20/kloc-0 On June 6th, July, the research group of Tang Fuqiao from the Biodynamic Optical Imaging Center of Peking University College of Life Sciences published a research paper entitled "Single Cell Multiomics Sequencing of Mouse Early Embryos and Embryonic Stem Cells" in Cell Research. For the first time in the world, the whole genome sequencing technology (single cell COOL-seq) was developed to simultaneously perform chromatin state, DNA methylation, genome copy number variation and chromosome ploidy on a single cell, and the key features of epigenome reprogramming during mouse preimplantation embryo development and the interaction between chromatin state and DNA methylation were systematically and deeply analyzed by using this technology at single cell resolution. ?
The existing research methods based on high-throughput sequencing to analyze the whole genome chromatin state usually need a large number of cells (such as ATAC-seq, DNase-seq, FAIRE-seq, MNase-seq, etc. ). Even if these methods can reach the single-cell resolution, it is impossible to study the interaction between various omics at the single-cell resolution. However, Tang Fuqiu's research team skillfully combined NOMe-seq (whole genome nucleosome localization and DNA methylation sequencing) technology with PBAT-seq technology, and optimized and improved the system, realizing the analysis of the genome and epigenome characteristics of the same single cell up to five levels. ? Using this newly established scCOOL-seq method, the research team systematically described the dynamic changes of multi-level epigenome during mouse preimplantation embryo development with single cell resolution. The study found that:
Within 12 hours after fertilization, male and female pronuclei from highly specialized eggs and sperm experienced large-scale genomic demethylation. In this process, the chromosome state of the parent genome opens rapidly, reaching a highly open state in the pronuclear stage of the fertilized egg, and then the chromatin openness drops sharply in the late stage of the fertilized egg, and gradually increases after the 2- cell stage, reaching the highest point in the blastocyst stage. ?
The heterogeneity of chromatin state in mouse preimplantation embryo development was analyzed by single cell resolution system for the first time. It was found that within 12 hours after fertilization, the promoter regions of most genes in fertilized eggs were rapidly reprogrammed from uniform closed state to uniform open state, which prepared for the subsequent transcription of fertilized eggs. ?
It is proved for the first time in single cell discrimination that maintaining the promoters of most genes in early embryos in an open state requires continuous transcription, chromatin state opening and transcription activity promote each other, and * * * maintains the stable expression of zygote genes. ?
It was found that the target gene binding sites of the pluripotent core factor Oct4 were opened at the 4-cell stage, much earlier than the blastocyst stage when pluripotency was really established, suggesting that these sites, as potential cis-regulatory elements, may participate in the fate determination process of early embryonic cells. ?
For the first time, the chromatin state and DNA methylation of the parent genome were deeply analyzed in a single cell. It was found that the chromatin state and DNA methylation were asynchronously reprogrammed after fertilization, and the chromatin state of the parent genome was rapidly reprogrammed, achieving an accurate balance in each single cell and maintaining it all the time. The reprogramming of DNA methylation is slow, and it keeps asymmetric distribution among parental genomes. ?
The similarities and differences of DNA methylation and chromatin state reprogramming of parental X chromosome in female embryonic cells were analyzed at single cell resolution for the first time. The results showed that after fertilization, the DNA methylation reprogramming speed of the inactivated paternal X chromosome in female embryos was significantly slower than that of the active maternal X chromosome, and the difference of DNA methylation between them gradually disappeared until the late blastocyst. However, in female embryos, the parental X chromosome is rapidly reprogrammed, and the precise balance of chromatin state between the parental X chromosomes is maintained throughout the preimplantation period. ?
The heterogeneity of epigenome of mouse preimplantation embryo development was first revealed at the single cell level. After fertilization, the gene with strong DNA methylation heterogeneity in the promoter region and the gene with strong chromatin state heterogeneity are two different types of genes. This indicates that the heterogeneity of chromatin state and DNA methylation in mouse embryo development before implantation may be regulated by different mechanisms. ?
For the first time, the cell cycle and chromatin state are linked in single cell resolution, and the ploidy and cell cycle stage of each single cell are accurately inferred. It was found that mouse embryos used basically the same set of DNA replication initiation sites as embryonic stem cells during in vivo development. ?
In this study, the precise and orderly changes of DNA methylation and chromatin state, the interaction between various genomics levels, and the reprogramming process of DNA methylation and chromatin state in the parental genome during the development of preimplantation embryos were systematically described. This work lays a foundation for people to continue to study the totipotency and pluripotency of early embryonic cells in mammals in the future, and provides a new idea for improving the efficiency of somatic cell cloning and the diagnosis and treatment of early embryonic dysplasia. ? Frant Gwo, postdoctoral fellow, Lin Li and Li Jingyun, doctoral students of Life Science Center of Peking University College of Life Sciences, are the first authors of this paper. Researcher Tang from Peking University College of Life Sciences and researcher from Sichuan University are the co-authors of this paper. The research work was jointly completed by Peking University and Sichuan University, supported by the National Natural Science Foundation, Beijing Future Gene Diagnosis High-tech Innovation Center and Peking University-Tsinghua Joint Center.