Single Cell Sequencing Identifies Key Epigenetic Regulators in Nuclear Transfer Mediated Reprogramming [RNA-seq]

Differentiated cell can be reprogrammed into totipotent embryo through somatic cell nuclear transfer (SCNT). However, this process is highly inefficient and most cloned embryos arrest at certain developmental stages. Through single cell sequencing combined with embryo biopsy, here we generate a global map of DNA methylome and RNA transcriptome for SCNT embryos with distinct developmental fates. We subsequently demonstrate that the unfaithful reactivation of two histone demethylases, Kdm4b and Kdm5b, accounts for the arrest of cloned embryos at 2-cell and 4-cell stage, respectively. Ectopic expression of Kdm4b and Kdm5b in SCNT can remove H3K9me3 barrier, restore the transcription profile and facilitate the blastocyst developmental efficiency over 95%. Moreover, these cloned embryos can further support full-term development and the derivation of SCNT-embryonic stem cells with greater efficiency. Our study reveals that histone methylation reset is crucial for the development of SCNT embryos, which provides a clue to further improve therapeutic cloning. For SCNT embryos or injected SCNT embryos 3-8 replicates were performed for each stage . As the control, 3-6 replicates were performed for each stage of wild type samples分化细胞可通过体细胞核移植（Somatic Cell Nuclear Transfer，SCNT）技术重编程为全能胚胎。然而该过程效率极低，绝大多数克隆胚胎会在特定发育阶段停滞发育。本研究结合单细胞测序与胚胎活检技术，构建了具有不同发育命运的体细胞核移植胚胎的全基因组DNA甲基化组与RNA转录组图谱。本研究随后证实，两种组蛋白去甲基化酶Kdm4b与Kdm5b的异常重激活，分别导致克隆胚胎在2细胞期与4细胞期停滞发育。在体细胞核移植胚胎中异位表达Kdm4b与Kdm5b，可清除H3K9me3修饰障碍，恢复正常转录组谱，并将囊胚发育效率提升至95%以上。此外，此类克隆胚胎可进一步支持足月发育，并能以更高效率获取体细胞核移植胚胎干细胞。本研究揭示，组蛋白甲基化重编程对于体细胞核移植胚胎的发育至关重要，为进一步优化治疗性克隆技术提供了新思路。针对体细胞核移植胚胎与显微注射后的体细胞核移植胚胎，每个发育阶段均设置3-8次生物学重复；作为对照的野生型样本，每个发育阶段均设置3-6次生物学重复。