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H3K27 dimethylation dynamics reveal stepwise establishment of facultative heterochromatin in early mouse embryos

Nature Cell Biology volume 27, pages 28–38 (2025)Cite this article

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Abstract

Facultative heterochromatin is formed by Polycomb repressive complex 2 (PRC2)-deposited H3K27 trimethylation (H3K27me3) and PRC1-deposited H2AK119 mono-ubiquitylation (H2AK119ub1). How it is newly established after fertilization remains unclear. To delineate the establishment kinetics, here we profiled the temporal dynamics of H3K27 dimethylation (H3K27me2), which represents the de novo PRC2 catalysis, in mouse preimplantation embryos. H3K27me2 is newly deposited at CpG islands (CGIs), the paternal X chromosome (Xp) and putative enhancers during the eight-cell-to-morula transition, all of which follow H2AK119ub1 deposition. We found that JARID2, a PRC2.2-specific accessory protein possessing an H2AK119ub1-binding ability, colocalizes with SUZ12 at CGIs and Xp in morula embryos. Upon JARID2 depletion, SUZ12 chromatin binding and H3K27me2 deposition were attenuated and H3K27 acetylation at putative enhancers was increased in morulae and subsequently H3K27me3 failed to be deposited in blastocysts. These data reveal that facultative heterochromatin is established by PRC2.2-driven stepwise H3K27 methylation along pre-deposited H2AK119ub1 during early embryogenesis.

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Fig. 1: Allelic H3K27me2 dynamics during mouse preimplantation development.
Fig. 2: Stepwise deposition of Polycomb marks at functional elements.
Fig. 3: JARID2 and SUZ12 are colocalized in morula embryos.
Fig. 4: JARID2 is required for SUZ12 chromatin binding and H3K27me2 deposition in morula embryos.
Fig. 5: Role of JARID2 and AEBP2 in H3K27me3 deposition in blastocyst embryos.

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Data availability

All sequencing libraries generated in this study are summarized in Supplementary Table 6 and have been deposited to the Gene Expression Omnibus database under accession number GSE264193 for CATCH-seq and CUT&RUN and GSE263441 for RNA-seq. The H3K27me1, H3K27me2 and H3K27me3 ChIP-seq datasets of mES cells were from GSE39496 and GSE51006 (ref. 35). The H3K27me3 datasets of two-cell, blastocyst and epiblast were from GSE76687 (ref. 16). The H3K27me3 and H2AK119ub1 CUT&RUN datasets of four-cell embryos were from GSE153531 (ref. 63). The H3K27me3 CUT&RUN and RNA-seq datasets of FGOs and morula embryos and H2AK119ub1 CUT&RUN of one-cell, two-cell and morula embryos were from GSE153496 (ref. 17). The H3K36me3 STAR ChIP-seq datasets of FGOs and eight-cell embryos were from GSE112835 (ref. 46). All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank L. Shen for sharing a detailed protocol of CATCH-seq, H. Mei for teaching some bioinformatics analyses, S. Matoba for sharing tips of Triple-CRISPR, S. Henikoff for sharing the protein A-MNase, J. Takano and H. Koseki for sharing the typical Polycomb target gene list, and the Kazusa DNA Research Institute and H. Sugishita for support with next-generation sequencing. We thank S. Namekawa, S. Ito, O. Masui, H. Koseki, T. Ishiuchi, and the Inoue laboratory members for critical reading of the manuscript. This project was partly supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research on Innovative Areas (19H05754 to A.I.), Japan Agency for Medical Research and Development PRIME (JP20gm6110012 to A.I.), intramural grants within RIKEN including the RIKEN Pioneering Project ‘Genome Building from TADs’ and ‘Long-timescale Molecular Chronobiology’ to A.I. and RIKEN JRA programme to M.M.

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Authors and Affiliations

  1. Laboratory for Epigenome Inheritance, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Masahiro Matsuwaka, Mami Kumon & Azusa Inoue

  2. Tokyo Metropolitan University, Hachioji, Japan

    Masahiro Matsuwaka & Azusa Inoue

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  1. Masahiro Matsuwaka
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  2. Mami Kumon
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  3. Azusa Inoue
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Contributions

A.I. conceived the project. M.M. and A.I. designed the experiments. M.M. performed the experiments of embryos and analysed all sequencing data. M.K. and A.I. generated CATCH-seq and CUT&RUN libraries. M.M. and A.I. interpreted the data and wrote the manuscript.

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Correspondence to Azusa Inoue.

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The authors declare no competing interests.

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Nature Cell Biology thanks Rob Klose and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Quality validation of H3K27me2 CATCH-seq datasets.

a. Quantification of H3K27me2 immunostaining data. The numbers of embryos examined (n) in a single experiment are indicated in the main figure [additionally, at least 3 independent experiments were performed to compare different embryonic stages (for example eight-cell versus morula)]. Zygotes at 11 hpf were quantified for the one-cell samples. The averaged signal intensity in maternal pronuclei of one-cell zygotes was set as 1.0. Error bars, s.d. b. Quantification of H3K27me2 immunostaining data in each hemisphere of two-cell embryos. The numbers of embryos examined (n) are indicated. The averaged signal intensity in a hemisphere with lower signal intensity was set as 1.0. Error bars, s.d. c. Scatter plots showing H3K27me2 enrichment (10-kb window) of our CATCH-seq datasets using 500 and 5,000 mouse embryonic stem cells (mESCs) compared to a public ChIP-seq dataset in mESCs35. Spearman correlation is also shown. d. Genome browser view of the H3K27me2 distribution. e. Heatmap showing the signal enrichment of H3K27me3, 2, and 1. f. Heatmap showing the H3K27me2 levels of highly expressed genes, intermediate genes, and repressed genes. The centre lines in the boxes represent median values. The box edges, upper, and lower whiskers indicate the interquartile range (IQR; from the 25th to 75th percentile), the largest value smaller than 1.5× the IQR above the 75th percentile, and the smallest value larger than 1.5× the IQR below the 25th percentile, respectively (n = 1).

Extended Data Fig. 2 Characterization of H3K27me2 distribution in early embryos.

a. Scatter-plot showing the correlation between biological duplicates of H3K27me2 CATCH-seq in FGOs, four-cell, eight-cell, and morula embryos. Spearman correlation is also shown. b. Averaged signal profiles of maternal and paternal H3K27me2 at gene bodies in four-cell, eight-cell, and morula embryos. The two groups represent active genes (FPKM > 5) and inactive genes (FPKM < 1), respectively. c. Genome browser views of H3K27me2, H3K27me3 and H3K36me3 distributions at paternal and maternal alleles. The H3K36me3 datasets of FGO and eight-cell embryos are from46. Those of four-cell and morula embryos were generated in this study. d. Heatmap showing the genome-wide enrichment of H3K27me2 and H3K36me3 (10-kb bins). e. Genome browser view at the Gab1 (top) and Runx1 loci (bottom) in morula embryos. M, maternal allele. P, paternal allele. f. Scatter plots showing H3K27me2 enrichment (10-kb window) in maternal and paternal alleles of four-cell, eight-cell, and morula embryos. Spearman correlation is also shown. g. Genome browser view of the parental alleles next to each other. The genomic location is identical to the main figure. h. Heatmap showing the allelic biases of H3K27me2 at allelic H3K27me3-retained regions. Each row represents a 10-kb bin that had enough signals (RPKM > 2). Upper and lower groups represent the regions where H3K27me3 are maternally and paternally biased, respectively, in morula embryos (FC > 2). The H3K27me3 dataset is from17. i. Averaged signal profiles of H3K27me2 at regions with different gene densities (gene number per 1 Mb sliding window with a 200-kb step) in four-cell, eight-cell and morula embryos. j. Genomic distribution of H3K27me2-persisted (group1, 3) and -gained loci (group2, 4). Promoters represent the regions of ±2.5 kb around transcription start sites.

Extended Data Fig. 3 Dynamics of H2AK119ub1, H3K27m2, and H3K27me3 during early development.

a. Heatmap showing the averaged signals of genome-wide 10-kb bins within each cluster at the maternal and paternal alleles. The H2AK119ub1 and H3K27me3 datasets are from 16,17,63. b. Genome browser view of each cluster. c, d. Representative images (c) and quantification (d) of H2AK119ub1 immunostaining analysis. The numbers of embryos examined (n) in a single experiment are indicated [additionally, at least 3 independent experiments were performed to compare different embryonic stages (for example one-cell versus two-cell)]. The averaged signal intensity in maternal pronuclei of one-cell zygotes was set as 1.0. Scale bars, 20 µm. Error bars, s.d. e. GO-term analysis of persisted and de novo clusters of typical Polycomb target genes. p, two-sided p value (non-adjusted). f. Heatmap showing the signal enrichment at putative enhancers. Putative enhancers were defined by non-TSS H3K27ac peaks (at least 2.5 kb far from TSSs) in 8 adult tissues and mESCs. g. TF motif enrichment analysis at H3K27me2-high, -gained, and -low clusters of putative enhancers. Gradient colour indicates gene expression levels in morula embryos. p value, hypergeometric test with Bonferroni correction, two-sided from HOMER. h. Heatmap showing the spike-in scaled H3K27me2 signal enrichment at the typical Polycomb target genes. i-l. Box plot showing the spike-in scaled H3K27me2 signal enrichment at the “de novo” cluster of typical Polycomb target genes (i), paternal X-chromosome (j), Xp-linked genes (k), and the “gained” cluster of putative enhancers (l). The centre lines in the boxes represent median values. The box edges, upper, and lower whiskers indicate the interquartile range (IQR; from the 25th to 75th percentile), the largest value smaller than 1.5× the IQR above the 75th percentile, and the smallest value larger than 1.5× the IQR below the 25th percentile, respectively (n = 1). p, two-sided Mann–Whitney U-test.

Source data

Extended Data Fig. 4 Characterization of JARID2 and SUZ12 distributions in morula embryos.

a. Heatmap showing Ribo-seq signals of PRC2 core and -accessory proteins and H3K27-specific demethylases in oocytes and preimplantation embryos. The Ribo-seq datasets are from54.b. Scatter plots showing the correlation between biological duplicates in JARID2 and SUZ12 CUR&RUN datasets. Spearman correlation is also shown. c. Pie chart showing the percentage of JARID2 and SUZ12 peaks that are overlapped with annotated CGIs. d. Scatter-plot showing the correlation between SUZ12, JARID2, and H3K27me2 across the paternal X-chromosome in morula embryos. Spearman correlation is also shown.

Extended Data Fig. 5 Characterization of Jarid2 KO morula embryos.

a. Preimplantation development of control and Jarid2 KO embryos. The embryos that reached the four-cell, eight-cell and morula stages in a timely fashion were counted at 48, 56 and 72 hours post-fertilization (hpf), respectively. The number of two-cell embryos was set as 100%. b. Representative images of H3K27me2 immunostaining analysis in control and Jarid2 KO morula embryos. Scale bar, 20 µm. c. Quantification of H3K27me2 signal intensities. The number of embryos examined (n) is indicated. The averaged signal intensity in CTR embryos was set as 1.0. The centre lines in the boxes represent median values. The box edges, upper, and lower whiskers indicate the interquartile range (IQR; from the 25th to 75th percentile), the largest value smaller than 1.5× the IQR above the 75th percentile, and the smallest value larger than 1.5× the IQR below the 25th percentile, respectively. p, two-sided Mann–Whitney U-test. d. Scatter-plot showing the correlation between biological duplicates. That of SUZ12 CUT&RUN in control morula embryos is shown in Extended Data Fig. 4b. Spearman correlation is also shown. e, f. Heatmap (e) and averaged plot (f) showing the SUZ12 and H3K27me2 signal enrichment at original JARID2 peak loci in control and Jarid2 KO morula embryos. g. Box plot showing H3K27me2 and SUZ12 levels across the paternal X-chromosome in morula embryos. The centre lines in the boxes represent median values. The box edges, upper, and lower whiskers indicate the interquartile range (IQR; from the 25th to 75th percentile), the largest value smaller than 1.5× the IQR above the 75th percentile, and the smallest value larger than 1.5× the IQR below the 25th percentile, respectively (n = 1, as biological replicates were combined). p, two-sided Mann–Whitney U-test. h. Heatmap showing the ratio of allelic read counts of single embryo RNA-seq in WT and Jarid2 KO female morula embryos. Ratio: paternal reads / (paternal + maternal reads). The X-linked gene list is from47, but only those that have >20 SNP-containing reads are shown (paternal + maternal reads > 20). i. Representative images of H2AK119ub1 immunostaining analysis. The numbers and percentage of embryos exhibiting clear H2AK119ub1 foci are indicated. The right images are enlarged blastomeres from the regions indicated by dotted lines in the left images. White arrowheads indicate putative inactive X-chromosome showing H2AK119ub1 foci. Scale bar, 20 µm.

Source data

Extended Data Fig. 6 Characterization of H3K27ac in Jarid2 KO morula embryos.

a. Pie charts showing the ratios of H3K27me2-lost (C1) and intermediate (C2) putative enhancers belonging to each group of H3K27me2 dynamics. b. Scatter-plot showing the correlation between biological duplicates. Spearman correlation is also shown. c. Heatmap showing the enrichment of allelic H3K27me3 in wild-type morula embryos and allelic H3K27ac in control and Jarid2 KO morula embryos at H3K27me2-lost (C1) putative enhancers. Loci that did not have enough SNP-containing reads ( < 20) were removed from the analysis. Averaged signal profiles at the right indicate H3K27ac levels at H3K27me2-lost putative enhancers that are devoid of H3K27me3. d. Box plots showing the expression levels (log2TPM) of the nearest genes from the putative enhancers of each cluster. The gene numbers (n) are indicated at the bottom. The centre lines in the boxes represent median values. The box edges, upper, and lower whiskers indicate the interquartile range (IQR; from the 25th to 75th percentile), the largest value smaller than 1.5× the IQR above the 75th percentile, and the smallest value larger than 1.5× the IQR below the 25th percentile, respectively. p, two-sided Mann–Whitney U-test.

Extended Data Fig. 7 H3K27me3 changes and H2AK119ub1 immunostaining in mutant blastocysts, and the expression of AEBP2.

a. Scatter-plot showing the correlation of H3K27me3 CATCH-seq between biological duplicates. Spearman correlation is also shown. b. Scatter plots showing H3K27me3 enrichment (10-kb window). Percentages at the lower right indicate the proportion of H3K27me3-lost bins [RPKM(CTR) > 1, FC(CTR/KO) > 2]. c, d. Heatmap (c) and averaged plot (d) showing H3K27me3 signals at SUZ12 peaks that had been defined in CTR morula embryos. e, f. Representative images (e) and quantification (f) of AEBP2 immunostaining analysis in preimplantation embryos. The numbers of embryos examined (n) in a single experiment are indicated. Embryos without XCI foci were quantified. The averaged signal intensity in morula embryos was set as 1.0. Scale bar, 20 µm. g. Representative images of AEBP2 immunostaining analysis for AEBP2 KO embryos. The number of embryos examined in three independent experiments is 42 (CTR) and 55 (KO). The numbers of embryos lacking AEBP2 staining signals are indicated. Scale bar, 20 µm. h. Heatmap showing pairwise Pearson correlations of H3K27me3 between each group. i. Heatmap of H3K27me3 signals at CTR and DKO blastocysts. Genes with FC(DKO/CTR) > 0.75 were considered as putative PRC2.2-independent targets (n = 31), and their representative genome browser snapshots are shown at right. j. Heatmap and averaged plot showing H3K27me3 signals at CGIs. k. Representative images of H2AK119ub1 immunostaining analysis in blastocysts. The numbers and percentage of embryos exhibiting clear H2AK119ub1 foci are indicated. The right images are enlarged blastomeres from the regions indicated by dotted lines in the left images. White arrowheads indicate putative inactive X-chromosome showing H2AK119ub1 foci. Scale bar, 20 µm.

Source data

Extended Data Fig. 8 Schematic model illustration of the stepwise establishment of facultative heterochromatin at CGIs, putative enhancers, and the paternal X-chromosome (Xp).

a. At CGI promoters of many Polycomb-targeted developmental genes, H2AK119ub1 is gained at the two-cell stage, and H3K27me2 and 3 are gained at the morula and the blastocyst stages, respectively, catalysed by PRC2.2. In Jarid2 KO morula embryos, H3K27me2 fails to be deposited at CGIs and distal regions, resulting in an increase of H3K27ac at putative enhancers. In Jarid2 KO and DKO blastocysts, H3K27me3 fails to be deposited with the DKO being more prominent. By contrast, H3K27me3 is deposited at CGIs in Aebp2 KO blastocysts. b. Across Xp, H2AK119ub1 is progressively accumulated from the eight-cell stage, while H3K27me2 and 3 are accumulated from the morula and the blastocyst stages, respectively, in a PRC2.2-dependent manner. H3K27me2 fails to accumulate at Xp in Jarid2 KO morula embryos. In both Jarid2 KO and Aebp2 KO blastocysts, H3K27me3 fails to accumulate, but it is locally deposited at CGIs in the Aebp2 KO. H3K27me3 deposition is abrogated in DKO blastocysts.

Extended Data Fig. 9 H3K27me3 distribution in JARID2 overexpressed (OE) two-cell and morula embryos.

a. Representative images of two-cell embryos that had been injected with Jarid2 mRNA (OE group). No-injection embryos were served as a control. Shown are immunostaining analysis with anti-JARID2 and anti-H3K27me3 antibodies. The numbers of embryos examined (n) in 3 independent experiments are 15 (no-injection) and 21 (OE). Scale bar, 20 µm. b. Heatmap showing the signal enrichment at promoters of typical Polycomb target genes. The dataset of wild-type (WT) blastocyst embryos was served as a positive control. CTR, no-injection control. J2OE, Jarid2 overexpression. c, d. Box plot showing the signal enrichment at the “de novo” cluster of typical Polycomb target genes (c) and Xp (d). The centre lines in the boxes represent median values. The box edges, upper, and lower whiskers indicate the interquartile range (IQR; from the 25th to 75th percentile), the largest value smaller than 1.5× the IQR above the 75th percentile, and the smallest value larger than 1.5× the IQR below the 25th percentile, respectively (n = 1). p, two-sided Mann–Whitney U-test. e. Genome browser view of H3K27me3 enrichment across Xp.

Source data

Supplementary information

Reporting Summary

Supplementary Tables 1, 3–6

Supplementary Table 1. Gene expression levels of Jarid2 KO morula embryos (bulk samples). Supplementary Table 3. Allelic TPM of single female embryo RNA-seq in Jarid2 KO morula embryos. Supplementary Table 4. H3K27me3 levels at typical Polycomb target genes in Jarid2/Aebp2 DKO blastocyst embryos. Supplementary Table 5. sgRNA and primer sequences. Supplementary Table 6. Summary of sequence library information.

Supplementary Table 2

Supplementary Table 2. Allelic read counts of single female embryo RNA-seq in Jarid2 KO morula embryos.

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Matsuwaka, M., Kumon, M. & Inoue, A. H3K27 dimethylation dynamics reveal stepwise establishment of facultative heterochromatin in early mouse embryos. Nat Cell Biol 27, 28–38 (2025). https://doi.org/10.1038/s41556-024-01553-1

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