Extensive epigenetic reprogramming occurs during preimplantation embryo development. However, it remains largely unclear how the drastic epigenetic reprogramming contributes to transcriptional regulatory network during this period. Here, we develop a single-cell multiomics sequencing technology (scNOMeRe-seq) that enables profiling of genome-wide chromatin accessibility, DNA methylation and RNA expression in the same individual cell. We apply this method to depict a single-cell multiomics map of mouse preimplantation development. We find that genome-wide DNA methylation remodeling facilitates the reconstruction of genetic lineages in early embryos. Further, we construct a zygotic genome activation (ZGA)-associated regulatory network and reveal coordination among multiple epigenetic layers, transcription factors and repeat elements that instruct proper ZGA. Cell fates associated cis-regulatory elements are activated stepwise in post-ZGA stages. Trophectoderm (TE)-specific transcription factors play dual roles in promoting the TE program while repressing the inner cell mass (ICM) program during the ICM/TE separation.

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Background: Epigenetic abnormalities caused by superovulation have recently attracted increasing attention. Superovulation with exogenous hormones may prevent oocytes from establishing an appropriate epigenetic state, and this effect may extend to the methylation programming in preimplantation embryos, as de novo DNA methylation is a function of developmental stage of follicles and oocyte size. Follicle-stimulating hormone (FSH) and human menopausal gonadotropin (hMG) are common gonadotropins used for superovulation, and appropriate concentrations of these gonadotropins might be necessary. However, no systematic study on the effects of DNA methylation alterations in oocytes associated with superovulation with different dosages of FSH/hMG at the singlecell level has yet been reported. In the current study, different dosages of FSH/hMG combined with human chorionic gonadotropin (hCG) were used in female mice to generate experimental groups, while naturally matured oocytes and oocytes superovulated with only hCG were respectively used as controls. Single-cell level DNA methylation sequencing was carried out on all these matured oocytes.Results: In this study, we revealed that the genome-wide methylation pattern and CG methylation level of the maternal imprinting control regions of all mature oocytes were globally conserved and stable. However, methylation alterations associated with superovulation were found at a specific set of loci, and the differentially methylated regions (DMRs) mainly occurred in regions other than promoters. Furthermore, some of the annotated genes in the DMRs were involved in biological processes such as glucose metabolism, nervous system development, cell cycle, cell proliferation, and embryo implantation and were altered in all dosages of FSH/hMG group (for example, Gfod2 and SYF2). Other genes were impaired only after high gonadotropin dosages (for instance, Sox17 and Phactr4).

17Extensive epigenetic reprogramming occurs during preimplantation embryo development and is 18 accompanied by zygotic genome activation (ZGA) and first cell fate specification. Recent studies 19 using single-cell epigenome sequencing techniques have provided global views of the dynamics of 20 different epigenetic layers during this period. However, it remains largely unclear how the drastic 21 epigenetic reprogramming contributes to transcriptional regulatory network. Here, we developed a 22 single-cell multiomics sequencing technology (scNOMeRe-seq) that enables profiling of genome-23 wide chromatin accessibility, DNA methylation and RNA expression in the same individual cell 24 with improved performance compared to that of earlier techniques. We applied this method to 25 analyze the global dynamics of different molecular layers and their associations in mouse 26 preimplantation embryos. We found that global DNA methylation remodeling facilitates the 27 reconstruction of genetic lineages in early embryos and revealed that the gradual increases in 28 heterogeneity among blastomeres are driven by asymmetric cleavage. Allele-specific DNA 29 methylation pattern is maintained throughout preimplantation development and is accompanied by 30 allele-specific associations between DNA methylation and gene expression in the gene body that 31 are inherited from oocytes and sperm. Through integrated analyses of the collective dynamics 32 between gene expression and chromatin accessibility, we constructed a ZGA-associated regulatory 33 network and revealed coordination among multiple epigenetic layers, transcription factors (TFs) 34 and repeat elements that instruct the proper ZGA process. Moreover, we found that inner cell mass 35 (ICM)/trophectoderm (TE) lineage-associated cis-regulatory elements are stepwise activated in 36 blastomeres during post-ZGA embryo stages. TE lineage-specific TFs play dual roles in promoting 37 the TE program while repressing the ICM program, thereby separating the TE lineage from the ICM 38 lineage. Taken together, our findings not only depict the first single-cell triple-omics map of 39 chromatin accessibility, DNA methylation and RNA expression during mouse preimplantation 40 development but also enhance the fundamental understanding of epigenetic regulation in early 41 embryos. 42 43