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H2A.Z reinforces maternal H3K4me3 formation and is essential for meiotic progression in mouse oocytes

Nature Structural & Molecular Biology (2025)Cite this article

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Abstract

Mammalian oocytes establish a unique landscape of histone modifications, some of which are inherited by early embryos. How histone variants shape the maternal histone landscape remains unknown. Here we map histone H2A variants in mouse fully grown oocytes (FGOs) and find that H2A.Z forms broad domains across intergenic regions, along non-canonical H3K4me3 (ncH3K4me3). During oocyte growth, H2A.Z progressively transitions from an active promoter-rich, canonical distribution to a non-canonical broad distribution (ncH2A.Z). Depletion of H2A.Z in oocytes partially impairs ncH3K4me3 formation and causes severe defects in meiotic progression, which resemble Mll2-knockout oocytes. Conversely, depletion of ncH3K4me3 by Mll2 knockout also causes a reduction of ncH2A.Z in FGOs. Thus, our study suggests that ncH2A.Z and ncH3K4me3 reinforce each other to form functional oocytes.

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Fig. 1: Distribution of H2A variants in FGOs.
Fig. 2: Shifting to ncH2A.Z distribution during oocyte growth.
Fig. 3: H2A.Z is dissociated from bivalent promoters in FGOs.
Fig. 4: H2A.Z contributes to MLL2-dependent ncH3K4me3 formation.
Fig. 5: ncH2A.Z and MLL2-dependent ncH3K4me3 reinforce each other.
Fig. 6: Both H2A.Z-DKO and Mll2-KO oocytes are defective in meiotic maturation.
Fig. 7: Illustration of H2A.Z dynamics in 5-day GOs and FGOs, and histone modification changes in H2A.Z-DKO and Mll2-KO FGOs.

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Data availability

All the sequencing datasets generated in this study have been deposited in the GEO database under accession numbers GSE262044 and GSE262049. The H2A.Z ChIP–seq datasets of mouse MII oocytes and preimplantation embryos were from GSE188590 (ref. 17). RNA-seq datasets during oogenesis were from GSE70116 (ref. 41). H3K4me3 ChIP–seq datasets of four stages of WT oocytes were from GSE93941 (ref. 7). H3K27ac ChIP–seq datasets of mouse oocytes were from GSE217970 (ref. 33). H3K4me3 ChIP–seq datasets of mouse MII oocytes and preimplantation embryos were from GSE73952 (ref. 6) and GSE72784 (ref. 8), respectively. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

The software and tools used in our study are all publicly available. Scripts used for data visualization are available upon request.

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Acknowledgements

We thank L. Shen (Zhejiang Univ.) for sharing a detailed protocol for CATCH-seq. We appreciate the Inoue Lab members for critical reading of the paper and D. Noda (Kumamoto Univ.) for advice about the flox knock-in construct. This project was partly supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Leading Initiative for Excellent Young Researchers Grant to A.I., Grant-in-Aid for Scientific Research on Innovative Areas (19H05754 to A.I.), Grant-in-Aid for Transformative Research Areas (A) (25H01355 to A.I.), Grant-in-Aid for Scientific Research (B) (23K27093 to A.I. and 23K28009 to C.K.), Grant-in-Aid for JSPS Fellows (20J21541 to R.H.), Japan Agency for Medical Research and Development PRIME (JP20gm6110012 to A.I.), and intramural grants within RIKEN including the RIKEN Pioneering Project ‘Genome Building from TADs’ (to A.I. and H.K.) and ‘Long-timescale Molecular Chronobiology’ (to A.I.).

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Author notes

  1. These authors contributed equally: Hailiang Mei, Ryoya Hayashi.

Authors and Affiliations

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

    Hailiang Mei, Ryoya Hayashi, Chisayo Kozuka, Mami Kumon & Azusa Inoue

  2. Tokyo Metropolitan University, Hachioji, Japan

    Ryoya Hayashi & Azusa Inoue

  3. Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Haruhiko Koseki

  4. Department of Molecular and Cellular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan

    Haruhiko Koseki

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  1. Hailiang Mei
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  6. Azusa Inoue
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Contributions

A.I. conceived of the project. H.M. and A.I. designed the experiments. R.H., C.K., M.K. and A.I. performed the experiments. H.M. analyzed sequence data. H.M. and A.I. interpreted the data. H.K. provided the H2afz and H2afv flox mouse lines. H.M. and A.I. wrote the paper.

Corresponding author

Correspondence to Azusa Inoue.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Patrick Murphy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Dimitris Typas, in collaboration with the Nature Structural and Molecular Biology team.

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Extended data

Extended Data Fig. 1 H2A.Z dynamics during oogenesis.

a, Scatter plots showing the correlations between biological replicates of H2A.Z CATCH-seq. b, Heatmaps showing the enrichment of H2A.Z signals at genic regions and expression levels of genes classified into 4 clusters. Actively expressed genes were defined by [RPKM > 1], while lowly expressed genes are [0.1 < RPKM < 1], and inactive genes are [RPKM < 0.1] in all stages, respectively. The remaining genes were defined as variably expressed genes. The RNA-seq datasets were from Veselovska et al.41. c, Box plots showing the H2A.Z signal intensity at the promoters of the 4 clusters. The center 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 maximal value smaller than 1.5 x the IQR above the 75th percentile, and the minimal value larger than 1.5 x the IQR below the 25th percentile, respectively. P-value was calculated by two-sided t-test. d, Transcription factor motifs identified at intergenic ncH2A.Z-H3K27ac dual-marked regions (left). Right bubble plots represent the expression level of TFs in FGOs and the statistical significance of the motif enrichment (P-value of hypergeometric test with one-sided). e, The proportion of the number of merged H2A.Z domains when merging adjacent peaks at each different distance from 1- to 12-kb. After summing the number of merged domains at each different distance from 1- to 12-kb, the number of merged domains at each distance was divided by the total number. f, Heatmap showing the global Pearson correlations between H2A.Z and H3K4me3 in four stages during oogenesis. The bin size is 5 kb. g, Violin plot showing the “skewing distance” of each H2A.Z peak. h, Representative images of 5-ethynyl uridine (EU) incorporation assay to validate transcriptional inhibition. 10d-GOs were first treated with 0.2% DMSO, 100 µM 5,6-dichlorobenzimidazole (DRB), or 100 µg/mL α-amanitin for 2 hrs, incubated with a medium containing EU and the respective inhibitors for additional 2 hrs, and then fixed. Bar plot indicates quantification of the EU signal intensity. The data are representative of a single time experiment using 10 (DMSO), 12 (DRB), and 10 (amanitin) oocytes. Scale bar, 20 µm. i, Genome browser views of H2A.Z and H3K4me3 landscapes in 5d-GO, 10d-GO, 15d-GO, and 28d-FGO oocytes, as well as their dynamic profiles in mouse preimplantation embryos. The H2A.Z ChIP-seq datasets are from Liu et al.17. The H3K4me3 ChIP-seq datasets in oocytes and preimplantation embryos are from Hanna et al.7 and Dahl et al.8, respectively. Broad ncH3K4me3 domains are highlighted.

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Extended Data Fig. 2 Characterization of H2A.Z loss in FGOs.

a, Volcano plots showing differentially modified regions of H2A.Z at two adjacent stages. H2A.Z-lost peaks are highlighted (FDR < 0.05, |FC | > 2). FDR is calculated by Benjamini-Hochberg adjusted p-value (two-sided) of glmLRT in ‘edgeR’. b, Genomic distribution of H2A.Z peaks that are lost or retained in FGOs compared with 15 d GOs. c, Transcription factor motifs identified at H2A.Z-lost promoters (left). Right bubble plots represent the expression level of TFs in FGOs and the statistical significance of the motif enrichment (P-value of hypergeometric test with one-sided). d, Box plots showing expression levels of the genes whose promoters lose or retain H2A.Z in FGOs. The center 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 maximal value smaller than 1.5 x the IQR above the 75th percentile, and the minimal value larger than 1.5 x the IQR below the 25th percentile, respectively (n = 1 as biological replicates were combined). The number of H2A.Z-lost or H2A.Z-retained genes are 1,799 and 10,567, respectively. e, Heatmaps showing the H2A.Z enrichment at bivalent promoters in DMSO-, DRB- and α-amanitin-treated GOs. f, Genome browser views showing the loss of H2A.Z at bivalent promoters in DRB- and α-amanitin-treated GOs.

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Extended Data Fig. 3 CATCH-seq in H2A.Z.1 and H2A.Z.2 double knockout oocytes.

a, Scheme of generation of the H2A.Z.1 and H2A.Z.2 conditional alleles. The location of the genotyping PCR primers is indicated by red arrows. b, Scatter plots showing the correlations between biological replicates of H2A.Z and H3K4me3 CATCH-seq in H2A.Z control and DKO FGOs. c, Volcano plots showing differential H3K4me3 changes between H2A.Z control and DKO FGOs. ‘H3K4me3 loss’ was defined by FDR < 0.05 & |FC | ≥ 2, while ‘Intermediate’ was defined by FDR < 0.05 & 1.5 < |FC | < 2. FDR is calculated by Benjamini-Hochberg adjusted p-value (two-sided) of glmLRT in ‘edgeR’. d, GO biological processes enriched for H3K4me3-lost intergenic regions in H2A.Z DKO FGOs. Adjusted P-value (two-sided) is calculated by a binomial test with FDR correction. e, Pie charts showing the proportion of H2A.Z, H3K4me3, and H3K36me2 modified chromatins in WT FGOs. f, Genome browser views showing the mutually exclusive distribution of H2A.Z and H3K36me2 in mouse FGOs. g, Average signal profiles of H2A.Z, H3K4me3, and H3K36me2 at intergenic H3K4me3 peaks. h, Scatter plots showing the correlations between biological replicates of H3K36me2 CATCH-seq in H2A.Z control and DKO FGOs. i, Density plot showing the change of H3K36me2 over H2A.Z-enriched bins between H2A.Z control and DKO FGOs. j, Genome browser views showing gain of H3K36me2 at H2A.Z/H3K4me3-lost regions in H2A.Z DKO FGOs. k, Box plots showing H3K4me3 levels at H3K36me2-up bins in H2A.Z control and DKO FGOs. The center 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 maximal value smaller than 1.5 x the IQR above the 75th percentile, and the minimal value larger than 1.5 x the IQR below the 25th percentile, respectively (n = 1 as biological replicates were combined). The number of H3K36me2-up bins in CTR and DKO are 15,008. P-value was calculated using a two-sided t-test. l, Bar charts showing the proportion of transcriptionally up- and down-regulated bins among H3K36me2-up & expressed bins (the number of intergenic RNA-seq reads >= 5 in either CTR or DKO).

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Extended Data Fig. 4 CATCH-seq in Mll2 conditional knockout oocytes.

a, Scheme of CRISPR/Cas9-mediated knock-in of the flox cassette at Mll2/Kmt2b. The location of the genotyping PCR primers is indicated by red arrows. b, Scatter plots showing the correlations between biological replicates of H2A.Z and H3K4me3 CATCH-seq in Mll2 WT and KO FGOs. c, Volcano plots showing differential H2A.Z changes between Mll2 WT and KO FGOs. ‘H2A.Z loss’ was defined by FDR < 0.05 & |FC | ≥ 2, while ‘Intermediate’ was defined by FDR < 0.05 & 1.5 < |FC | < 2. FDR is calculated by Benjamini-Hochberg adjusted p-value (two-sided) of glmLRT in ‘edgeR’. d, Scatter plots of genome-wide non-TSS bins of H2A.Z levels in Mll2 WT and KO FGOs (left) and H3K4me3 levels in H2A.Z CTR and DKO FGOs (right). Green and red dots represent H3K4me3-lost bins in the H2A.Z DKO and H2A.Z-lost bins in the Mll2 KO, respectively. Shown are 50,000 randomly selected bins (2-kb).

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Extended Data Fig. 5 Development of H2A.Z DKO oocytes and characterization of differentially expressed genes.

a, Representative images at 24- and 96- hours post-fertilization (hpf), which correspond to the time points of 2-cell and blastocyst stages, respectively, in CTR. Scale bar, 100 µm. Two independent experiments were performed with similar results. b, The ratios of preimplantation development. The total number of zygotes examined in 2 independent experiments was 106 (H2A.Z CTR) and 160 (H2A.Z DKO). c, d, Scatter plots showing the correlations of RNA-seq data between biological duplicates. e, f, MA plot of log2 FC in gene expression of H2A.Z DKO (e) and Mll2 KO (f) FGOs versus WT. Significantly up- and down-regulated genes in statistics (Benjamini-Hochberg-adjusted P-value (two-sided) < 0.01) are highlighted. g, Gene ontology terms enriched for the down- and up-regulated DEGs overlapped between H2A.Z DKO and Mll2 KO FGOs. P-value is calculated by accumulative hypergeometric test (two-sided).

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Extended Data Fig. 6 Effect of H2A.Z DKO in H2AK119ub1 and H3K27me3 in FGOs.

a, b, Scatter plots showing the correlations between biological duplicates of H3K27me3 (a) and H2AK119ub1 (b) CATCH-seq in H2A.Z control and DKO FGOs. c, Heatmaps showing the signal intensity of H2A.Z, H3K4me3, H3K27me3, and H2AK119ub1 at all genes in H2A.Z control and H2A.Z DKO FGOs. Actively expressed genes are defined by [RPKM > 1], while lowly expressed genes are [0.1 < RPKM < 1], and inactive genes are [RPKM < 0.1] in all stages, respectively. The remaining genes are defined as variably expressed genes. The RNA-seq datasets are from Veselovska et al.41. d, Box plots showing the signal intensity of H2A.Z, H3K4me3, H3K27me3, and H2AK119ub1 at promoters in H2A.Z control and DKO FGOs. The center 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 maximal value smaller than 1.5 x the IQR above the 75th percentile, and the minimal value larger than 1.5 x the IQR below the 25th percentile, respectively (n = 1 as biological replicates were combined). P-value was calculated by two-sided t-test. e, Heatmaps (left panel) showing the signal intensity of indicated histone modifications at H2AK119ub1/H3K4me3-harbored promoters. The line plots at right show the average signal profiles of H2K119ub1 and H3K4me3 around TSSs in H2A.Z control and DKO FGOs. f, Genome browser views of representative genes that show a reduction of H2AK119ub1 at active genes in H2A.Z DKO FGOs. g, Heatmaps showing the signal intensity of indicated histone modifications at ‘H3K4me3-lost’, ‘Intermediate’, and ‘H3K4me3-unchanged’ promoters in H2A.Z control and DKO FGOs. h, Box plots showing the signal intensity of H2AK119ub1 and H3K4me3 at ‘H3K4me3-lost’, ‘Intermediate’, and ‘H3K4me3-unchanged’ regions in H2A.Z control and DKO FGOs. The definitions of lines, box edges, whiskers, and n are the same as panel d. P-value was calculated by two-sided t-test.

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Supplementary information

Reporting Summary

Supplementary Tables 1–4

Table 1: List of all genes and DEGs of RNA-seq in H2A.Z CTR and DKO FGOs. Table 2: List of all genes and DEGs of RNA-seq in Mll2 WT and KO FGOs. Table 3: Primers and oligo DNA information. Table 4: Sequence data summary

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Mei, H., Hayashi, R., Kozuka, C. et al. H2A.Z reinforces maternal H3K4me3 formation and is essential for meiotic progression in mouse oocytes. Nat Struct Mol Biol (2025). https://doi.org/10.1038/s41594-025-01573-x

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