5hmC and 5mC kinetics during mouse zygotic development. 5mC (a) and 5hmC (b) enrichment in mouse zygotes at different developmental stages as in assessed by immunofluorescence using 5mC and 5hmC specific antibodies. DNA is stained using PI. Representative images are shown and correspond to the 5mC and 5hmC signals quantification presented in (c). (c) Quantification of 5mC (red line, left axis) and 5hmC (green line, right axis) staining is shown as a ratio between signal from paternal pronucleus relative to the signal from maternal pronucleus. Values are plotted against the area of the mid-sections of the paternal pronuclei. Each data point represents a zygote. Experiment reproduced 3 times (n>100) (d) Loss of paternal 5mC and accumulation of 5hmC are temporally separated. Early PN3 zygotes do not show any detectable 5mC or 5hmC in paternal pronucleus. 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; PN, pronuclei; PI, propidium iodide; ♀, female pronucleus; ♂, male pronucleus; pb, polar body. (Scale bars, 5um.)

Small molecule inhibition of Tet protein activity abrogates 5hmC formation but does not prevent DNA demethylation. (a) 5mC and 5hmC staining of control and DMOG-treated zygotes (IVF). Quantification of both DNA modifications is represented as a ratio between the pronuclear signals (pat/mat). For 5mC staining, n=18 PN3 zygotes and n=40 PN4-5 zygotes; for 5hmC staining, n=17 PN3 zygotes and n=48 PN4-5 zygotes. This experiment has been replicated 4 times independently. (b) Quantification of 5mC/dG and 5hmC/dG ratio in sperm, MII oocytes, and in zygotes without polar bodies (control or treated with DMOG) by LC/MS (n=3 independent experiments with 2 technical replicates each, except for DMOG-treated zygotes; replicate of this experiment in ). Limits of quantification are summarised in . For peaks below quantification limit, an overestimation of 5hmC/dG ratio is calculated based on the limit of detection of 5hmC. (c) Quantification of DNA modifications in 2-cell embryos derived from DMOG-treated or control zygotes analysed by LC/MS (n=2 independent experiments and 2 technical replicates for each point). Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. DMOG, dimethyloxallyl glycine; ♀, female pronucleus; ♂, male pronucleus. n.d., non-detectable; ***, p<0.001. (Scale bars, 5um.)

Tet3 is not required for loss of 5mC in early zygote. (a) Scheme of the targeting strategy used to generate Tet3 conditional mice. (b) RT-qPCR analysis of Tet3 mRNA (exon3 and exon11) in control (Tet3^mat+) and Tet3-depleted oocytes (Tet3^mat−). Results are normalised to endogenous H3f3a and to control (Tet3^mat+). Bars represent the mean of 3 technical replicates. (c) Tet3^WT (n=10) and Tet3^mat−/pat+ (n=8) zygotes were stained for Tet3 protein. Quantification is represented as the mean of intensity on the paternal pronuclei after background subtraction. (d) Tet3^WT and Tet3^mat−/pat+ zygotes were co-stained for 5mC and 5hmC at different time points post-fertilisation. Quantification of both DNA modifications is presented as a ratio of paternal over maternal signal intensity. Each data point represents an independent zygote (n=5 PN3, n=6 PN3L and n=13 Tet3^WT zygotes; n=5 PN3, n=6 PN3L and n=19 Tet3^mat−/pat+ zygotes; 2 independent experiments). (e) Quantification of 5mC/dG and 5hmC/dG in Tet3^WT and Tet3^mat−/pat+ zygotes (with polar bodies) by LC/MS. Each point represents the mean of 2 technical replicates of a pool of about 100 oocytes or embryos. (f) Quantification of DNA modifications in 2-cell embryos derived from Tet3^WT or Tet3^mat−/pat+ zygotes analysed by LC/MS. Each point represents the mean of 2 technical replicates of a pool of about 50 embryos. Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. PN3L, late PN3; ♀, female pronucleus; ♂, male pronucleus. n.d., non-detectable; *, p<0.05; **, p<0.01; ****, p<0.0001 (Scale bars, 5um.)

Hydroxylation targets newly deposited 5mC generated by Dnmt3a and Dnmt1. (a) Isotope-labelled 5mC (5mC*) quantified by LC/MS after incubation of zygotes with heavy methionine (¹³C,d₃-methyl) in the presence or absence of aphidicolin (IVF). Each point represents a biological replicate (n=2). An example of the 5mC* peak detected by LC/MS is depicted for each condition and further confirms the existence of both maintenance and de novo DNA methylation in zygotes. Note that the observed signal represents only a fraction of new zygotic 5mC due to the endogenous pool of unlabelled S-adenosyl-methionine. (b) Inhibition of new zygotic DNA methylation by 5-azadeoxycytidine (azadC) (IVF) affects accumulation of paternal 5hmC as assessed by staining using 5mC and 5hmC specific antibodies. Only zygotes with a paternal mid-section area > 200um² (~PN4-5 zygotes) were considered to avoid developmental staging bias. Quantification of 5mC and 5hmC is represented as signal intensity in paternal and maternal pronuclei (left axis) or as a ratio between the pronuclei signal (pat/mat) (right axis). (n=6 control and n=18 treated zygotes; experiment replicated twice independently). 5mC and 5hmC staining in PN4-5 zygotes (paternal mid-section area >200um²) with maternal (c) Dnmt3a ([♀Dnmt3a^2lox/2lox, Zp3-Cre] × ♂WT) (n=12 WT and n=10 KO zygotes; experiment reproduced twice independently) or (d) Dnmt1 ([♀Dnmt1^2lox/2lox, Zp3-Cre × ♂WT]) (n=13 WT and n=11 KO zygotes; experiments reproduced twice independently) deletion. Note that only total signal intensity is plotted in (c) as Dnmt3a deletion affects 5mC and 5hmC level in maternal PN. (e, f) Tet3 localisation and signal intensity is identical between (e) WT (n=4) and Dnmt3a KO (n=3) or (f) WT (n=5) and Dnmt1 KO (n=3) zygotes. Quantification is represented as the mean of intensity on the paternal pronuclei after background subtraction. Statistical analysis was carried out using Student’s t-test (two-sided). Error bars indicate s.d. *, p<0.05; **, p<0.01; ***, p<0.001. ♀, female pronucleus; ♂, male pronucleus; azadC, 5-azadeoxycytidine. (Scale bars, 5um.)

De novo DNA methylation activity is present in the oocyte and generates a target for hydroxylation in SCNT experiment. (a) Schematic of the somatic cell nuclear transfer (SCNT) experiment using wild-type (WT) or triple Dnmt knockout [Dnmt1^−/− Dnmt3a^−/− Dnmt3b^−/−] (TKO) ES cells. 5mC and 5hmC staining following SCNT into enucleated oocyte using WT (left panel) or a TKO (right panel) ES cells. Staining was carried out 5 and 14 hours post-activation of the reconstituted embryos. As a control, staining of maternal genome (indicated by an arrow) in embryos following SCNT into non-enucleated oocyte (lower panel). The increase of 5mC and 5hmC intensity on the TKO nuclei 14hrs post-activation reflects de novo DNA methylation activity in the oocyte, targeted by Tet3-hydroxylation. Representative images are shown (n=7 and n=6 WT-ESC and TKO pseudopronuclei respectively) (Scale bars, 5um.) (b) Following genome-wide loss of spermatic 5mC in the male pronucleus of mouse zygote, newly deposited 5mC produced by zygotic Dnmt1 and Dnmt3a is hydroxylated by Tet3 (model).