Active demethylation in mouse zygotes involves cytosine deamination and base excision repair
© Santos et al.; licensee BioMed Central Ltd. 2013
Received: 22 August 2013
Accepted: 30 October 2013
Published: 14 November 2013
DNA methylation in mammals is an epigenetic mark necessary for normal embryogenesis. During development active loss of methylation occurs in the male pronucleus during the first cell cycle after fertilisation. This is accompanied by major chromatin remodelling and generates a marked asymmetry between the paternal and maternal genomes. The mechanism(s) by which this is achieved implicate, among others, base excision repair (BER) components and more recently a major role for TET3 hydroxylase. To investigate these methylation dynamics further we have analysed DNA methylation and hydroxymethylation in fertilised mouse oocytes by indirect immunofluorescence (IF) and evaluated the relative contribution of different candidate factors for active demethylation in knock-out zygotes by three-dimensional imaging and IF semi-quantification.
We find two distinct phases of loss of paternal methylation in the zygote, one prior to and another coincident with, but not dependent on, DNA replication. TET3-mediated hydroxymethylation is limited to the replication associated second phase of demethylation. Analysis of cytosine deaminase (AID) null fertilised oocytes revealed a role for this enzyme in the second phase of loss of paternal methylation, which is independent from hydroxymethylation. Investigation into the possible repair pathways involved supports a role for AID-mediated cytosine deamination with subsequent U-G mismatch long-patch BER by UNG2 while no evidence could be found for an involvement of TDG.
There are two observable phases of DNA demethylation in the mouse zygote, before and coincident with DNA replication. TET3 is only involved in the second phase of loss of methylation. Cytosine deamination and long-patch BER mediated by UNG2 appear to independently contribute to this second phase of active demethylation. Further work will be necessary to elucidate the mechanism(s) involved in the first phase of active demethylation that will potentially involve activities required for early sperm chromatin remodelling.
KeywordsEpigenetic reprogramming DNA methylation Hydroxymethylation AID BER UNG2 TET3
DNA methylation is an important epigenetic mark involved in gene silencing, X chromosome and transposon inactivation, genomic imprinting, and chromosome stability. DNA methylation is subject to reprogramming during development, involving both demethylation (active and passive) and de novo methylation phases. To date, the most clear examples of active DNA demethylation take place during the very early steps of mammalian development, namely in the zygote where the paternal genome undergoes a massive wave of loss of 5-methylcytosine (5mC) right after fertilisation [1–4].
Within 1 h of fertilisation, the paternal genome goes through major chromatin remodelling, loses protamines and is re-packaged by maternal nucleosomal histones, forming the paternal pronucleus [5, 6]. Post-fertilisation development can be defined by the pronuclear stages PN0/1 to PN5; PN0-PN2 embryos are in the G1 phase, PN3 and PN4 embryos are largely in S phase, replicating both the paternal and maternal genomes, and PN5 embryos are mostly in the post-replicative G2 phase [3, 7–9]. Several reports have shown that the paternal genome undergoes genome-wide DNA demethylation via an active mechanism before replicating its DNA [1–4]. The search for enzymes responsible for this demethylation has produced numerous candidates and reaction mechanisms [10–13]. These fall within three main groups: (1) direct removal of the methyl group from the 5-C position of cytosine; (2) DNA repair, either base excision repair (BER) or nucleotide excision repair (NER); and (3) iterative enzymatic oxidation leading to conversion of 5mC to 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).
The methyl-CpG-binding domain protein 2 (MBD2) was reported to possess direct demethylase activity , but the result could not be reproduced by others. DNA glycosylases have been described in plants that can remove 5mC, leaving an abasic site that is repaired by the BER machinery but mammalian glycosylases (e.g., thymine DNA glycosylase (TDG) and methyl-CpG-binding domain protein 4 (MBD4)) show weak activity on 5mC in vitro. However, both MBD2 and MBD4 null fertilised oocytes undergo paternal loss of DNA methylation indistinguishable from matched controls .
An alternative to direct removal of 5mC by a DNA glycosylase is enzymatic deamination of 5mC to thymine, followed by T-G mismatch specific BER that replaces thymine with cytosine . Two classes of enzymes have been proposed to be capable of carrying out the first step in this process: cytosine deaminases and DNA methyltransferases (reviewed in ). Cytosine DNA deaminases convert cytosine to uracil in nucleic acids and are well known from their roles in RNA editing, viral defence and antibody diversification . Recently a series of results have pointed to an involvement of activation-induced deaminase (AID) mediated cytosine deamination in DNA demethylation in primordial germ cells (PGCs) and induced pluripotent stem (iPS) cell reprogramming, in cancer and embryonic stem (ES) cell gene expression [19–21]. Furthermore, overexpression of AID and MBD4 have been described to cause general demethylation of the zebrafish embryo genome, suggesting that deamination of 5mC followed by BER of T-G mismatches results in demethylation . Gadd45, a p53-inducible gene involved in a variety of cellular processes, seems to facilitate this process and it has also been shown to interact with nucleotide excision repair (NER) components [22, 23]. Models have been proposed for Gadd45 mediated demethylation of DNA either by deamination followed by BER or NER, or even by a combination involving consecutive NER and BER mechanisms (reviewed in ). AID's best defined activity is in B lymphocytes, where deamination of cytosines leading to uracil initiates both somatic hypermutation and immunoglobin class switch recombination [25–27]. However, its expression in mouse oocytes as well as in ES cells and PGCs , make it a potential candidate for performing global demethylation. In vitro assays have shown AID has 5mC deaminase activity, resulting in thymine and, therefore, T-G mismatches in DNA, which can be effectively repaired through the BER pathway . Cytosine and 5-methylcytosine can also be enzymatically deaminated by DNA methyltransferases (DNMTs), primarily known as enzymes that transfer a methyl group to the C-5 position of cytosine from the methyl donor S-adenosylmethionine (SAM- reviewed in ). Recent work in mammalian cell lines has led to the proposal that deamination by the Dnmt3a and Dnmt3b DNA methyltransferases could be a means of achieving fast, active DNA demethylation at promoters undergoing transcriptional cycling, by generating thymine, which is repaired via TDG and other enzymes [12, 30–32]. More recently, studies have suggested that loss of DNA methylation in the paternal genome in the zygote is primarily dependent on TET3 [4, 33], a member of the ten-eleven translocation (TET) family of DNA dioxygenases, which are capable of converting 5mC to 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) through iterative oxidation , suggesting that the main mechanism involved in active genome-wide demethylation is via oxidation of 5mC. Although these various models for active loss of DNA methylation from the paternal pronucleus have been proposed, they have, for the most part, overlooked the evidence that this process occurs in two phases - before and coincident with DNA replication. To investigate this DNA methylation dynamics we have analysed fertilised mouse oocytes, by indirect immunofluorescence (IF) of DNA methylation and hydroxymethylation, and evaluated the relative contribution of different activities to active demethylation, by three-dimensional (3D) imaging and IF semi-quantification.
Results and discussion
We have previously characterised the dynamics of DNA methylation loss during the first cell cycle in the mouse by indirect immunofluorescence [3, 35]. Since then, several similar studies have been published, more recently including other modifications of DNA methylation, namely hydroxymethylation, and attempts have been made to semi-quantify the immunofluorescence signals in order to get a sense of the kinetics of demethylation [4, 8, 33, 36, 37]. These approaches have provided clear evidence for the oxidation of 5mC in the zygote, specifically in the paternal pronucleus, seemingly explaining the concomitant decrease in DNA methylation.
DNA methylation (5mC) average paternal to maternal ratios (indirect immunofluorescence)
Iqbal et al., 20111
Wossidlo et al., 20111
BDF1 (C57BL/6 x DBA)
Inoue and Zhang, 2011
BDF1 (C57BL/6 x DBA)
Salvaing et al., 20121
F1 (C57BL/6 x CBA)
DNA hydroxymethylation (5hmC) average paternal to maternal ratios (indirect immunofluorescence)
Iqbal et al., 20111
Wossidlo et al., 20111
BDF1 (C57BL/6 x DBA)
Inoue and Zhang, 2011
BDF1 (C57BL/6 x DBA)
Salvaing et al., 20121
F1 (C57BL/6 x CBA)
According to recent reports, deamination of cytosine and 5-methylcytosine could also be mediated by DNA methyltransferases, namely the Dnmt3a and Dnmt3b de novo methyltransferases (reviewed in [11, 12]) and more recently that DNA could be directly demethylated by the same enzymes, at least in an in vitro system . Moreover, results from ES cells suggest that TET and Dnmt3 enzymes may interact at common binding sites . These prompted us to investigate whether Dnmt3a null oocytes were capable of demethylating a control (B6) sperm using a conditional (Zp3-Cre) Dnmt3a knock-out mouse model . Oocytes null for Dnmt3a are severely depleted of DNA methylation  which precluded the use of the paternal/maternal ratio as a measure of demethylation as the maternal pronucleus showed only residual levels of 5mC staining (Additional file 7A); instead we directly compared the total levels of immunofluorescence signal in the paternal pronuclei of Dnmt3a null, control (B6) and AID null oocytes fertilised by B6 sperm (Additional file 7B). No significant difference, in either 5mC or 5hmC levels, between paternal pronuclei in control and Dnmt3a null fertilised oocytes was observed, whereas both are significantly different from AID null in 5mC but not in 5hmC levels (Additional file 7B). These results do not support a role for Dnmt3a in paternal active demethylation in the mouse one-cell embryo.
As a whole, our results support a role for both 5mC hydroxymethylation and cytosine deamination, followed by LP BER, as demethylation mechanisms in the mouse zygote. Both these mechanisms seem to contribute independently to a decrease in paternal DNA methylation coincident, but not reliant, on DNA replication. Furthermore, an initial loss of paternal DNA methylation, prior to S-phase, seems to take place, for which none of the activities investigated in this work seem to supply an explanation. It is likely that this early demethylation is related to the need for major chromatin remodelling of the sperm within the first hours post fertilisation and may involve other pathways of DNA repair. NER has been suggested as a candidate for DNA demethylation (reviewed in [10, 11]), removes bulky DNA lesions and is a multistep process involving the action of as many as 20 to 30 proteins working in a well-defined sequence . Other repair pathways, such as non-homologous end joining (NHEJ) and homologous recombination (HR), could also play a role in this first phase of paternal loss of methylation . The complexity, and possible redundancy, of these alternative pathways will require considerable research effort in order to elucidate this early DNA methylation loss.
Our analysis shows that there are two observable phases of active DNA demethylation in the mouse zygote, before and coincident with DNA replication. We further show that TET3 seems to be involved only in the second phase of loss of methylation. Cytosine deamination and uracil BER seem to also independently contribute to this second phase of active demethylation. Together these findings allow us to conclude that demethylation is achieved by at least two parallel mechanisms which may or may not be partially redundant. Our results highlight the dynamic nature of DNA demethylation, with two apparent distinct stages (Phase I and Phase II). The major increase in 5hmC (Phase II) seems to be uncoupled from the initial loss of DNA methylation (Phase I) which was not previously acknowledged. Still, further work will be necessary to elucidate the mechanism(s) involved in the first phase of demethylation, likely to involve activities required for early chromatin remodelling on fertilisation and perhaps other types of DNA repair.
To our knowledge, this is the first report of possible involvement of LP BER in DNA demethylation, opening new avenues of investigation not formerly considered.
Mice and sample collection
All experimental procedures were approved by the Animal Welfare, Experimentation and Ethics Committee (AWEEC) at the Babraham Institute and were performed under licenses by the Home Office (UK) in accordance with the Animals (Scientific Procedures) Act 1986.
Fertilised mouse oocytes were collected on the day of plugging from naturally mated inbred C57BL/6 J (B6) mice supplied from breeding colonies in the Biological Support Unit at the Babraham Institute. Mechanistic investigation into the loss of DNA methylation was made possible through the generation of conditional deletion of key activities, Dnmt3a  and TDG  (rescued by a floxed TDG minigene), derived by breeding female mice homozygous for floxed alleles together with the Zp3 Cre recombinase transgene . Conditionally deleted oocytes generated in this way were referred to as maternal knock-outs (MAT KO). In all cases these MAT KO generating females were bred to wild-type B6 control males. Constitutively deleted activities were derived for AID  and UNG2 [51, 55] from homozygous females null for the respective enzymes.
Individual zygote pronuclear staging was performed as previously described . The cell-cycle state of these different stages has been characterised according to the literature, establishing that zygotes between PN1 and PN2 will be in G1 and from PN3 to PN5 in S-G2 .
Generation of TET3 conditional deletion
C57BL/6 N Tac ES cells (TaconicArtemis) were targeted with a vector introducing LoxP sites around exon 5 of Tet3 RefSeq NM_183138.2 (sequence: CCGGACCTGTGCTTGCCAAGGCAAAGACCCTAACACCTGCGGTGCCTCCTTCTCCTTCGGCTGTTCCTGGAGCATGTACTTCAACGGCTGCAAATATGCTCGGAGCAAGACGCCACGAAAGTTCCGCCTCACGGGAGACAATCCGAAGGAG) which encodes residues required for chelation of Fe(II) and is upstream of exons containing other key catalytic residues . Expression of Cre recombinase results in excision of this region and a frame-shift from exon 6 affecting all downstream exons until a premature stop codon in exon 7. Animals heterozygous for the floxed allele were bred to a transgenic mouse line containing Zp3 Cre on a B6 background  and homozygous mice were generated by inter-crossing to give females of the appropriate genotype.
Immunofluorescence and confocal microscopy
Antibody staining of DNA methylation (Eurogentec, BI-MECY) and hydroxymethylation (Active Motif, 39769) was performed as previously described  with modifications. Briefly, zygotes were fixed with 4% PFA for 15 min and, after permeabilisation with 0.5% Triton X-100, the samples were treated with 4 N HCl for 10 min at room temperature, washed in PBS/Tween and blocked overnight; simultaneous incubation with both primary antibodies followed by simultaneous secondary detection (AlexaFluor, Molecular probes, Invitrogen) was used. To allow for full 3D sample capture the samples were mounted in fibrin clots . Image acquisition was performed with a LSM 510 Meta confocal laser scanning microscope (Carl Zeiss) equipped with a ‘Plan-Apochromat’ 63x/1.40 DIC oil-immersion objective and an Olympus FV1000 equipped with a UPLSAPO 60x/1.35 DIC oil-immersion objective. DNA was counterstained with YOYO1™ (Molecular Probes, Life Technologies). Z-stacks of 20 to 65 optical sections were collected from each zygote (700x700, pixel size; z-step, 0.50 μm). At least 10 zygotes of each group were imaged from at least two biological replicates. Images were pseudo-coloured using Adobe Photoshop CS4.
Semi-quantification and statistics
Fluorescence semi-quantification analysis (total sum, 3D rendering) was performed as follows, 3D reconstruction of confocal image stacks was performed using Volocity 5.5 (Improvision), after which regions of interest (ROIs) were defined around each pronucleus and total voxel count signal intensity (SUM) for each channel was computed. Examples of each of these steps can be found in Additional file 1. The data were then exported into Excel and the individual maternal to paternal ratios (male/female ratios) for each zygote calculated as this measure is widely used in the field and allows for a good degree of comparison between studies. Statistical analysis (analysis of variance (ANOVA), Mann–Whitney or unpaired t test) and whisker-plot graphs were performed with GraphPad Prism 5 and 6.
Wolf Reik and Wendy Dean are senior authors.
Analysis of variance
Apurinic-apyrimidinic endonuclease 1
C57BL/6 J mouse strain
Base excision repair
- ES cells:
Embryonic stem cells
DNA (cytosine-5)-methyltransferase 3a
DNA (cytosine-5)-methyltransferase 3b
- iPS cells:
induced pluripotent stem cells
- MAT KO:
Methyl-CpG binding Domain 2
Methyl-CpG binding Domain 4
Nucleotide excision repair
Non-homologous end joining
poly-ADP-ribose polymerase 1
Proliferating cell nuclear antigen
Primordial germ cells
Regions of interest
Thymine DNA glycosylase
Ten-eleven translocation 3
Uracil-DNA glycosylase 2
X-ray cross-complementing group 1.
This work was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC), the Medical Research Council (MRC) and The Wellcome Trust. The authors would like to thank Dr Hiro Sasaki for providing the Dnmt3a mice used in this study, Dr Primo Schär for the TDG materials and Dr Gabriella Ficz for the initial design of the TET3 knock-out.
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