Molecular Machanisms Of Epigenetics During Mouse Pre-implantation Embryonic Development And Somatic Cell Nuclear Transfer

Histone modifications play critical roles in regulating the expression of developmental genes during embryo development in mammals. However, genome-wide analyses of histone modifications in pre-implantation embryos have been impeded by technical difficulties and scarcity of the required materials. In the first section of this thesis, by using a published ultra-low-input micrococcal nuclease-based native ChIP (ULI-NChIP) method, for the first time, we mapped the genome-wide profile of histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), which are associated with gene activation and repression, respectively, in mouse pre-implantation embryos. We found that the re-establishment of H3K4me3, especially on promoter regions, occurs much more rapidly than that of H3K27me3 following fertilization, which is consistent with the major wave of zygotic genome activation (ZGA) at the 2-cell stage. Furthermore, H3K4me3 and H3K27me3 possess distinct features of sequence preference and dynamics in pre-implantation embryos. Although H3K4me3 modifications exist constantly on transcription start site (TSS) regions, the breadth of the H3K4me3 domain is a highly dynamic feature. Interestingly, the broad H3K4me3 (wider than 5 kb) is associated with higher transcription activity and cell identity not only in pre-implantation embryos but also in the process of deriving embryonic stem cells (ESCs) from the inner cell mass (ICM) and trophoblast stem cells (TSCs) from the trophectoderm (TE). Compared to ESCs, we found that the bivalency (co-occurrence of H3K4me3 and H3K27me3) in early embryos is much less and unstable. Taken together, our results provide a genome-wide map of H3K4me3 and H3K27me3 modifications in pre-implantation embryos, facilitating further exploration of the epigenetic regulation mechanism in early embryo development.Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer (SCNT). However, most cloned embryos arrest at early stages and the underlying molecular mechanism remains largely unexplored. In the second section of this thesis, we developed a SCNT embryo biopsy system at 2-or 4-cell stage, which allows us to trace the developmental fate of the biopsied embryos precisely. Through single-cell transcriptome sequencing of SCNT embryos with different developmental fates, we identified Kdm4b, which can reset the histone methylation barrier previously reported responsible for 2-cell arrest. Moreover, we discovered another histone demethylase Kdm5b, accounts for the arrest of cloned embryos at 4-cell stage. Co-injection of Kdm4b and Kdm5b during SCNT can reset histone methylation, reduce DNAmethylation level, restore transcriptional profiles, and improve the blastocyst development (over 95%) as well as the production of cloned mice. Our study provides an effective approach to identify key factors determining the fate of cloned embryos and provides a new view for the insight of molecular mechanism of SCNT or other embryo developmental process.