The picture was designed based on ‘Ba Gua’, which represents the fusion of parental genome after fertilization. The chromatin of the paternal genome (golden snake) is more open than that of maternal genome (red snake) and this state is maintained until the 4-cell stage.

On June 18, 2018, Fuchou Tang's lab from Beijing Advanced Innovation Center for Genomic and Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, in collaboration with Jie Qiao's lab from Peking University Third Hospital, published a research paper entitled ‘Single-cell Multi-omics Sequencing of Human Early Embryos’ in Nature Cell Biology. Using single cell multi-omics sequencing technology, single-cell COOL-seq (Cell Research, 2017), the research group developed a genome-wide map of DNA methylation and chromatin accessibility at single-cell resolution during human preimplantation development.

 

Professor Fuchou Tang and Professor Jie Qiao have been working closely for a long time to study the epigenetic regulation mechanism of gene expression during human preimplantation development. In 2013, the research team used the single-cell RNA-seq technology developed by professor Fuchou Tang to draw a complete transcriptome map of human preimplantation embryo (Nature Structural & Molecular Biology, 2013). Then, they developed the low-input and high-throughput DNA methylation sequencing technology to study DNA methylation reprogramming during human preimplantation development in 2014 (Nature, 2014). In 2018, the team used single cell high-throughput DNA methylation sequencing technology to study this process in a higher resolution (Nature Genetics, 2018), which revealed that the genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused re-methylation.

 

In order to analyze the chromatin state during the DNA methylation reprogramming in this process, the research group used the single-cell multi-omics sequencing technology (scCOOL-seq) to systematically describe the dynamic change of each epigenetic layers in single-base and single-cell resolution. For samples with a high proportion of aneuploidy (such as human preimplantation embryos, human cancer samples, etc.), the result from traditional sequencing methods using a small number of cells, such as ChIP-seq, ATAC-seq, DNase-seq, may be confused by abnormal cells mixed in the sample. Therefore the research team used scCOOL-seq technology, which enabled the discrimination of aneuploid cells from euploid ones, to accurately reflect the dynamic of epigenome in this process. The main findings of the study are:

 

(1) The most drastic chromatin remodeling was completed within the first 19 h after fertilization. Once fertilized, the average chromatin accessibility of the genome became less open and reached its lowest level at the 8-cell stage. After zygotic gene activation (ZGA), the global chromatin accessibility pattern increased and reached its highest point at the morula stage (34.2% mean GCH methylation level in zygotes, 31.9% at the 2-cell stage, 30.3% at the 4-cell stage, 26.9% at the 8-cell stage, and 39.6% at the morula stage, Fig 1a). Then, 61,403 proximal NDRs were used to perform t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis and unsupervised hierarchical clustering analysis. We found that proximal NDRs had clear stage-specific features during preimplantation development. The most dramatic chromatin remodeling of proximal NDRs occurred between the 4-cell stage and the 8-cell stage during ZGA (Fig 1b).

Fig1. Chromatin accessibility landscapes across each stage of human preimplantation embryos (a). t-SNE (t-Distributed Stochastic Neighbor Embedding) analysis of the chromatin accessibility of 61,403 proximal NDRs (NDRs within 2 kb upstream and downstream of the TSS) called from oocytes to hESCs (b).

 

(2) The research team used SNPs to separately trace the epigenetic reprogramming of parental genomes in each individual cell (Fig 2a). Unlike in mice, shortly after fertilization, the chromatin of the paternal genome in human embryos was reprogrammed to a more open state than that of the maternal genome, which was maintained to the 4-cell stage (Fig 2b). Afterwards, the parental genomes reached comparable chromatin states in humans. While in mice, the chromatin of the paternal genome became comparable after fertilization and maintained onward.

 

(3) They found that there were distinct features of chromatin accessibility in parental genomes during mouse and human preimplantation development (Fig 2c). Compared to mouse embryos at the same stage, human embryos had much more accessible chromatin. The chromatin accessibility of human paternal genome decreased gradually and reached its lowest level at the 8-cell stage, whereas that of mouse paternal genome dramatically decreased to the lowest level at the 2-cell stage. The differences mentioned above were probably due to the different times of ZGA between species.

 

(4) The research analyzed the chromatin accessibility and DNA methylation of the parental X chromosome in each female blastomere during human preimplantation development (Fig 2d). Similar to the global pattern of differential epigenetic reprogramming, the paternal X chromosome demethylated and reactivated quickly after fertilization. After the 2-cell stage, DNA methylation of the maternal X chromosome was already higher than that of the paternal one, and this methylation difference was maintained. For the chromatin state, the paternal X chromosome was more open than the maternal one from zygote to the 4-cell stage.

Fig 2. Differential DNA methylation and chromatin accessibility between parental genomes within each individual blastomere during human preimplantation development (a, b). Distinct features of chromatin accessibility between human and mouse embryos (c). The difference in DNA methylation levels and chromatin accessibility between parental X chromosomes in female embryos (d).

 

(5) During human preimplantation development, DNA demethylation and chromatin remodeling were unsynchronized in different genomic regions (Fig 3a, b). Interestingly, after fertilization, genes showing high variations in DNA methylation and those with high variations in chromatin accessibility tended to be two different sets. For example, from the 4-cell stage to the morula stage, genes showing high chromatin accessibility variations but low DNA methylation variations were strongly enriched in genes related to chromosome organization, chromatin modification and cell cycle. This finding indicated that the chromatin states of the promoters of chromatin remodeling-associated genes were heterogeneous at these stages. However, from zygote to the blastocyst stage, genes showing high DNA methylation variations but low chromatin accessibility variations were strongly enriched in GO terms such as inflammatory response and the detection of chemical stimulus involved in sensory perception; these genes were not involved in early embryogenesis and showed unsynchronized DNA demethylation.

Fig3. Heat map showing the median variance of DNA methylation levels (a) or chromatin accessibility (b) among individual cells in different regions at each developmental stage. Density plot showing the relationship between DNA methylation variance and chromatin accessibility variance of the promoter regions among individual cells (c).

 

(6) The research blocked RNA polymerase II-mediated transcription by α-Amanitin. Compared to the control group, 1,797 out of 5,155 wide proximal NDRs were unable to be maintained when transcription was inhibited. Among the nearest genes of these 1,797 transcription-dependent proximal NDRs, many of them played a key role in embryogenesis. These results indicated that continual transcription was functionally crucial for a large set of zygotic genes to maintain their promoters’ openness as a feedback mechanism.

Fig4. Continual transcription is crucial for human preimplantation chromatin remodeling

 

(7) The research performed de-novo prediction of enhancers using information from both open chromatin and low-methylated regions (LMRs) at each stage. However, few overlaps were found based on human-mouse homologous regions. The distal NDRs in ICM were enriched for motifs of ZNF281, while that in TE specifically were enriched for motifs of CDX2, AP2-gamma and GATA2.

 

The research applied scCOOL-seq to six critical stages of human preimplantation development and provided insights towards a deeper understanding of epigenetic reprogramming during human preimplantation development. In summary, this research offers a new possibility to decipher highly complex, yet orderly and orchestrated epigenomic reprogramming processes and their impacts on gene expression in human early embryonic development.

 

PhD students Lin Li and Yun Gao from Beijing Advanced Innovation Center for Genomic of Peking University, professor Fan Guo from Sichuan University, and PhD student Yixin Ren from Peking University Third Hospital are co-lead authors of this paper. The corresponding authors of this paper are professor Fuchou Tang and professor Jie Qiao. The project is supported by the National Natural Science Foundation of China, the National Major Scientific Research Program of China, Beijing Municipal Commission of Science and Technology, Beijing Advanced Innovation Center for Genomic (ICG), and the Joint Center for Life Sciences (CLS).

 

Link：https://www.nature.com/articles/s41556-018-0123-2

 

Reference：

Guo, F., Li, L., Li, J., Wu, X., Hu, B., Zhu, P., Wen, L., and Tang, F. (2017). Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells. Cell research.

Yan, L., Yang, M., Guo, H., Yang, L., Wu, J., Li, R., Liu, P., Lian, Y., Zheng, X., Yan, J., et al. (2013). Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 20, 1131-1139.

Guo, H., Zhu, P., Yan, L., Li, R., Hu, B., Lian, Y., Yan, J., Ren, X., Lin, S., Li, J., et al. (2014). The DNA methylation landscape of human early embryos. Nature 511, 606-610.

Zhu, P., Guo, H., Ren, Y., Hou, Y., Dong, J., Li, R., Lian, Y., Fan, X., Hu, B., Gao, Y., et al. (2018). Single-cell DNA methylome sequencing of human preimplantation embryos. Nature genetics.