Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.

中文翻译：

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小鼠早期胚胎和胚胎干细胞的单细胞多组学测序。

最近已经开发了单细胞表观基因组测序技术。然而，尚未实现单个细胞中表观基因组测序的不同层的组合。在这里，我们开发了一种单细胞多组学测序技术（单细胞COOL-seq），可以从同一哺乳动物细胞中同时分析染色质状态/核小体定位，DNA甲基化，拷贝数变异和倍性。我们使用这种方法来分析小鼠植入前胚胎中染色质状态和DNA甲基化的重编程。我们发现在受精的12小时内，每个单独的细胞都经历了全局基因组去甲基化，同时母体和父本基因组都快速且全局地重新编程为高度开放的染色质状态。随后在合子晚期后开放度降低。此外，从晚期合子到4细胞阶段，残留的DNA甲基化优先保留在每个卵裂球中父本等位基因的基因间区域和母本等位基因的基因内区域。但是，从合子晚期到胚泡期，每个单个细胞的父本和母本等位基因之间的染色质可及性相似。从2细胞阶段开始，几种多能性调节剂的结合基序在远端核小体耗尽的区域富集。这表明这种靶基因的顺式调控元件已经从2-细胞阶段起被引发为开放状态，远远早于在胚泡的ICM中最终建立了多能性。基因可以分为均质开放，基于单个细胞中启动子区域的染色质可及性，实现均匀闭合和发散状态。这可以追溯到植入前发育过程中的逐步过渡。我们的研究为早期小鼠胚胎中单基因组分辨率的基因组规模染色质状态和DNA甲基化动力学提供了首个单细胞和父母等位基因特异性分析，并为表观基因组重编程的异质性但高度有序的特征提供了新的见解。过程。