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. 2017 Oct 1;31(19):1927-1932.
doi: 10.1101/gad.304113.117.

Genomic imprinting of Xist by maternal H3K27me3

Azusa Inoue  1   2   3 , Lan Jiang  1   2   3 , Falong Lu  1   2   3 , Yi Zhang  1   2   3   4   5
Affiliations

Genomic imprinting of Xist by maternal H3K27me3

Azusa Inoue et al. Genes Dev. .

Abstract

Maternal imprinting at the Xist gene is essential to achieve paternal allele-specific imprinted X-chromosome inactivation (XCI) in female mammals. However, the mechanism underlying Xist imprinting is unclear. Here we show that the Xist locus is coated with a broad H3K27me3 domain that is established during oocyte growth and persists through preimplantation development in mice. Loss of maternal H3K27me3 induces maternal Xist expression and maternal XCI in preimplantation embryos. Our study thus identifies maternal H3K27me3 as the imprinting mark of Xist.

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

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Figures

Figure 1.
Figure 1.
Ectopic removal of H3K9me3 does not induce maternal Xist expression. (A) Representative images of zygotes stained with anti-H3K9me3 antibody. (M) Maternal pronucleus; (P) paternal pronucleus. The bar graph shows relative H3K9me3 signal intensity of the maternal pronuclei. The average signal intensity of noninjected zygotes was set as 1.0. The total numbers of embryos examined were eight (no injection), nine (1000 ng/µL Kdm4b), and nine (2600 ng/µL Kdm4b). Error bars indicate SE. (***) P < 0.001; (*) P < 0.05, two-tailed Student's t-test. (B) Representative images of Xist RNA FISH (magenta) in Kdm4b-injected four-cell embryos. The gender of each embryo was assessed by simultaneous DNA FISH against the Rnf12 locus (green). Arrows indicate the blastomeres enlarged in the bottom panels. (C,D) The ratio of blastomeres that show the indicated number of Xist RNA clouds and spots in male (C) and female (D) embryos. Each bar represents an individual embryo. The numbers of embryos examined were six (no injection), five (1000 ng/µL Kdm4b), and 12 (2600 ng/µL Kdm4b) males and six (no injection), six (1000 ng/µL Kdm4b), and eight (2600 ng/µL Kdm4b) females.
Figure 2.
Figure 2.
Maternal H3K27me3 coats Xist in oocytes and preimplantation embryos. (A) Genome browser view of H3K27me3 ChIP-seq (Zheng et al. 2016), DNase I-seq (Inoue et al. 2017), and DNA methylation levels (Kobayashi et al. 2012) at the Xist locus. (Oo) MII oocyte; (Sp) sperm; (7 d) growing oocytes collected from 7-d-old females; (14 d) growing oocytes collected from 14-d-old females; (GV) fully grown GV stage oocytes. (B) Genome browser view of allelic H3K27me3 in one-cell, two-cell, and blastocyst embryos and E6.5 epiblast at the Xist locus. The highlighted square indicates a computationally determined region where the maternal allele bias of H3K27me3 enrichment is retained in blastocyst embryos. (Mat) Maternal allele; (Pat) paternal allele. The H3K27me3 ChIP-seq data sets were from Zheng et al. (2016). (C) A higher-resolution view of the Xist locus in blastocyst embryos. The maternal allele-biased H3K27me3 domain is highlighted.
Figure 3.
Figure 3.
Loss of H3K27me3 induces maternal Xist expression. (A) Representative images of zygotes stained with anti-H3K27me3 antibody. (M) Maternal pronucleus, (P) paternal pronucleus. The bar graph indicates the relative H3K27me3 signal intensity of maternal pronuclei. The average signal of Kdm6bMUT-injected zygotes was set as 1.0. The total numbers of embryos examined were 15 (Kdm6bMUT) and 13 (Kdm6bWT). Error bars indicate SE. (***) P < 0.001, two-tailed Student's t-test. (B) Genome browser view of the Xist locus showing loss of maternal H3K27me3 ChIP-seq signal in Kdm6bWT-injected morula embryos. (Mat) Maternal allele; (Pat) paternal allele. (C) Representative images of Xist RNA FISH (magenta) in Kdm6b-injected four-cell embryos. The gender of each embryo was assessed by simultaneous DNA FISH against the Rnf12 locus (green). (D,E) The ratio of blastomeres showing the indicated number of Xist RNA clouds and spots in male (D) and female (E) four-cell embryos. Each bar represents an individual embryo. The numbers of embryos examined were eight (Kdm6bMUT) and 15 (Kdm6bWT) for males and eight (Kdm6bMUT) and 12 (Kdm6WT) for females.
Figure 4.
Figure 4.
Loss of H3K27me3 induces maternal XCI. (A) Illustration depicting maternal XCI caused by Kdm6WT-mediated maternal Xist expression. (B) Representative images of Xist RNA FISH (magenta) in Kdm6b-injected morula embryos. The gender of each embryo was assessed by simultaneous DNA FISH against the Rnf12 locus (green). (C,D) The ratio of blastomeres showing the indicated number of Xist RNA clouds in male (C) and female (D) morula embryos. Each bar represents an individual embryo. The numbers of embryos examined were 19 (Kdm6bMUT) and 35 (Kdm6bWT) for males and 34 (Kdm6bMUT) and 35 (Kdm6bWT) for females. (E) Box plot showing the relative expression of genes on individual maternal chromosomes between Kdm6bMUT- and Kdm6bWT-injected blastocysts. Genes with enough SNP reads (SNP reads > 10, RPM [reads per million] > 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 < 0.001, Mann-Whitney-Wilcoxon test.
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