Diverse epigenetic mechanisms maintain parental imprints within the embryonic and extraembryonic lineages

Genomic imprinting and X chromosome inactivation (XCI) require epigenetic mechanisms to direct allele-specific expression. Despite their critical roles in embryonic development, how universal epigenetic regulators coordinate these specific tasks at single loci or across chromosome scales remains understudied. Here, we systematically disrupted essential epigenetic pathways within polymorphic F1 embryos to examine canonical and non-canonical genomic imprinting as well as X chromosome inactivation. We find that DNA methylation and Polycomb group repressors are both indispensable for autosomal imprinting, albeit at distinct gene sets. Moreover, the extraembryonic ectoderm relies on a broader spectrum of imprinting mechanisms, including non-canonical targeting of maternal endogenous retrovirus (ERV) driven promoters by the H3K9 methyltransferase G9a. We further utilize our data to identify Polycomb dependent and independent gene clusters on the imprinted X chromosome, which appears to reflect distinct domains of Xist-mediated suppression. From our data, we assemble a comprehensive inventory of the epigenetic mechanisms utilized in eutherian mammals to maintain parent-specific imprinting, including an expanded view of the placental lineage that comprises multiple unique pathways. For this study, we disrupted the DNA methyltransferase Dnmt1, the histone 3 lysine 9 (H3K9) methyltransferases G9a and Glp (target genes Ehmt1 and Ehmt2, double KO), as well as the PRC1 and PRC2 complexes individually (target genes: Rnf2 and Eed, respectively) in BDF1xCAST zygotes. Epiblast and ExE samples were collected from 30 knockout embryos on E6.5 and processed for bulk RNA sequencing.基因组印记（Genomic imprinting）与X染色体失活（X chromosome inactivation, XCI）依赖表观遗传机制（epigenetic mechanisms）以实现等位基因特异性表达（allele-specific expression）。尽管二者在胚胎发育（embryonic development）中发挥关键作用，但通用表观遗传调控因子（universal epigenetic regulators）如何在单个基因座或整条染色体尺度上协调这些特异性过程，目前仍未得到充分研究。本研究系统性敲除多态性F1胚胎中的核心表观遗传通路，以探究经典与非经典基因组印记（canonical and non-canonical genomic imprinting）以及X染色体失活的调控机制。我们发现，DNA甲基化（DNA methylation）与Polycomb家族阻遏因子（Polycomb group repressors）均为常染色体印记（autosomal imprinting）维持所必需，尽管二者作用的基因集存在差异。此外，胚外外胚层（extraembryonic ectoderm）依赖更为广泛的印记调控机制，包括由H3K9甲基转移酶G9a（H3K9 methyltransferase G9a）介导的母源内源性逆转录病毒（endogenous retrovirus, ERV）驱动型启动子的非经典靶向调控。我们进一步利用本研究数据，在印记X染色体上鉴定出Polycomb依赖与非依赖的基因簇，这一结果反映了Xist介导的基因沉默（Xist-mediated suppression）的不同结构域。基于本研究数据，我们构建了真兽类哺乳动物（eutherian mammals）中维持亲本特异性印记（parent-specific imprinting）的表观遗传调控机制的完整目录，其中涵盖了包含多种独特调控通路的胎盘谱系（placental lineage）的拓展认知。本研究在BDF1×CAST受精卵中分别开展了以下基因敲除操作：DNA甲基转移酶Dnmt1（DNA methyltransferase Dnmt1）、组蛋白3赖氨酸9（histone 3 lysine 9, H3K9）甲基转移酶G9a与Glp（靶基因为Ehmt1与Ehmt2，构建双敲除模型），以及PRC1与PRC2复合物（PRC1 and PRC2 complexes，靶基因分别为Rnf2与Eed）。收集胚胎发育第6.5天（E6.5）的30份敲除胚胎的上胚层（epiblast）与胚外外胚层（extraembryonic ectoderm，缩写ExE）样本，进行批量RNA测序（bulk RNA sequencing）分析。