Maternal H3K27me3 controls DNA methylation-independent imprinting

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Abstract

Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashions. After fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci, including imprinting control regions. How parental chromatin becomes equalized and how imprinting control regions escape from this reprogramming is largely unknown. Here we profile parental allele-specific DNase I hypersensitive sites in mouse zygotes and morula embryos, and investigate the epigenetic mechanisms underlying these allelic sites. Integrated analyses of DNA methylome and tri-methylation at lysine 27 of histone H3 (H3K27me3) chromatin immunoprecipitation followed by sequencing identify 76 genes with paternal allele-specific DNase I hypersensitive sites that are devoid of DNA methylation but harbour maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induces maternal allele expression. H3K27me3-dependent imprinting is largely lost in the embryonic cell lineage, but at least five genes maintain their imprinted expression in the extra-embryonic cell lineage. The five genes include all paternally expressed autosomal imprinted genes previously demonstrated to be independent of oocyte DNA methylation. Thus, our study identifies maternal H3K27me3 as a DNA methylation-independent imprinting mechanism.

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Figure 1: Allelic DHSs in zygotes mark allelic gene expression at ZGA.
Figure 2: Oocyte-specific H3K27me3 prevents maternal chromatin accessibility at DNA hypomethylated regions.
Figure 3: Genes with H3K27me3-marked AG-DHSs are paternally expressed in morula embryos.
Figure 4: Maternal H3K27me3 serves as an imprinting mark.
Figure 5: Cell-lineage-specific dynamics of H3K27me3-dependent genomic imprinting.

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Change history

  • 26 July 2017

    The label for the colour scale in Fig. 3d was corrected.

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Acknowledgements

We thank S. Matoba for technical advice and L. Tuesta for reading the manuscript. This project was supported by the Howard Hughes Medical Institute. F.L. is supported by a Charles A. King Trust Postdoctoral Research Fellowship. Y.Z. is an Investigator of the Howard Hughes Medical Institute.

Author information

A.I. and Y.Z. conceived the project and designed the experiments. A.I. performed the experiments; L.J. analysed sequencing datasets. F.L. performed liDNase-seq and RNA-seq; T.S. assisted in embryo manipulation experiments; A.I., L.J., and Y.Z. interpreted the data. A.I. and Y.Z. wrote the manuscript.

Correspondence to Yi Zhang.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks R. Feil and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Identification of parental allelic DHSs.

Related to Fig. 1. a, Correlation of DHSs between three biological replicates in paternal and maternal pronuclei (PN). b, Bi-allelic DHSs (grey), Ps-DHSs (blue), and Ms-DHSs (red). The cutoffs used to define these DHS groups are indicated. c, Averaged DHS signals of Ps-DHSs and Ms-DHSs within ±5 kb around DHSs. d, Genomic distribution of DHSs. Promoters represent ±1 kb around transcription start sites. ‘Random’ indicates the percentages of each genomic element of the mouse genome. e, Percentages of DHSs located at CpG islands (CGIs). The genomic locations of CGIs are defined previously9. f, Genome browser view of Ps-DHSs at ICRs of representative imprinted genes. The genomic locations of ICRs were referred in ref. 9. g, List of genes harbouring promoter Ps-DHSs or Ms-DHSs in zygotes. h, Genome browser view of representative allelic promoter DHSs at genes not previously known to be imprinted.

Extended Data Figure 2 Correlation between allelic ZGA in two-cell embryos and allelic DHSs in zygotes.

Related to Fig. 1. a, Schematic for identifying parental allele-specific gene expression at ZGA. Androgenetic embryos and gynogenetic embryos were produced by pronuclear transfer. Androgenetic two-cell embryos contained paternally expressed nascent transcripts and maternally stored transcripts. Gynogenetic two-cell embryos contained maternally expressed nascent transcripts and maternally stored transcripts. α-Amanitin-treated (Ama) two-cell embryos contained maternally stored transcripts only. b, Correlation between biological duplicate of two-cell RNA-seq samples. c, Flowchart for avoiding maternally stored transcripts and identifying nascent allelic transcripts at ZGA. d, Nascent transcripts in androgenetic and gynogenetic two-cell embryos. For each gene, the FPKM value in α-amanitin-treated embryos was subtracted from that in androgenetic and gynogenetic embryos, respectively. Androgenetic- and gynogenetic-specific DEGs (fold change > 10) are indicated in blue and red, respectively. Known imprinted genes are indicated in green. e, f, DHS allelic bias at promoters (±0.5 kb at transcription start sites) of androgenesis- (e) and gynogenesis- (f) specific DEGs. Fold change > 2 was considered as ‘bias’ (blue or red).

Extended Data Figure 3 Zygotic Ms-DHSs are inherited from oocyte DHSs.

Related to Fig. 2. a, Correlation between three biological replicates of liDNase-seq for germinal vesicle nuclei isolated from fully grown oocytes. b, Genome browser view of sperm DHSs passed on to paternal pronuclei of zygotes. The nearest gene names are indicated at the top of each panel. c, Heat map showing Ps-DHSs. Each row represents the liDNase-seq signal intensity at DHS ± 5 kb. Note that Ps-DHSs are largely absent in both sperm and oocytes. d, Genome browser view of representative Ps-DHSs. e, Heat map showing Ms-DHSs. Note that Ms-DHSs are mostly already present in oocytes. f, Genome browser view of representative Ms-DHSs. g, Heat map showing bi-allelic DHSs. h, Genome browser view of representative bi-allelic DHSs.

Extended Data Figure 4 Distinct epigenetic features of Kdm6b- and Kdm4d-affected Ps-DHSs.

Related to Fig. 2. a, Percentages of Ps-DHSs that overlap (black) or are associated (grey) with oocyte gDMRs within ±100 kb. Oocyte gDMR was defined by >80% methylation in oocytes and <20% methylation in sperm. b, Percentages of Ps-DHSs organized on the basis of their oocyte DNA methylation levels. c, H3K27me3 signal levels at Ps-DHSs ± 1 kb in gametes (left) and zygotes (right). Ps-DHSs were divided into oocyte DNA hypomethylated (0–20%, n = 296) and hypermethylated groups (80–100%, n = 305). Middle lines in the boxes represent the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively. d, Representative images of Kdm6b- or Kdm4d-injected zygotes stained with anti-Flag antibody, using non-injected zygotes as negative controls. e, Representative images of zygotes stained with anti-H3K27me3 antibody. M, maternal pronucleus; P, paternal pronucleus. The bar graph on the right represents relative immunostaining signal intensity of maternal pronuclei. The averaged signal of non-injected zygotes was set as 1.0. The total numbers of embryos examined were 8 (No injection), 13 (Kdm6bWT), and 10 (Kdm6bMUT). Error bars, s.d. ***P < 0.001 (two-tailed Student’s t-test). NS, statistically not significant. f, Representative images of zygotes stained with anti-H3K9me3 antibody. The bar graph on right represents relative immunostaining signal intensity in the maternal pronuclei. The averaged signal of non-injected zygotes was set as 1.0. The total numbers of embryos examined were 5 (No injection), 5 (Kdm4dWT), and 7 (Kdm4dMUT). Error bars, s.d. ***P < 0.001 (two-tailed Student’s t-test). NS, statistically not significant. g, Correlation between biological duplicates of liDNase-seq for maternal (Mat) and paternal pronuclei (Pat) of Kdm6bWT- and Kdm6bMUT-injected zygotes. h, Correlation between biological duplicates of liDNase-seq for maternal and paternal pronuclei of Kdm4dWT- and Kdm4dMUT-injected zygotes. i, Genome browser view of representative Ps-DHSs affected by Kdm4dWT. j, H3K27me3 signals at Kdm6b- or Kdm4d-affected Ps-DHSs ± 1 kb in gametes (left) and zygotes (right). Middle lines in the boxes indicate the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively.

Extended Data Figure 5 Androgenetic- and gynogenetic-specific DHSs in morula embryos.

Related to Fig. 3. a, Correlation between biological duplicates of liDNase-seq for androgenetic and gynogenetic morula embryos. b, Averaged SNP-tracked liDNase-seq signal intensity of paternal and maternal alleles in hybrid morula embryos. The data were obtained from morula embryos of a BDF1 and JF1 cross7. Plots from the biological duplicates are shown. Note that paternal (JF1), but not maternal (BDF1), SNP reads are enriched in AG-DHSs (left), while neither SNP read is enriched in GG-DHSs (right). c, Genome browser view of DHSs at known ICRs. The genomic locations of ICRs were defined previously9. d, AG-DHSs grouped on the basis of their oocyte DNA methylation levels.

Extended Data Figure 6 Allelic gene expression in morula embryos and allelic H3K27me3 at non-canonical and canonical imprinted genes.

Related to Fig. 3. a, Correlation between biological duplicates of RNA-seq samples. b, Gene expression levels in androgenetic and gynogenetic morula embryos. Androgenetic- and gynogenetic-specific DEGs (fold change > 10) are indicated in blue and red, respectively. Paternally and maternally expressed known imprinted genes are indicated in green and orange, respectively. c, Genome browser views of allelic H3K27me3 levels in non-canonical imprinted genes. Sp; sperm. Oo; MII-stage oocyte. Paternal (Pat) and maternal (Mat) allele signals in one-cell and ICM were based on SNP analyses. d, Genome browser views of allelic H3K27me3 levels in representative canonical imprinted genes. Known ICRs are indicated at the bottom of each imprinted gene.

Extended Data Figure 7 The effect of Kdm6b mRNA injection on maternal allele expression and accessibility.

Related to Fig. 4. a, Developmental ratio of Kdm6bWT- and Kdm6bMUT-injected parthenogenetic (PG) embryos. The total embryo numbers examined were 60 (WT) and 58 (MUT). b, Correlation between biological duplicates of RNA-seq for Kdm6bWT- and Kdm6bMUT-injected parthenogenetic embryos. c, Relative gene expression levels of canonical imprinted genes expressed in androgenetic (AG) morula embryos (RPKM > 0.5). Note that none are de-repressed by Kdm6bWT injection. d, Correlation between biological duplicates of liDNase-seq for Kdm6bWT- and Kdm6bMUT-injected parthenogenetic embryos. e, f, Wide genome browser views of non-canonical (e) and canonical (f) imprinted genes. Arrowheads indicate AG-DHSs at which chromatin accessibility is gained in Kdm6bWT-injected parthenogenetic embryos (shown in Fig. 4e). Known ICRs are indicated above each panel of canonical imprinted genes (f). g, Genome browser view of AG-DHSs at ICRs of representative canonical imprinted genes.

Extended Data Figure 8 Genomic imprinting in E6.5 embryos.

Related to Fig. 5. a, Expression levels of marker genes for trophectoderm (Cdx2) and ICM (Sox2) in the samples. b, Correlation between biological duplicates of the E6.5 epiblast (EPI), visceral endoderm (VE), and extra-embryonic ectoderm (EXE) RNA-seq samples from both B6 × PWK and PWK × B6 crosses. c, Expression levels of marker genes for epiblast (Pou5f1 and Nanog), visceral endoderm genes (Gata6 and Gata4) and extra-embryonic ectoderm (Elf5 and Gata3) in the samples. d, Heat map showing PEGs and MEGs in epiblast, visceral endoderm, and extra-embryonic ectoderm of E6.5 embryos. All genes exhibiting parental allele-specific expression (fold change > 2 in both B6/PWK (B × P) and PWK/B6 (P × B)) in each sample are shown. Genes not previously known to be imprinted are indicated in bold. e, Genome browser view of RNA-seq data of newly identified imprinted genes. D7Ertd715e and Smoc1 are paternally expressed, and Mas1 is maternally expressed.

Extended Data Figure 9 Sample preparation and quality verification.

Related to Fig. 5. a, Experimental scheme of placenta cell purification. Sperm or oocytes were collected from B6GFP mice, and in vitro fertilized with the counterparts collected from the PWK strain. Embryos were transplanted into surrogate mothers. The placentae were harvested at E9.5 and dissociated into single cells by trypsin treatment before FACS of GFP-positive cells. b, Correlation between biological duplicates of RNA-seq samples. c, Total numbers of the paternal and maternal SNP reads in the purified placental cells.

Extended Data Figure 10 Genomic imprinting in E9.5 placentae.

Related to Fig. 5. a, Heat map showing PEGs and MEGs in E9.5 placentae. All genes exhibiting parental allele-specific expression (fold change > 2 in both B6/PWK and PWK/B6) are shown. Genes not previously known to be imprinted are indicated in bold type. b, Genome browser view of RNA-seq data of newly identified imprinted genes. D7Ertd715e and Smoc1 are paternally expressed, and Cbx7 and Thbs2 are maternally expressed.

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Inoue, A., Jiang, L., Lu, F. et al. Maternal H3K27me3 controls DNA methylation-independent imprinting. Nature 547, 419–424 (2017) doi:10.1038/nature23262

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