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H2AK119ub1 guides maternal inheritance and zygotic deposition of H3K27me3 in mouse embryos

Abstract

Parental epigenomes are established during gametogenesis. While they are largely reset after fertilization, broad domains of Polycomb repressive complex 2 (PRC2)-mediated formation of lysine 27–trimethylated histone H3 (H3K27me3) are inherited from oocytes in mice. How maternal H3K27me3 is established and inherited by embryos remains elusive. Here, we show that PRC1-mediated formation of lysine 119–monoubiquititinated histone H2A (H2AK119ub1) confers maternally heritable H3K27me3. Temporal profiling of H2AK119ub1 dynamics revealed that atypically broad H2AK119ub1 domains are established, along with H3K27me3, during oocyte growth. From the two-cell stage, H2AK119ub1 is progressively deposited at typical Polycomb targets and precedes H3K27me3. Reduction of H2AK119ub1 by depletion of Polycomb group ring finger 1 (PCGF1) and PCGF6—essential components of variant PRC1 (vPRC1)—leads to H3K27me3 loss at a subset of genes in oocytes. The gene-selective H3K27me3 deficiency is irreversibly inherited by embryos, causing loss of maternal H3K27me3-dependent imprinting, embryonic sublethality and placental enlargement at term. Collectively, our study unveils preceding dynamics of H2AK119ub1 over H3K27me3 at the maternal-to-zygotic transition, and identifies PCGF1/6–vPRC1 as an essential player in maternal epigenetic inheritance.

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Fig. 1: H2AK119ub1 is established in a noncanonical distribution along with H3K27me3 in mouse oocytes.
Fig. 2: Remodeling of H2AK119ub1 precedes that of H3K27me3.
Fig. 3: Cotransmission of maternal Polycomb domains and sequential establishment of zygotic Polycomb domains.
Fig. 4: PCGF1/6–PRC1 deficiency causes gene-selective loss of H3K27me3 in oocytes.
Fig. 5: Gene-selective loss of H3K27me3 is irreversible and causes loss of H3K27me3-dependent imprinting in Pcgf1/6 matKO embryos.
Fig. 6: Maternal PCGF1/6–PRC1 deficiency causes placental enlargement at term.

Data availability

All of the CUT&RUN and RNA-seq datasets generated in this study are summarized in Supplementary Table 6 and have been deposited to the Gene Expression Omnibus database under accession number GSE153496. The H2AK119ub1 ChIP-seq datasets of mESCs were from GSE119620 (ref. 41). The H3K27me3 and H3K4me3 ChIP-seq and whole-genome bisulfite sequencing datasets of FGOs were from GSE93941 (ref. 19). The H3K27me3 ChIP-seq datasets of 7-d GOs, MII oocytes, zygotes, early two-cell embryos, late two-cell embryos, inner cell masses of blastocysts and E6.5 epiblasts were from GSE76687 (ref. 18). The H3K27me3 CUT&RUN datasets of morula embryos were from GSE116713 (ref. 26). The H3K27me3 ChIP-seq datasets in mESCs were from GSE119620 (ref. 41). The RNA-seq datasets of Ring1a/1b KO FGOs and Dnmt3l matKO and Eed matKO morula embryos were from GSE132156 (ref. 56), GSE130115 (ref. 29) and GSE116713 (ref. 26), respectively. The PCGF1 ChIP-seq datasets in mESCs were from GSE119620 (ref. 41).

Code availability

The code developed for this study is available at https://github.com/Azusa-lab/Intergenerational-epigenetic-inheritance.

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Acknowledgements

We thank all of the Koseki laboratory members for helpful discussion and the Kazusa DNA Research Institute for support with next-generation sequencing. The protein A-MNase for CUT&RUN was a kind gift from S. Henikoff. We appreciate T. Ishiuchi (Kyushu University), S. Ito (RIKEN) and S. Yamaguchi (Osaka University) for critical reading of the manuscript and K. Yamamoto and T. Yamamoto (RIKEN) for support as the young chief investigator program directors. 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.), Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) (18H02359 to A.I. and 16H02622 to H.K.), Grants-in-Aid for Scientific Research on Innovative Areas (19H05754 to A.I. and 19H05745 to H.K.), Grant-in-Aid for Early-Career Scientists (19K17971 to C.K.), Grant-in-Aid for JSPS Fellows (20J21541 to R.H.), Japan Agency for Medical Research and Development (PRIME; JP18gm6110012 to A.I.), AMED-CREST (JP18gm0510016 to H.K.), the Uehara Memorial Foundation (to A.I.) and intramural grants within RIKEN, including the All-RIKEN ‘Epigenome Manipulation Project’ (to A.I. and H.K.).

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Authors

Contributions

A.I. conceived the project and designed the experiments. H.M. analyzed the sequencing data. A.I., C.K., R.H. and M.K. performed the experiments. R.H. helped with the data analysis. A.I. and H.M. interpreted the data. H.K. provided the Pcgf1 and Pcgf6 floxed mouse lines. A.I. and H.M. wrote the manuscript with contributions from H.K.

Corresponding author

Correspondence to Azusa Inoue.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature Genetics thanks Neil Brockdorff, Maxim Greenberg 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 Characterization of H2AK119ub1 distribution in mouse oocytes.

a, Scatter plots showing H2AK119ub1 enrichment (10 kb window) between our low-input CUT&RUN datasets using 250, 500, 1,000, 5,000 mouse embryonic stem cells (mESCs) and a public ChIP-seq dataset in mESCs41. Spearman correlation is also shown. b, Genome browser view of H2AK119ub1 in our low-input CUT&RUN datasets and a public ChIP-seq dataset of mESCs. c, Scatter plot showing the correlation between biological duplicate of H2AK119ub1 CUT&RUN in FGOs. d, Pie chart showing the percentage of H2AK119ub1 (H2Aub)-covered regions that overlap H3K27me3 (K27me3) or/and H3K4me3 (K4me3). e, Genome browser view showing local association of H2AK119ub1 and H3K4me3 at promoters in FGOs. The H3K4me3 and H3K27me3 ChIP-seq datasets are from19. f, Heatmap showing the enrichment of H2AK119ub1, H3K27me3, and H3K4me3 at promoters harboring H2AK119ub1 peaks in FGOs. Reads density (normalized RPKM with different scales) are plotted at ±5 kb of the transcription start sites (TSS). The promoters are clustered into 3 groups according to the relative enrichment of the histone PTMs. The H3K27me3 and H3K4me3 ChIP-seq datasets are from19. The rightmost column represents the expression levels of genes corresponding to individual rows in FGOs. The RNA-seq dataset is from16. g, Box plot showing gene expression levels of the 3 clusters of panel f. The middle lines in the boxes represent the medians. Box edges, the upper, and the lower whiskers indicate the interquartile range (IQR, from the 25th to 75th percentile), the largest value smaller than 1.5 x IQR above the 75th percentile, and the smallest value larger than 1.5 x IQR below the 25th percentile, respectively (n = 1, as biological replicates were combined). *** p-value = 1.43e-71 (two sided Mann-Whitney U test). h, MA plot of log2FC(KO/CTR) in gene expression of Ring1a/1b KO FGOs. Cluster C genes in panel f are highlighted by red dots. The percentages of up- and down-regulated differentially expressed genes (DEGs) and non-DEGs with the fold change>2 cutoff are indicated. i, Scatter plot showing the correlation between biological duplicates of H2AK119ub1 CUT&RUN in 7d-GOs. j, Additional genome browser views of H2AK119ub1 and H3K27me3 distributions in 7d-GOs and FGOs. The H3K27me3 ChIP-seq datasets are from18.

Extended Data Fig. 2 Characterization of allelic H2AK119ub1 distribution at the maternal-to-zygotic transition.

a, Scatter plot showing the correlations between biological duplicates of H2AK119ub1 CUT&RUN in individual samples. b, The ratios of SNP-containing reads assigned to the paternal and maternal genomes. Note that MII oocytes have almost no paternal SNP reads, supporting the validity of our SNP analysis pipeline. c, Averaged signal profiles of H2AK119ub1 at regions with different gene densities (gene number per 1 Mb sliding window with a 2kb step) in zygotes (n=1, as biological replicates were combined). The shaded areas represent the 95% confidence interval for the fitted LOESS curve. Note that the H2AK119ub1 enrichment at the paternal allele is negatively correlated with gene density. d, Representative images of anti-H2AK119ub1 and anti-H3.3 immunostaining analysis for fertilized oocytes. Dotted circles, the rims of fertilized oocytes. M, maternal genome. P, paternal genome. Scale bar, 20 µm. Ten fertilized oocytes were examined in each time point. e, Enlarged images of the paternal genomes in panel d. DAPI-dense loci are pointed by arrows. DAPI was shown in blue in the merged images. Scale bar, 10 µm. f, Genomic distribution of H2AK119ub1 peaks. M, maternal allele. P, paternal allele. Promoters represent the regions of ±2.5 kb around transcription start sites.

Extended Data Fig. 3 H2AK119ub1 dynamics and gene expression during the maternal-to-zygotic transition.

a, Genome browser views of H2AK119ub1 and H3K27me3 dynamics at gene deserts. MII, MII oocyte. 1C, 1-cell. E2C, early 2-cell. L2C, late 2-cell. Mor, morula. Bl, blastocyst. Epi, E6.5 epiblast. b, c, Heatmaps showing the H2AK119ub1 and H3K27me3 enrichment at promoters (b) and gene bodies (c) in the maternal allele of the indicated samples. Genes with few SNP reads [RPKM(H2AK119ub1) < 1 in all samples] were filtered out from this analysis. The gene expression levels are shown in the right RNA-seq heatmap. The H3K27me3 ChIP-seq datasets are from18. The RNA-seq datasets are from16. d, Pie chart showing the proportion of putative H3K27me3-dependent imprinted genes27 in Group A/B/C of the panel c. Genes with few SNP reads [RPKM(H2AK119ub1) < 1 in all samples] were filtered out from this analysis. The total number of genes in this pie chart is 62. e, Pie chart showing the proportion of typical Polycomb (PcG) target genes in Group A/B/C of the panel b. Genes with few SNP reads [RPKM(H2AK119ub1) < 1 in all samples] were filtered out from this analysis. The total number of genes in this pie chart is 1,405. f, Genome browser views of H2AK119ub1 and H3K27me3 dynamics at typical PcG targets. The genomic length of each view is indicated at the top. ESC, embryonic stem cells.

Extended Data Fig. 4 Generation of oocyte-specific Pcgf1/6 knockout mice.

a, The expression levels of the Pcgf family genes during oogenesis and preimplantation development. The RNA-seq datasets are from16. The exact RPKM values of Pcgf1 over 200 are indicated at the top of the bars. D10-and D14-GO, growing oocytes (GO) from postnatal day 10 (D10) and day 14 (D14) females. FGO, fully-grown oocytes. MII, MII-stage oocytes. ICM, inner cell mass of blastocysts. mESC, mouse embryonic stem cell. vPRC1, variant PRC1 components. cPRC1, canonical PRC1 components. b, Construct for targeted disruption of Pcgf1. Black boxes indicate the coding exons. Red arrows indicate genotyping primers. c, Genotyping of Pcgf1 and Pcgf6 flox alleles. The F2/R1 primer set was used to detect WT and flox alleles. d, Construct for targeted disruption of Pcgf6. e, Sanger sequencing to confirm the deletions of floxed exons in Pcgf1/6 KO FGOs. After cDNA preparation by reverse transcription of total RNA from WT or KO FGOs, the targeted regions were PCR amplified and sequenced. This confirmed that exons 2–7 (Pcgf1) and 2–3 (Pcgf6), which encode the Ring finger domains of PCGF1 and PCGF6, respectively, were successfully deleted in KO FGOs.

Extended Data Fig. 5 Characterization of Pcgf1/6 KO fully-grown oocytes (FGOs).

a, Heatmap showing the enrichment of H2AK119ub1 CUT&RUN signals in replicate 1 (not scaled) and 2 (scaled by spike-in chromatin). Reads densities are plotted at the peaks ±2 kb flanking regions. b, c, Scatter plots showing the correlations between biological duplicates of H2AK119ub1 (b) and H3K27me3 (c) CUT&RUN in CTR and KO FGOs. d, Heatmap showing the enrichment of H3K27me3 CUT&RUN signals. Reads density are plotted at the peaks ±2 kb flanking regions. e, Scatter plots showing the correlations between biological duplicates of RNA-seq in CTR and KO FGOs. f, Scatter plots showing the correlations between biological duplicates of H3K27me3 CUT&RUN in Pcgf1 and Pcgf6 single KO FGOs. g, Box plot showing the enrichment of PCGF1 binding at H3K27me3-lost, -intermediate, and -unchanged genes defined in Fig. 4e. PCGF1 ChIP-seq datasets in mESCs are from41. ***p = 1.8e-105 (two sided Mann-Whitney U test). h, Box plot showing CpG density at promoters of the 3 groups of genes. ***p = 3.9e-100 (two sided Mann-Whitney U test). i, Box plot showing H2AK119ub1 and H3K27me3 enrichment at gene bodies of the 3 groups in 7-day growing oocytes (7d-GOs). ***p = 2.3e-69 (two sided Mann-Whitney U test).

Extended Data Fig. 6 Preimplantation development of Pcgf1/6 maternal KO (matKO) embryos.

a, The averaged numbers of MII oocytes following superovulation. The numbers of females examined were 17 (CTR) and 14 (KO). Error bars, SD. b, Preimplantation development of Pcgf1/6 CTR and matKO embryos. The embryos that reached at the 2-cell, 4-cell, morula, and blastocyst stages in a timely fashion were counted at 24, 48, 72, and 96 hours post-fertilization (hpf), respectively. The numbers of 1-cell zygotes were set as 100%. The numbers of embryos examined are 127 (CTR) and 171 (matKO) from 6 biologically independent experiments. ***p < 0.001 (Chi-squired test). c, Representative images of preimplantation embryos at the indicated time points. The expected stages at these time points are indicated in parentheses. The arrowheads indicate embryos that had not reached the expected stages in a timely manner. Note that the other Pcgf1/6 matKO embryos form grossly normal blastocysts at 96 hpf. The experiment was repeated 6 times. Scale bar, 100 µm. d, Quantifications of H2AK119ub1 and H3K27me3 immunostaining analysis in Pcgf1/6 CTR and matKO embryos. The numbers of embryos examined (n) are indicated in Fig. 5a. The averaged signal intensity of CTR was set as 1.0 in each stage. Maternal pronuclei were quantified for 1-cell zygotes. Bars overlaid on the plots indicate mean. ***p < 0.001 (two-tailed Student’s t-test).

Extended Data Fig. 7 Characterization of Pcgf1/6 maternal KO (matKO) morula embryos.

a, b, Scatter plots showing the correlations between biological duplicates of RNA-seq in B6xPWK and PWKxB6 (a) and Pcgf1/6 CTR and matKO (b) morula embryos. c, The ratio of blastomeres showing the indicated numbers of Xist RNA clouds. Each bar represents an individual embryo. d, Box plot showing the relative expression of genes on individual maternal chromosomes between CTR and matKO morula embryos. Box edges, the upper, and the lower whiskers indicate the interquartile range (IQR, from the 25th to 75th percentile), the largest value smaller than 1.5 x IQR above the 75th percentile, and the smallest value larger than 1.5 x IQR below the 25th percentile, respectively (n=1, as biological replicates were combined). ***p < 2.2e-16 (One-way ANOVA test). e, Scatter plots showing the correlations between biological triplicates of H3K27me3 CUT&RUN in CTR and matKO morula embryos. f, Additional genome browser views of H3K27me3 distributions in Pcgf1/6 CTR and KO FGOs, and CTR and matKO morula embryos. Mat, maternal allele. Pat, paternal allele. No views in embryos are shown at Pnliprp2 and Gm32885 that have few SNPs. g, Heatmap showing the CUT&RUN signal enrichment of maternal H3K27me3 (scaled) in late 2-cell embryos. Reads density were plotted at the peaks ±2 kb flanking regions. h, Averaged signal profiles of maternal H3K27me3 (scaled) at their peaks ±2 kb flanking regions in late 2-cell embryos. i, Heatmap showing the enrichment of the H3K27me3 intensity in FGOs, late 2-cell, and morula embryos. The list and the order of genes are the same as Fig. 5d. Heatmaps for the corresponding paternal allele are also shown. The rightmost heatmap indicates H2AK119ub1 signal intensity at the maternal allele of wild-type preimplantation embryos.

Extended Data Fig. 8 Characterization of Pcgf1/6 maternal KO (matKO) embryos at E6.5.

a, Summary table of E6.5 dissection. b, Pictures of all of 4 CTR and 8 matKO litters at E6.5. Scale bar, 1 mm. c, Representative images of H3K27me3 immunostaining of E6.5 embryos. H3K27me3 spots are indicators of X chromosome inactivation (XCI). Note that single XCI and no XCI is observed in female and male embryos, respectively, in both CTR and matKO. This indicates that aberrant XCI in matKO embryos is restored by E6.5. Oct4-positive and -negative cells represent epiblast and extra-embryonic ectoderm, respectively. The number of embryos examined was 6 (CTR) and 9 (matKO) females and 9 (CTR) and 5 (matKO) males. Scale bar, 50 µm. d, Expression levels of cell lineage marker genes in CTR and matKO extraembryonic ectoderm (ExE) samples. RNA-seq datasets of wild-type epiblast (EPI), visceral endoderm (VE), and ExEs are from27. e, Correlation between biological replicates of RNA-seq samples.

Extended Data Fig. 9 Characterization of Pcgf1/6 double and respective single maternal KO (matKO) fetuses and placentae at term.

a, Summary table of Caesarean sections at E18.5. b, Experimental scheme of mixed embryo transfer. Genotyping of the ΔPcgf1 or ΔPcgf6 allele allows distinguishing between CTR and matKO fetuses. c, d, Placental weights (c) and body weights (d) of Pcgf1/6 CTR and matKO fetuses dissected from surrogated mothers at E19.5. Bars overlaid on the plots indicate mean±SD. The number of placentae and fetuses examined was 16 (CTR) and 8 (matKO) from 3 litters. ***p < 0.0001 (two-tailed Student’s t-test). e, Ratios of wild-type (WT), Pcgf1 heterozygous (Het), Pcgf6 Het, Pcgf1/6 double Het fetuses derived from WT females that had been mated with Pcgf1/6 double Het males. A total of 80 fetuses obtained from 9 litters were examined at E18.5. f, Placental weights of the indicated genotypes. Bars overlaid on the plots indicate mean±SD. p, two-tailed Student’s t-test. g, Summary table of Caesarean sections at E18.5 for Pcgf1 and Pcgf6 single matKO, respectively. h, i, j, The numbers of implantation (h), the litter sizes (i) and the placental weights (j) of the indicated matKO embryos. The numbers of litters and placentae examined are 11 and 85 (CTR), 10 and 24 (Pcgf1/6 matKO), 7 and 48 (Pcgf1 matKO), and 10 and 69 (Pcgf6 matKO), respectively. Bars overlaid on the plots indicate mean ± SD. ***p < 0.0001 (two-tailed Student’s t-test).

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Mei, H., Kozuka, C., Hayashi, R. et al. H2AK119ub1 guides maternal inheritance and zygotic deposition of H3K27me3 in mouse embryos. Nat Genet 53, 539–550 (2021). https://doi.org/10.1038/s41588-021-00820-3

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