On June 16^th, 2017, Cell Research published ‘Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells’ from the laboratories of Fuchou Tang working in Biodynamic Optical Imaging Center, College of Life Sciences, Peking University. In this study, they 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. Then, they used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos.

Existing methods for analyzing chromatin state/nucleosome positioning based on high-throughput sequencing require hundreds of thousands of cells as starting material(such as ATAC-seq, DNase-seq, FAIRE-seq, MNase-seq).Even if these methods can achieve single-cell resolution, it is impossible to study the interaction between Multi-omics. However, Fuchou Tang Labs combined NOMe-seq (Nucleosome Occupancy and Methylome Sequencing) and PBAT-seq (Post-Bisulfite Adaptor Tagging Sequencing) methods and systematically modified them(Figure 1).It can analyze the genomic and epigenetic characteristics of the same single cell at up to five levels.

 

Figure 1 Diagram of the single-cell COOL-seq method.

 

This team systematically described the dynamic changes of the epigenome at multiple levels during the development of mouse preimplantation embryos at single cell resolution by the newly established scCOOL-seq method. The study found:

Within < 12 h of fertilization, male and female pronuclei from eggs and sperm undergo 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. Then, at the 2-cell stage, the degree of openness gradually increased again, reaching the highest point at the blastocyst stage (Figure 2).

Figure 2 Dynamic changes of endogenous DNA methylation (A) and chromatin accessibility (B), and heterogeneity of chromatin accessibility (gene promoter region: Homogeneously open, Divergent, Homogeneously closed) (C), (D) during mouse preimplantation embryos development.

 

For the first time, the heterogeneity of chromatin accessibility was analyzed during the development of mouse preimplantation embryos in a single-cell resolution. This work found that the promoter regions of most genes coming from sperm rapidly reprogrammed from closed states to open within < 12 h of fertilization, preparing for the subsequent transcription of zygotic genes (Figure 2).
It is the first to prove that continuous transcription is necessary to maintain the open of the promoters of most genes in the early embryo at single-cell resolution. The opened chromatin and transcription activities promote each other to maintain the stable expression of zygotic genes together (Figure 3).

 

Figure 3 The maintenance of the opened chromatin by transcription and the regulation of transcription factors on the opened region of chromatin.

This group found that the target gene binding sites of the pluripotency master regulators Oct4, are open at the 4-cell stage, much earlier than the blastocyst stage, indicating that these sites may be involved the fate determination of early embryonic cells as potential cis-acting elements.
It is the first to analysis the dynamics of DNA methylation and chromatin accessibility of parental genomes in each individual cell. As shown by this study, it is asymmetry of DNA methylation and chromatin accessibility between the parental genomes after fertilization. The chromatin accessibility between parental genomes is rapidly reprogrammed and reach and maintain a precise balance in each individual cell, but DNA methylation is slowly and maintain asymmetry.

Figure 4 Asymmetry of DNA methylation between parental genomes during mouse preimplantation development.

 

It is the first time to analyze the DNA methylation dynamics and chromatin accessibility of paternal and maternal X chromosomes within the same female blastomere. Group found that the DNA methylation speed of the inactivated paternal X chromosome is significantly slower than the active maternal X chromosome after fertilization. When the blastocyst stage had been reached, the DNA methylation levels of both parental X chromosomes became comparable. However, chromatin accessibility between parental X chromosomes keep synchronization after fertilization, and maintain balance of chromatin states between the parental X chromosomes before the preimplantation (Figure 5).

 

Figure 5 DNA methylation and chromatin accessibility dynamics of parental X chromosomes within each individual cell during mouse female preimplantation development.

 

The heterogeneity of epigenome during mouse preimplantation embryo development was revealed at single cell resolution for the first time. After fertilization, the genes of the promoter region with significant DNA methylation heterogeneity and heterogeneity of chromatin accessibility are different. This indicates that they may be regulated by different mechanisms during mouse preimplantation embryos development.
For the first time, cell cycle and chromatin accessibility were linked at single cell resolution, the ploidy and cell cycle stage of each individual cell were accurately inferred, and it was found that mouse preimplantation embryos used the same DNA origin of replication as embryonic stem cells during in vivo development.

Figure 6 Diagram of DNA methylation and chromatin remodeling during mouse preimplantation embryos development.

 

This study contain that systematic description for the precise and orderly changes in DNA methylation and chromatin accessibility during post-fertilization highly specialized gametes reprogram to developmental totipotent fertilized eggs and further development into pluripotent embryos, the interaction between various taxonomic levels, and the reprogramming process of DNA methylation and chromatin accessibility of parental genomes during preimplantation embryo development (figure 6).This work lays a foundation for the further study of totipotency and pluripotency of mammalian early embryonic cells, and provides a new idea for the improvement of efficiency of human somatic cell cloning and the diagnosis and treatment of  abnormalities of early embryonic development.

 

Postdoctoral Dr. Fan Guo (now professor at Sichuan University) and postgraduate students Lin Li, Jingyun Li of the BIOPIC, College of Life Sciences, Peking University are the co-first authors of the paper. Fuchou Tang, professor at the College of Life Sciences, Peking University, and Fan Guo , professor at Sichuan University, are the co-correspondent authors of this paper. The work was jointly completed by Peking University and Sichuan University, and was supported by the National Natural Science Foundation of China, Beijing Advanced Innovation Center for Genomics, and Peking-Tsinghua Center for Life Sciences.