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De novo DNA methylation drives 5hmC accumulation in mouse zygotes

Nature Cell Biology volume 18, pages 225–233 (2016)Cite this article

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Abstract

Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs 1,2,3,4). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.

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Figure 1: 5hmC and 5mC kinetics during mouse zygotic development.
Figure 2: Small-molecule inhibition of Tet protein activity abrogates 5hmC formation but does not prevent DNA demethylation.
Figure 3: Tet3 is not required for loss of 5mC in the early zygote.
Figure 4: Tet3 targets newly deposited 5mC generated by Dnmt3a and Dnmt1.
Figure 5: De novo DNA methylation activity is present in the oocyte and generates a target for hydroxylation in the SCNT experiment.

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Acknowledgements

We are grateful to the members of the Hajkova laboratory (especially K. McEwen) and to N. Navaratnam for discussions and revision of the manuscript. We thank T. Carell for providing the isotope-labelled synthetic nucleosides. We would like to acknowledge the MRC CSC Microscopy facility for help with imaging of the embryos, and M. Woodberry, D. Hardy and J. Glegola for mouse husbandry. We would like to thank MRC transgenic facility for their help regarding IVF. The LC-MS analysis was conducted in collaboration with Agilent Technologies, whom we would like to thank for generous support and help. This work was supported by MRC (MC_ US_A652_5PY70) and EpigeneSys network funding to P.H. R.A. was a recipient of the Marie Curie Intra-European Fellowship (FP7). B.N. was a recipient of the Marie Curie Incoming-European Fellowship (FP7).

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Author notes

  1. Vesela Encheva

    Present address: Present address: Cancer Research UK, Clare Hall Laboratories, South Mimms EN6 3LD, UK.,

  2. Buhe Nashun, Kenjiro Shirane and Shoma Nakagawa: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 0NN, UK

    Rachel Amouroux, Buhe Nashun, Peter W. S. Hill, Zelpha D’Souza, Aleksandra Turp, Elodie Ndjetehe, Vesela Encheva & Petra Hajkova

  2. Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan

    Kenjiro Shirane & Hiroyuki Sasaki

  3. Institute of Reproductive and Developmental Biology, Imperial College London, London W12 0NN, UK

    Shoma Nakagawa & Nobuaki R. Kudo

  4. Department of Technology Development, Chromosome Engineering Team, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan

    Manabu Nakayama

  5. Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan

    Masashi Matsuda & Haruhiko Koseki

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Contributions

P.H. and R.A. conceived the study and wrote the manuscript with assistance from H.S. and N.R.K. R.A. performed the experiments with the help of Z.D’S. and P.W.S.H.; R.A. and P.H. analysed the data. B.N. carried out micromanipulation of zygotes, and provided technical assistance. S.N. performed SCNT with assistance from B.N. and R.A. R.A. and A.T. performed the LC-MS experiment and analysed the data with the help of V.E. H.S. provided the Dnmt1, Dnmt3a and Dnmt3L KO mice and K.S. performed the IVF experiments. E.N. carried out Tet3 targeting in ESCs and generated Tet3 chimaera. M.N., M.M. and H.K. provided the Tet3 KO mice.

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Correspondence to Petra Hajkova.

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Integrated supplementary information

Supplementary Figure 1

(a) Tet3 staining in zygotes shows an accumulation of Tet3 in the paternal pronucleus from PN2 and a gradual enrichment from PN3 to PN5. Tet3 protein is not detectable following the 2-cell stage. Representative images are shown (replicates indicated on the figure). (b) Expression of Tet1/2/3 in mouse preimplantation embryos (based on 39). (c) 5fC and 5caC stainings in PN3 and PN4 zygotes. Note that in the early zygote 5fC (and 5caC) is observed in the maternal pronucleus only (see also Supplementary Fig. 2c, d). DNA stained with PI. Representative images are shown (replicates indicated on the figure). (d) Dot blot analysis of C-, 5mC- and 5hmC- containing PCR products (amplified with dCTP, 5mdCTP and 5hmdCTP, respectively) to assess the sensitivity and cross-reactivity of anti-5mC and anti-5hmC antibodies. 5hmC antibody shows about 10,000 fold greater sensitivity than the 5mC antibody. (e) Competition assay to assess specificity of 5hmC antibody using genomic DNA from 3T3 cells overexpressing Tet1 or Tet3 protein. Competitor nucleosides (200-fold excess) are 5-methyldeoxycytidine (5mC) and 5-hydroxymethyldeoxycytidine (5hmC). , female pronucleus; , male pronucleus; 5fC, 5formylcytosine; 5caC, 5carboxylcytosine; DAPI, 4′,6′-diamidino-2-phenylindole; PI, propidium iodide; RPKM, reads per kilobase of transcript per million mapped reads. (Scale bars, 5 μm).

Supplementary Figure 2

(a) TET1 in vitro activity assay in the presence of DMOG. Conversion of 5mC to 5hmC is assessed by dot blot. (b) Dynamics of 5mC and 5hmC in mouse zygotes treated with DMOG. Note the initial disappearance of paternal 5mC despite the absence of 5hmC in DMOG-treated zygotes. The accumulation of 5mC in DMOG-treated zygotes is detectable only in the late PN stages and correlates with the appearance of 5hmC in control zygotes (n = 192 zygotes). 5fC (n = 8 PN3 and n = 9 PN4 control zygotes; n = 8 PN3 and n = 6 PN4 DMOG-treated zygotes) (c) and 5caC (n = 6 PN3 and n = 8 PN4 control zygotes; n = 8 PN3 and n = 7 PN4 DMOG-treated zygotes) (d) stainings in DMOG-treated PN3 and PN4 zygotes. Quantification is represented as a ratio between the pronuclear signals (pat/mat). Note that in the early zygote 5fC (and 5caC) is observed in the maternal pronucleus only (see also Supplementary Fig. 1c). (Scale bars, 5 μm.) (e) 5hmC staining in pre-implantation embryos derived from in vitro fertilisation in the presence of DMOG. 5hmC is not detectable at 2-cell stage following DMOG treatment which is in agreement with the absence of detectable Tet3 protein at this stage (Supplementary Fig. 1a). 5hmC is gradually detectable from 4-cell stage onwards. (Scale bars, 10 μm.) (f) Lack of zygotic 5hmC (DMOG treatment during IVF) does not affect the developmental potential of mouse embryos (n > 20). , female pronucleus; , male pronucleus; 5fC, 5formylcytosine; 5caC, 5carboxylcytosine; DMOG, dimethyloxallyl glycine; PI, propidium iodide. Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Supplementary Figure 3

(a) Histone modification asymmetry is maintained in DMOG-treated zygotes as assessed by staining with specific antibodies for H3K4me2 (n = 5 zygotes), H3K9me2 (n = 5 zygotes), and H3K36me3 (n = 5 zygotes) and H3K27me3 (n = 8 zygotes). Representative images are shown. (b) TET1 in vitroactivity assay in the presence of Fe2+ chelator deferoxamine (DFX). Conversion of 5mC to 5hmC is assessed by dot blot. (c) Inhibition of Tet activity using DFX leads to decrease in paternal 5hmC without affecting the loss of paternal 5mC. Values are represented as a ratio between the signals in parental pronuclei (pat/mat). For 5mC staining, n = 8 control and n = 8 DFX-treated zygotes; for 5hmC staining, n = 8 control and n = 8 DFX-treated zygotes. Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. (d) Bisulphite sequencing analysis of the 5′UTR of Line1 repetitive elements (L1Md_Tf and L1Md_Gf subtypes) indicating the percentage of methylated and hydroxymethylated CpG dinucleotides in zygotes without polar bodies (n = 22 and n = 20 amplified clones derived from control and DMOG-treated zygotes, respectively at PN4, 10hpf; 2 independent biological replicates are shown) and in 2-cell embryos (n = 24 and n = 21 amplified clones derived from control and DMOG-treated embryos, respectively). Note that bisulphite sequencing does not distinguish between 5mC and 5hmC. (e) Bisulphite sequencing analysis of the 5′UTR of Line1 repetitive elements (L1Md_Tf and L1Md_Gf subtypes) on the paternal pronuclei of control and DMOG-treated zygotes (late PN3-PN4, 7-10hpf) extracted by micromanipulation (n = 44 and n = 42 paternal pronuclei extracted from control and DMOG-treated zygotes, respectively; 2 independent biological replicates are shown). (f) Bisulphite sequencing analysis of Line1 repetitive elements (L1Md_Tf and L1Md_Gf subtypes) in wild-type (Tet3WT) and Tet3 maternally depleted (Tet3mat−/pat+) zygotes recovered at PN4, 9hpf (n = 31 and n = 31 amplified clones, respectively). For bisulphite experiments, p values were calculated using Mann-Whitney U-test. ∗∗, p < 0.01. , female pronucleus; , male pronucleus; DMOG, dimethyloxallyl glycine; DFX, deferoxamine; PI, propidium iodide. (Scale bars, 5 μm.)

Supplementary Figure 4

(a) Table summarising limit of detection (LOD), limit of quantification (LOQ) and signal-to-noise (S/N) ratio (in brackets) of LOQ peak for analysed nucleosides in our LC/MS method. (b) Parthenogenetically activated oocytes were incubated for a short pulse with EdU post-activation to control for the completion of S-phase. (n = 4 parthenotes.) Representative images are shown. (Scale bars, 5 μm.) (c) LC/MS quantification of 5mC/dG ratio in MII oocytes and parthenogenetically activated oocytes blocked for replication, showing relative stability of genome-wide DNA methylation on the maternal genome (2 biological replicates and 2 technical replicates, 100 embryos (or oocytes) used per measurement). (d) Calculation of paternal 5mC levels based on the assumption that 5mC signal on the maternal pronucleus remains stable in the zygote (according to measurements in Fig. 2b). Error bars indicate s.d. (e) LC/MS measurement of 5mC/dG and 5hmC/dG ratio in sperm, control and DMOG-treated zygotes (with polar bodies) confirming results shown in Fig. 2b. Each value represents the average of 2 independent experiments with 2 technical replicates; 100 zygotes used per measurement. For peaks below quantification limit, an overestimation of 5hmC/dG ratio is calculated based on the limit of detection of 5hmC. (f) Inhibition of replication in aphidicolin-treated zygotes assessed by EdU incorporation. (g) 5mC and 5hmC staining in control and aphidicolin-treated zygotes. Loss of 5mC and increase in 5hmC signals occur independently of DNA replication. Each data point represents an independent zygote (n = 9 control and n = 12 aphidicolin-treated zygotes). Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. (h) LC/MS measurement of 5mC/dG ratio in aphidicolin-treated zygotes (with polar bodies). Each value represents the average of 2 independent experiments with 2 technical replicates. 100 zygotes used per measurement. (i) 5mC kinetics in WT and Tet3 maternally depleted zygotes (Tet3mat−/pat+). (j) Bisulphite sequencing of the 5′UTR of Line1 repetitive elements (L1Md_Tf and L1Md_Gf subtypes) indicating the percentage of modified CpG dinucleotides in control (DMSO) and azadC-treated zygotes after removal of both polar bodies (PN4, 9hpf). p value was calculated using Mann–Whitney U-test. , female pronucleus; , male pronucleus; EdU, 5-ethynyl deoxyuridine. (Scale bars, 5 μm.)

Supplementary Figure 5

(a) Dnmt3a and Dnmt3L are detectable in both zygotic pronuclei from early PN3. DNA counter-stained with DAPI. Representative images are shown (n = 9 zygotes) (Scale bars, 5 μm.) (b) Analysis of DNA modifications in zygotes derived from in vitro fertilisation of [Dnmt3L−/−] oocytes by wild-type sperm. Quantification is represented as signal intensity in parental pronuclei. Each data point represents an independent zygote. (n = 11 WT and n = 11 KO zygotes). Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. ∗∗∗, p < 0.001. (Scale bars, 5 μm.) (c) Immunostaining of wild-type (WT) or triple Dnmt knockout [Dnmt1−/−Dnmt3a−/−Dnmt3b−/−] (TKO) ES cells with 5mC and 5hmC antibodies. (Scale bars, 15 μm.) (d) 5mC and 5hmC staining following SCNT into enucleated oocyte using WT (left panel) or TKO (right panel) ES cells 14 h post-activation. Detail of a mid-section shows that pericentric regions (yellow arrows) gain 5mC in WT-ESC and TKO-ESC pseudopronuclei but are protected from de novo hydroxymethylation. DNA counterstained by propidium iodide. (Scale bars, 15 μm.) (e) Confocal sections of paternal and maternal pronuclei in mouse zygotes co-stained with 5mC and 5hmC. Note the asymmetry in 5mC/5hmC modifications between parental pronuclei. DNA counterstained by propidium iodide. (Scale bars, 5 μm). (f) Total (-pre-extraction) and chromatin-bound (+ Triton pre-extraction) XRCC1 in control and DMOG-treated zygotes reflecting the activation of BER pathway independently of 5hmC accumulation. DNA counter-stained with DAPI. Representative images are shown. (g) LC/MS quantification of isotope-labelled dC (dC∗) ratio over dG in early pre-replicative PN3 zygotes and PN3 zygotes treated with aphidicolin (5.5hpf). Experiment carried out in technical and biological duplicates. (h) Replication kinetics visualised by EdU pulse in pre-replicative (5.5hpf) and post-replicative (6.5hpf) zygotes. Representative images are shown (n = 8). (Scale bars, 5 μm.) , female pronucleus; , male pronucleus; DMOG, dimethyloxallyl glycine; hpf, hours post-fertilisation, n.d., non-detected.

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Amouroux, R., Nashun, B., Shirane, K. et al. De novo DNA methylation drives 5hmC accumulation in mouse zygotes. Nat Cell Biol 18, 225–233 (2016). https://doi.org/10.1038/ncb3296

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