Large-scale H3.1/H3.2 - H3K9me3 deposition defines the epigenetic signature of extraembryonic lineage cells [Repli-seq]. Large-scale H3.1/H3.2 - H3K9me3 deposition defines the epigenetic signature of extraembryonic lineage cells [Repli-seq]

The extraembryonic lineage in mammals is unique in its unipotent differentiation ability and gestation-limited development. Here we identified the epigenomic characters of mouse trophoblast stem cells (TSCs) focusing on the genome-wide enrichment of histone H3.1/H3.2 and H3K9me3. Comparative ChIP-sequencing (ChIP-seq) analysis of TSCs and embryonic stem cells (ESCs) revealed that the TSC genome uniquely contained large (over 1Mb) H3.1/H3.2-H3K9me3 domains in the intergenic regions. This feature was common to extraembryonic cells in mice and humans. Depletion of CAF1, a H3.1/H3.2 chaperone, led to downregulation of TSC marker genes such as Cdx2 and Elf5 and ectopic expression of Oct3/4, an ESC marker. Nuclear transfer cloning using TSCs resulted in extremely poor embryonic development, but removal of H3K9me3 from their genome resulted in birth of the first TSC-cloned mice. Thus, the signature of the extraembryonic epigenome is safeguarded by the H3.1/H3.2-H3K9me3 enrichment, which protects the genome from cell fate transition. Overall design: ChIP-seq analysis of H3K9me3, H3.1/H3.2, and H3.3 in ESCs, TSCs, control TSCs, and P150 knockdown TSCs. ATAC-seq analysis of ESCs and TSCs. Hi-C analysis and Repli-seq analysis of TSCs. RNA-seq analysis of control TSCs and P150 knockdown TSCs.

哺乳动物的胚外谱系具有独特的单能分化能力与妊娠限制性发育特性。本研究聚焦组蛋白H3.1/H3.2及H3K9me3的全基因组富集特征，鉴定了小鼠滋养层干细胞（trophoblast stem cells, TSCs）的表观基因组图谱。通过对TSCs与胚胎干细胞（embryonic stem cells, ESCs）开展比较ChIP测序（ChIP-sequencing, ChIP-seq）分析，发现TSC基因组在基因间区域独有长度超过1Mb的H3.1/H3.2-H3K9me3结构域，该特征在小鼠与人类的胚外细胞中均保守存在。敲降H3.1/H3.2的分子伴侣CAF1后，TSC标志性基因如Cdx2与Elf5的表达出现下调，同时胚胎干细胞标志性基因Oct3/4发生异位表达。利用TSCs进行细胞核移植克隆时，胚胎发育能力极度低下，但去除其基因组中的H3K9me3后，成功获得了首例TSC克隆小鼠。综上，胚外表观基因组的特征由H3.1/H3.2-H3K9me3富集所维系，该机制可保护基因组免受细胞命运转换的干扰。整体实验设计：对ESCs、TSCs、对照TSCs及P150敲降TSCs开展H3K9me3、H3.1/H3.2与H3.3的ChIP-seq分析；对ESCs与TSCs进行ATAC-seq分析；对TSCs开展Hi-C分析与Repli-seq分析；对对照TSCs与P150敲降TSCs进行RNA-seq分析。