A small proportion of promoters initiate de novo DNA methylation by 8-cell stage.

(A) The mRNA expression levels of Dnmt3s from the 2-cell to blastocyst stage. The left ordinate represents FPKM values of Dnmt3s from published transcriptome data (Fan et al., 2015), and the right ordinate represents relative expression levels detected by qRT–PCR. (B) Immunofluorescent staining of DNMT3B in mouse preimplantation embryos. Scale bars: 50 µm. (C-D) DNA methylation changes of promoters (C) and gene bodies (D) during the transition from the 8-cell to blastocyst stage. Left panel: heat map of DNA methylation levels at promoters or gene bodies. Right panel: general trends of promoters or gene bodies undergoing de novo methylation or demethylation, and their relative contribution to differentially methylated regions. (E-F) DNA methylation dynamics of promoters subject to demethylation (E) or de novo methylation (F) during the transition from the 8-cell to blastocyst stage (red and green lines, average methylation levels; colored space, 10th/ 90th percentile). (G) Bisulfite sequencing analysis of Yy2 in the 8-cell embryos and blastocysts. Black and white circles represent methylated cytosines and unmethylated cytosines respectively. (H) A model illustrating two waves of de novo DNA methylation before and after the blastocyst stage, and their potential functions.

Minor de novo methylation preferentially occurs on the X chromosome and plays an important role in iXCI.

(A) The chromosome-wide distribution of de novo methylated promoters with different fold changes (FC) during the transition from the 8-cell to blastocyst stage. (B) Percentage of de novo methylated promotors on each chromosome during the transition from the 8-cell to blastocyst stage. (C) Representative immunostaining for H3K27me3 (red) in the nuclei (DAPI) of NC and Dnmt3b-KD female blastocysts colabeled with CDX2 (green)-positive trophoblast cells. Scale bars: 50 µm. (D) The ratio of trophoblast cells classified by the number of H3K27me3 domains in each NC and Dnmt3b-KD female blastocysts. Each bar represents one female embryo. All data are presented as the mean ± SD of at least three independent experiments. **P < 0.01. ns, not significant.

Minor de novo DNA methylation co-regulates iXCI via the interaction between DNMT3B and PRC2 core components.

(A) Representative localization of Xist expression detected by RNA-FISH in the nuclei (DAPI) of NC and Dnmt3b-KD female morulae. (B) The ratio of blastomere with Xist signal to the total number of blastomeres in each NC and Dnmt3b-KD female morulae. Each bar represents one female embryo. (C) Rnf12 and Xist expression levels in individual Dnmt3b-KD female 8-cell embryos and morulae. (D) Expression of H3K27me3 methylases (Ezh2, Suz12 and Eed) and demethylases (Kdm6a and Kdm6b) in individual Dnmt3b-KD female morulae. (E) Validation of the interaction between DNMT3B and PRC2 core components, i.e., EZH2, SUZ12, EED, via in situ PLA. (F) Representative immunostaining for H3K27me3 (red) in the nuclei (DAPI) of wild-type, Dnmt3b-KO, and Dnmt3a/3b double knockout (DKO) female ES cells differentiated for 7 days, as well as the ratio of ES cells classified by the number of H3K27me3 domains. (G) Western blot showing immunoprecipitation of DNMT3B and EZH2, SUZ12, EED from differentiated female ES cells. All data are presented as the mean ± SD of at least three independent experiments. **P < 0.01. ns, not significant. Scale bars: 50 µm (A, E); 20 µm (F).

Minor de novo DNA methylation participates in embryonic proliferation, differentiation and affects developmental potential.

(A) EdU staining of NC and Dnmt3b-KD blastocysts. Right panel: the percentage of EdU-positive cells. (B) The total cell number (left panel) and the ratio of ICM/TE (right panel) in NC and Dnmt3b-KD blastocysts. (C) The total ATP production (left panel) and OXPHOS-dependent ATP production (right panel) in NC and Dnmt3b-KD blastocysts. (D-E) Implantation rate (D) and live birth rate (E) in NC and Dnmt3b-KD groups. All data are presented as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01. ns, not significant. Scale bars: 50 µm (A, B). (F) A model illustrating a wave of minor de novo DNA methylation that initiates during the transition from the mouse 8-cell to blastocyst stage. Minor de novo DNA methylation co-regulates iXCI via the interaction between DNMT3B and PRC2 core components, and fine-tunes the processes of lineage commitment, proliferation and metabolic homeostasis before blastocyst formation.

(A) Dynamics of global methylation during preimplantation embryos (blue line, average methylation levels; colored space, 10th/ 90th percentile). (B) General trends of CpG islands undergoing de novo methylation or demethylation, and their relative contribution to differentially methylated regions. (C) Number of de novo methylated promoters with different fold changes (FC) at each consecutive transition. (D) DNA methylation dynamics of promoters with different CpG-densities during consecutive transitions.

(A) The distribution of de novo methylated and demethylated promoters cross the X chromosome during the transition from the 8-cell to blastocyst stage. (B) The chromosome-wide distribution of de novo methylated promoters with different fold changes (FC) during the transition from the blastocyst to post-implantation stage. (C) Percentage of de novo methylated promoters on each chromosome during the transition from the blastocyst to post-implantation stage.

(A) Relative expression levels of Dnmt3b in NC and Dnmt3b-KD morulae and blastocysts. (B-C) Immunofluorescent staining (C) and quantification (D) of DNMT3B in NC and Dnmt3b-KD morulae and blastocysts. (D-E) Immunofluorescent staining (D) and quantification (E) of 5-mC in NC and Dnmt3b-KD morulae and blastocysts. (F) The ratio of trophoblast cells classified by the number of H3K27me3 domains in each wild-type and Dnmt3b-KO female blastocysts. Each bar represents one female embryo. (G) The ratio of trophoblast cells classified by the number of H3K27me3 domains in female blastocysts exposed or not exposed to 5-aza-2’-deoxycytidine (5-aza-dC) from the 8-cell to blastocyst stage. Each bar represents one female embryo. (H) The fold changes of promoter methylation levels of X-linked non-escaping genes from the 8-cell to blastocyst stage. All data are presented as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01. ns, not significant. Scale bars: 50 µm (B, D).

The ratio of blastomere with Xist signal to the total number of blastomeres in each wild-type and Dnmt3b-KO female morulae. Each bar represents one female embryo.

(A) H3K27me3 enrichment cross the X chromosome from the 8-cell to blastocyst stage based on the previously published data (Liu et al., 2016). (B) Concurrent increase in H3K27me3 enrichment (left panel) and DNA methylation levels (right panel) at selected X-linked promoters that undergo minor de novo DNA methylation.

(A) Schematic illustration of generation of Dnmt3b-KO female ES cells via the CRISPR/Cas9n system. (B) Detection of deletion introduced by sgRNA-Cas9n targeting Dnmt3b via PCR with genomic DNA from wild-type and Dnmt3b-KO female ES cells. (C) Relative expression levels of Dnmt3b in wild-type and Dnmt3b-KO female ES cells. (D) Schematic illustration of generation of DKO female ES cells via the CRISPR/Cas9n system. (E) Detection of deletion introduced by sgRNA-Cas9n targeting Dnmt3a/3b via PCR with genomic DNA from wild-type and DKO female ES cells. (F) Relative expression levels of Dnmt3a and Dnmt3b in wild-type and DKO female ES cells. (G) Representative immunostaining for H3K27me3 (red) in the nuclei (DAPI) of wild-type, Dnmt3b-KO, and DKO female ES cells differentiated for 5 or 9 days, as well as the ratio of ES cells classified by the number of H3K27me3 domains. All data are presented as the mean ± SD of at least three independent experiments. **P < 0.01. Scale bars: 20 µm.

(A) A scatter plots of changes in promoter DNA methylation and gene expression from the 8-cell embryos to ICM. Red plots indicate putative genes that undergo minor de novo DNA methylation (FC > 2) and are downregulated (FC < 0.5). (B-C) Gene ontology analysis (B) and phenotype annotation (C) of genes transcriptionally regulated by minor de novo DNA methylation. Blue squares indicate genes that are related to developmental or lethal phenotypes. (D) Gene ontology analysis of genes transcriptionally regulated by DNA demethylation from the 8-cell embryos to ICM.

(A) EdU staining of blastocysts exposed to 5-aza-dC from the 8-cell to blastocyst stage. Right panel: the percentage of EdU-positive cells. (B) The total cell number (left panel) and the ratio of ICM/TE (right panel) of blastocysts treated with or without 5-aza-dC. (C) The total ATP production (left panel) and OXPHOS-dependent ATP production (right panel) of blastocysts treated with or without 5-aza-dC. All data are presented as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01. ns, not significant. Scale bars: 50 µm (A, B).