Maternal Eed knockout causes loss of H3K27me3 imprinting and random X-inactivation in the extra-embryonic cells

Genomic imprinting is essential for mammalian development. Recent studies have revealed that maternal histone H3 lysine 27 tri-methylation (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 KO (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 extra-embryonic cell lineage. Thus, our study not only demonstrates an essential role of Eed in H3K27me3 imprinting establishment but also reveals a unique XCI dynamics in the absence of Xist imprinting. mRNA profiling was performed for morula embryos and E6.5 extra-embryonic ectoderm (ExE) of Eed control and maternal knockouts. For morula embryos, two replicates were generated for each group. For E6.5 ExE samples, four replicates were produced for the control group (2 males and 2 females) and nine replicates were generated for the maternal KO group (5 males and 4 females). H3K27me3 profiling for Eed CTR and matKO morula embryos were performed using CUT&RUN.基因组印记（genomic imprinting）对哺乳动物发育至关重要。近期研究表明，母源组蛋白H3赖氨酸27三甲基化（H3K27me3）可介导不依赖DNA甲基化的基因组印记。然而，这一新型印记机制的调控机制与生物学功能尚未得到充分阐明。本研究证实，作为多梳蛋白复合体2（PRC2）的核心组分，母源Eed基因对于建立H3K27me3印记不可或缺。研究发现，包括Xist在内的所有H3K27me3印记基因，在Eed母源敲除（matKO）胚胎中均丧失了印记特异性表达模式，进而引发雄性偏向性胚胎致死。令人意外的是，尽管因Xist印记缺失，Eed matKO胚胎在植入前阶段即发生母源X染色体失活（XmCI），但该失活过程在围植入期得到逆转。最终，在随机X染色体失活（XCI）启动前，胚胎细胞谱系内的两条X染色体均被重新激活；而在胚外细胞谱系中，仅单条X染色体发生随机XCI。综上，本研究不仅明确了Eed在建立H3K27me3印记中的关键作用，同时揭示了缺失Xist印记时独特的X染色体失活动态调控模式。本研究针对Eed对照组与母源敲除组的桑椹胚及胚胎第6.5天（E6.5）胚外外胚层（ExE）样本开展了mRNA表达谱分析：其中桑椹胚每组设置2个生物学重复；对于E6.5 ExE样本，对照组共获得4个生物学重复（2雄、2雌），母源敲除组共获得9个生物学重复（5雄、4雌）。此外，本研究通过CUT&RUN技术对Eed对照组与matKO组桑椹胚的H3K27me3进行了表达谱分析。