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.
This is a preview of subscription content
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Burton, A. & Torres-Padilla, M. E. Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis. Nat. Rev. Mol. Cell Biol. 15, 723–735 (2014)
Inoue, A. & Zhang, Y. Nucleosome assembly is required for nuclear pore complex assembly in mouse zygotes. Nat. Struct. Mol. Biol. 21, 609–616 (2014)
Zhou, L. Q. & Dean, J. Reprogramming the genome to totipotency in mouse embryos. Trends Cell Biol. 25, 82–91 (2015)
Ferguson-Smith, A. C. Genomic imprinting: the emergence of an epigenetic paradigm. Nat. Rev. Genet. 12, 565–575 (2011)
Boyle, A. P. et al. High-resolution mapping and characterization of open chromatin across the genome. Cell 132, 311–322 (2008)
Stergachis, A. B. et al. Developmental fate and cellular maturity encoded in human regulatory DNA landscapes. Cell 154, 888–903 (2013)
Lu, F. et al. Establishing chromatin regulatory landscape during mouse preimplantation development. Cell 165, 1375–1388 (2016)
Wu, J. et al. The landscape of accessible chromatin in mammalian preimplantation embryos. Nature 534, 652–657 (2016)
Kobayashi, H. et al. Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet. 8, e1002440 (2012)
Deaton, A. M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011)
Zheng, H. et al. Resetting epigenetic memory by reprogramming of histone modifications in mammals. Mol. Cell 63, 1066–1079 (2016)
Agger, K. et al. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature 449, 731–734 (2007)
Matoba, S. et al. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell 159, 884–895 (2014)
Borensztein, M. et al. Xist-dependent imprinted X inactivation and the early developmental consequences of its failure. Nat. Struct. Mol. Biol. 24, 226–233 (2017)
Okae, H. et al. Re-investigation and RNA sequencing-based identification of genes with placenta-specific imprinted expression. Hum. Mol. Genet. 21, 548–558 (2012)
Okae, H. et al. RNA sequencing-based identification of aberrant imprinting in cloned mice. Hum. Mol. Genet. 23, 992–1001 (2014)
Varmuza, S. & Miri, K. What does genetics tell us about imprinting and the placenta connection? Cell. Mol. Life Sci. 72, 51–72 (2015)
Wang, X., Soloway, P. D. & Clark, A. G. A survey for novel imprinted genes in the mouse placenta by mRNA-seq. Genetics 189, 109–122 (2011)
Babak, T. et al. Genetic conflict reflected in tissue-specific maps of genomic imprinting in human and mouse. Nat. Genet. 47, 544–549 (2015)
Kuzmin, A. et al. The PcG gene Sfmbt2 is paternally expressed in extraembryonic tissues. Gene Expr. Patterns 8, 107–116 (2008)
Blakeley, P. et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165 (2015)
Bartolomei, M. S., Webber, A. L., Brunkow, M. E. & Tilghman, S. M. Epigenetic mechanisms underlying the imprinting of the mouse H19 gene. Genes Dev. 7, 1663–1673 (1993)
Ferguson-Smith, A. C., Sasaki, H., Cattanach, B. M. & Surani, M. A. Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature 362, 751–755 (1993)
Stöger, R. et al. Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73, 61–71 (1993)
Mager, J., Montgomery, N. D., de Villena, F. P.-M. & Magnuson, T. Genome imprinting regulated by the mouse Polycomb group protein Eed. Nat. Genet. 33, 502–507 (2003)
Lewis, A. et al. Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation. Nat. Genet. 36, 1291–1295 (2004)
Umlauf, D. et al. Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes. Nat. Genet. 36, 1296–1300 (2004)
Terranova, R. et al. Polycomb group proteins Ezh2 and Rnf2 direct genomic contraction and imprinted repression in early mouse embryos. Dev. Cell 15, 668–679 (2008)
Pandey, R. R. et al. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell 32, 232–246 (2008)
Yamasaki-Ishizaki, Y. et al. Role of DNA methylation and histone H3 lysine 27 methylation in tissue-specific imprinting of mouse Grb10. Mol. Cell. Biol. 27, 732–742 (2007)
Sanz, L. A. et al. A mono-allelic bivalent chromatin domain controls tissue-specific imprinting at Grb10. EMBO J. 27, 2523–2532 (2008)
Duffié, R. et al. The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals. Genes Dev. 28, 463–478 (2014)
Monk, D. et al. Comparative analysis of human chromosome 7q21 and mouse proximal chromosome 6 reveals a placental-specific imprinted gene, TFPI2/Tfpi2, which requires EHMT2 and EED for allelic-silencing. Genome Res. 18, 1270–1281 (2008)
Henckel, A. et al. Histone methylation is mechanistically linked to DNA methylation at imprinting control regions in mammals. Hum. Mol. Genet. 18, 3375–3383 (2009)
Kobayashi, H. et al. Imprinted DNA methylation reprogramming during early mouse embryogenesis at the Gpr1-Zdbf2 locus is linked to long cis-intergenic transcription. FEBS Lett. 586, 827–833 (2012)
Saitou, M., Kagiwada, S. & Kurimoto, K. Epigenetic reprogramming in mouse pre-implantation development and primordial germ cells. Development 139, 15–31 (2012)
Pires, N. D. & Grossniklaus, U. Different yet similar: evolution of imprinting in flowering plants and mammals. F1000Prime Rep. 6, 63 (2014)
Moreno-Romero, J., Jiang, H., Santos-González, J. & Köhler, C. Parental epigenetic asymmetry of PRC2-mediated histone modifications in the Arabidopsis endosperm. EMBO J. 35, 1298–1311 (2016)
Inoue, A., Nakajima, R., Nagata, M. & Aoki, F. Contribution of the oocyte nucleus and cytoplasm to the determination of meiotic and developmental competence in mice. Hum. Reprod. 23, 1377–1384 (2008)
Ohnishi, Y. et al. Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development. Nucleic Acids Res. 38, 5141–5151 (2010)
Inoue, A., Akiyama, T., Nagata, M. & Aoki, F. The perivitelline space-forming capacity of mouse oocytes is associated with meiotic competence. J. Reprod. Dev. 53, 1043–1052 (2007)
Sugimoto, M. et al. A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by Wnt inhibition. Stem Cell Reports 4, 744–757 (2015)
Xiang, Y. et al. JMJD3 is a histone H3K27 demethylase. Cell Res. 17, 850–857 (2007)
Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)
John, S. et al. Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat. Genet. 43, 264–268 (2011)
Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010)
Ramírez, F. et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44 (W1), W160–W165 (2016)
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)
Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014)
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)
Song, Q. et al. A reference methylome database and analysis pipeline to facilitate integrative and comparative epigenomics. PLoS ONE 8, e81148 (2013)
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008)
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.
The authors declare no competing financial interests.
Reviewer Information Nature thanks R. Feil and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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).
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.
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.
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.
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.
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.
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.
About this article
Cite this article
Inoue, A., Jiang, L., Lu, F. et al. Maternal H3K27me3 controls DNA methylation-independent imprinting. Nature 547, 419–424 (2017). https://doi.org/10.1038/nature23262
Nature Metabolism (2022)
Nature Reviews Genetics (2022)
Nature Reviews Genetics (2022)
BrewerIX enables allelic expression analysis of imprinted and X-linked genes from bulk and single-cell transcriptomes
Communications Biology (2022)
Epigenetic changes induced by in utero dietary challenge result in phenotypic variability in successive generations of mice
Nature Communications (2022)