组蛋白修饰对基因的表达起着很重要的调控作用，在植入前胚胎发育过程中，不正常的组蛋白修饰会导致胚胎发育异常甚至胚胎死亡。但是由于技术以及早期胚胎细胞数量的限制，对植入前胚胎全基因组水平上组蛋白修饰的检测至今尚未实现，这极大地阻碍了我们对早期胚胎表观修饰机制的研究。在本论文的第一部分中，我们对最近发表的适用于微量细胞的染色体免疫共沉淀技术进行适当的改进，并利用该技术第一次对小鼠植入前胚胎的组蛋白H3K4me3和H3K27me3修饰进行了全基因组水平上的检测。我们发现在基因的启动子区域，组蛋白H3K4me3修饰的建立更加迅速，而H3K27me3修饰则是在胚胎发育的过程中逐渐建立起来的。并且这两种修饰对DNA序列以及DNA甲基化修饰的偏好性都是明显不同的。非常有趣的是，虽然组蛋白H3K4me3修饰在基因启动子区域的存在是比较稳定的，但是这种修饰的长度却是在不断变化的，而这种长度变化跟基因的表达水平呈现明显的正相关。值得注意的是，我们发现在植入前胚胎中，宽的（broad）H3K4me3修饰的比例要远远高于体外培养的细胞系，而这种宽的H3K4me3修饰无论是在早期胚胎中还是在体外培养的细胞系中都与细胞特异表达的基因有关。另外，在对二价基因的探讨中，我们发现早期胚胎中的二价基因含量更少并且不稳定。通过这些研究，我们第一次建立起了小鼠植入前胚胎发育过程中的组蛋白H3K4me3和H3K27me3修饰图谱，为进一步研究早期胚胎的表观遗传调控机制提供了很好的基础。

通过体细胞核移植技术可以将完全分化的体细胞重编程为具有全能性的胚胎。但是到目前为止，体细胞核移植的效率都很低，大部分的核移植胚胎会在发育的早期被阻滞，然而核移植胚胎被阻滞的分子机制还不是很清楚。在本论文的第二部分中，我们建立了基于单细胞测序的核移植胚胎活检技术。通过这种技术我们可以在追踪胚胎发育命运的同时对胚胎的基因表达和DNA甲基化水平进行检测。通过比较不同发育命运的核移植胚胎的基因表达情况，我们找到了一些可能会导致胚胎被阻滞的关键基因，其中过表达组蛋白去甲基化酶Kdm4b，可以修复核移植胚胎组蛋白H3K9me3修饰的缺陷，帮助胚胎克服2-细胞阻滞的障碍。过表达另一个组蛋白去甲基化酶Kdm5b，可以帮助核移植胚胎克服4-细胞阻滞的障碍。同时过表达这两个基因可以使核移植胚胎在组蛋白修饰、DNA甲基化修饰以及基因表达水平上的缺陷得到极大的改善，重要的是过表达这两个基因可以将核移植胚胎的囊胚发育率提高到95%以上，并且对克隆小鼠的出生率也有极大的提高。这些结果说明，通过我们的方法可以有效地识别影响克隆胚胎发育命运的关键因子，同时，我们的研究结果也为进一步了解胚胎发育以及体细胞核移植的分子机制提供了新的视角。

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 DNA methylation 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.