Maternal Eed knockout causes loss of H3K27me3 imprinting and random X inactivation in the extraembryonic cells

Abstract

Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 Lys27 trimethylation (H3K27me3) can mediate DNA methylation-independent genomic imprinting. However, the regulatory mechanisms and functions of this new imprinting mechanism are largely unknown. Here we demonstrate that maternal Eed, an essential component of the Polycomb group complex 2 (PRC2), is required for establishing H3K27me3 imprinting. We found that all H3K27me3-imprinted genes, including Xist, lose their imprinted expression in Eed maternal knockout (matKO) embryos, resulting in male-biased lethality. Surprisingly, although maternal X-chromosome inactivation (XmCI) occurs in Eed matKO embryos at preimplantation due to loss of Xist imprinting, it is resolved at peri-implantation. Ultimately, both X chromosomes are reactivated in the embryonic cell lineage prior to random XCI, and only a single X chromosome undergoes random XCI in the extraembryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamic in the absence of Xist imprinting.

Keywords: H3K27me3; Polycomb; X chromosome inactivation; genomic imprinting; mouse early development.

Figure 1.

Maternal Eed knockout induces loss of H3K27me3 imprinting. (A) Representative images of zygotes and preimplantation embryos immunostained with an anti-H3K27me3 antibody. (M) Maternal pronucleus; (P) paternal pronucleus. Bar, 20 µm. (B) Quantification of the H3K27me3 signal intensity. The average signal intensity of CTR embryos was set as 1.0. The total numbers of embryos examined were eight CTR and 12 matKO for one-cell embryos, six CTR and five matKO for two-cell embryos, seven CTR and six matKO for four-cell embryos, nine CTR and nine matKO for eight-cell embryos, and 10 CTR and eight matKO for morula embryos, respectively. Error bars indicate SD. (***) P < 0.001, two-tailed Student t-test. (C) Scheme for assessing allele-specific gene expression and H3K27me3 enrichment in morula embryos. (+) Eed wild-type allele; (–) Eed knockout allele. (D) Heat map showing the allelic expression bias of H3K27me3-dependent and known DNA methylation-dependent PEGs in morula embryos with biological duplicates. Genes with >20 single-nucleotide polymorphism (SNP) reads in both replicates are shown. (E) Genome browser views showing loss of maternal H3K27me3 domains at the representative H3K27me3-dependent imprinted loci in CTR and Eed matKO morula embryos. (P) Paternal allele; (M) maternal allele.

Figure 2.

Loss of maternal H3K27me3 induces XmCI. (A) Representative images of Xist RNA FISH in female (XX) and male (XY) morula embryos. The gender of each embryo was assessed by simultaneous DNA FISH against the Rnf12 locus (green spots). (Blue) DAPI. Bars: merged images, 20 µm; enlarged images, 5 µm. (B) The ratio of blastomeres showing the indicated number of Xist RNA clouds. Each bar represents an individual embryo. The numbers of embryos examined were 25 CTR and 12 matKO for females and 16 CTR and 18 matKO for males. (C) Box plot showing the relative expression of genes on individual maternal chromosomes between CTR and Eed matKO morula embryos. Genes with enough SNP reads (SNP reads per kilobase per million mapped reads [RPKM] >0.5) were analyzed. The middle lines in the boxes represent the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively. (***) P < 1.2 × 10⁻²³, Mann-Whitney-Wilcoxon test. (D) The relative expression levels of X-linked genes between CTR and Eed matKO morula embryos. The expression levels of the maternal allele were analyzed. Each dot represents an individual gene with enough SNP reads (SNP RPKM >0.5). Known escapees were excluded. Dashed lines indicate a differential expression change of twofold.

Figure 3.

Loss of H3K27me3 imprinting in E6.5 Eed matKO embryos. (A) Heat map showing the allelic expression bias of H3K27me3-dependent PEGs in ExE at E6.5 embryos. The CTR samples include two females (F) and two males (M). The Eed matKO samples include four females and five males. (B,C) Heat maps showing the allelic expression bias of known DNA methylation-dependent PEGs (B) and MEGs (C) in ExE of E6.5 embryos. Genes with >20 SNP reads are shown.

Figure 4.

Aberrant XCI is restored in E6.5 Eed matKO embryos. (A) Box plot showing the relative expression of autosomes and X-linked genes between CTR and Eed matKO of morula embryos and E6.5 ExEs. The middle lines in the boxes represent the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively. (***) P < 1.2 × 10⁻⁴⁴; (**) P < 5.5 × 10⁻¹⁸; (*) P < 4.1 × 10⁻⁷, Mann-Whitney-Wilcoxon test. (B) Representative images of Xist RNA FISH in ExEs of female (XX) and male (XY) E6.5 embryos. (Blue) DAPI. Bars: merged images, 20 µm; enlarged images, 5 µm. (Xa) Active X chromosome. (C) The ratio of blastomeres showing the indicated number of Xist RNA clouds. Each bar represents an individual embryo. The numbers of embryos examined were nine CTR and 11 matKO for females and 16 CTR and seven matKO for males. (D) Heat map showing the allelic expression bias of X-linked genes in ExEs of female E6.5 embryos. CTR and Eed matKO groups contained two and four ExE samples, respectively. Genes with >20 SNP reads are shown. The right panel indicates a zoomed-in sector of Xist to demonstrate the anti-correlation of allelic expression of Xist and the other X-linked genes. The numbers inside the Xist row represent the actual values of log₂ fold change (paternal/maternal).

Figure 5.

Aberrant XCI is restored in Eed matKO peri-implantation embryos. (A) Representative images of Xist RNA FISH (magenta) in female (XX) E3.5 and E4.0 embryos. (Green) Immunostaining signal of Nanog, a marker of pre-Epi cells; (blue) DAPI. Bars: merged images, 20 µm; enlarged images, 5 µm. (B) The ratio of blastomeres showing the indicated number of Xist RNA clouds. Each bar represents an individual embryo. The numbers of embryos examined were 13 CTR and eight matKO for E3.5 and nine CTR and nine matKO for E4.0. (C) Representative images of Xist RNA FISH in male (XY) E3.5 and E4.0 embryos. (D) The ratio of blastomeres showing the indicated number of Xist RNA clouds. Each bar represents an individual embryo. The numbers of embryos examined were eight CTR and eight matKO for E3.5 and 15 CTR and 11 matKO for E4.0.

Figure 6.

Model illustrating XCI dynamics in the absence of Xist imprinting. (Epi) Epiblast; (N.D.) “not determined” in this study.