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) DNA methylation changes of promoters during the transition from the 8-cell to blastocyst stage. Left panel: heat map of DNA methylation levels at promoters. Right panel: general trends of promoters undergoing de novo methylation or demethylation, and their relative contribution to differentially methylated regions. (D-E) DNA methylation dynamics of promoters subject to demethylation (D) or de novo methylation (E) during the transition from the 8-cell to blastocyst stage (red and green lines, average methylation levels; colored space, 10th/ 90th percentile). (F) A model illustrating two waves of de novo DNA methylation before and after the blastocyst stage, and their potential functions.

(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 promoters that gain or loss DNA methylation in female and male embryos during the transition from the 8-cell to blastocyst stage. (D) DNA methylation changes of promoters that gain DNA methylation during the transition from the 8-cell to blastocyst stage in female or male embryos. (E) DNA methylation dynamics of promoters with different CpG-densities during consecutive transitions.

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

(A) Schematic diagram of the experimental design. (B) EdU staining of NC and Dnmt3b-KD blastocysts. Right panel: the percentage of EdU-positive cells. (C) The total cell number (left panel) and the ratio of ICM/TE (right panel) in NC and Dnmt3b-KD blastocysts. (D) The total ATP production (left panel) and OXPHOS-dependent ATP production (right panel) in NC and Dnmt3b-KD blastocysts. (E-F) Implantation rate (E) and live birth rate (F) 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).

(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 (ΔDNA methylation > 0.01) and are downregulated (ΔDNA methylation < -0.01). (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) 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. 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).

(A) Schematic diagram of the experimental design. (B) EdU staining of blastocysts exposed to 5-aza-dC from the 8-cell to blastocyst stage. Right panel: the percentage of EdU-positive cells. (C) The total cell number (left panel) and the ratio of ICM/TE (right panel) of blastocysts treated with or without 5-aza-dC. (D) 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 (B, C).

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 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) Schematic diagram of the experimental design and the molecular processes of iXCI initiation and establishment. Dnmt3b knockdown was performed by microinjecting Dnmt3b siRNA into zygotes. Immunostaining assay for H3K27me3 was performed and quantified in trophoblast cells in each female blastocyst. (D) 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. The arrowhead indicates the H3k27me3 domain and the blank arrowhead indicates the blastomere without the H3k27me3 domain. (E) 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. (F) The percentage of H3K27me3-positive and H3K27me3-negative trophoblast cells (TCs) to total trophoblast cells in female blastocysts. All data are presented as the mean ± SD of at least three independent experiments. **P < 0.01. ns, not significant.

(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 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) DNA methylation changes of promoters that gain DNA methylation during the transition from the 8-cell to blastocyst stage in chromosome X and autosomes of female embryos. (B) DNA methylation changes of promoters that gain DNA methylation during the transition from the 8-cell to blastocyst stage in chromosome X and autosome of male embryos.

(A) Schematic diagram of the experimental design. Dnmt3b-KO female embryos were collected by intercrossing Dnmt3b+/- male and female mice. Immunostaining assay for H3K27me3 was performed and quantified in trophoblast cells in each female homozygous blastocyst. (B) Representative immunostaining for H3K27me3 (red) in the nuclei (DAPI) of wild-type and Dnmt3b-KO female blastocysts colabeled with CDX2 (green)-positive trophoblast cells. The arrowhead indicates the H3k27me3 domain and the blank arrowhead indicates the blastomere without the H3k27me3 domain. (C) 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. (D) The percentage of H3K27me3-positive and H3K27me3-negative trophoblast cells (TCs) to total trophoblast cells in female blastocysts. (E) Schematic diagram of the experimental design. Zygotes were cultured in vitro and transiently exposed to 5-aza-dC during the window of minor de novo DNA methylation (the 8-cell to blastocyst stage) or not. Immunostaining assay for H3K27me3 was performed and quantified in trophoblast cells in each female blastocyst. (F) Representative immunostaining for H3K27me3 (red) in the nuclei (DAPI) of 5-aza-dC treated or untreated female blastocysts colabeled with CDX2 (green)-positive trophoblast cells. Scale bars: 50 µm. The arrowhead indicates the H3k27me3 domain and the blank arrowhead indicates the blastomere without the H3k27me3 domain. (G) The ratio of trophoblast cells classified by the number of H3K27me3 domains in female blastocysts exposed or not exposed to 5-aza-dC from the 8-cell to blastocyst stage. Each bar represents one female embryo. (H) The percentage of H3K27me3-positive and H3K27me3-negative trophoblast cells (TCs) to total trophoblast cells in female blastocysts. (I) The changes of promoter methylation levels of X-linked non-escaping genes from the 8-cell to blastocyst stage. (J) The expression level changes of de novo methylated X-linked non-escaping genes during the transition from 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, E).

(A) Genome browser views of the allelic expression, DNA methylation and H3K27me3 enrichment cross the X chromosome in WT and DKO female ExE based on the previously published data (Chen et al., 2019). (B) Genome browser views of the paternal expression of selected X-linked non-escaping genes that undergo minor de novo DNA methylation based on the previously published data (Chen et al., 2019).

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. The arrowhead indicates Xist RNA domain and the blank arrowhead indicates the blastomere without Xist RNA domain. (C) Rnf12 and Xist expression levels in individual Dnmt3b-KD female 8-cell embryos and morulae. (D) Expression of H3K27me3 modifiers 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. 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). (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) Representative localization of Xist expression detected by RNA-FISH in the nuclei (DAPI) of wild-type and Dnmt3b-KO female morulae. The arrowhead indicates Xist RNA domain and the blank arrowhead indicates the blastomere without Xist RNA domain. Scale bars: 50 µm. (B) 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. All data are presented as the mean ± SD of at least three independent experiments. ns, not significant.

(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, 7 or 9 days, as well as the ratio of ES cells classified by the number of H3K27me3 domains. Scale bars: 20 µm. (H) 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.