Young Investigator (EURYI) Award from the European Union to
D.B. R.D. is the recipient of an Institut Curie PhD fellowship. M.G. is the
recipient of a DIM-Stem Pole post-doctoral fellowship.
DEVELOPMENT
570
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.089268/-/DC1
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