转录因子CTCF在核移植胚胎发育中的调控机制研究

Somatic cell nuclear transfer (SCNT) enables cloning of differentiated cells by reprogramming their nuclei to a totipotent state. However, successful full-term development of SCNT embryos is a low-efficiency process and cloned embryos frequently exhibit severe abnormalities. Recent technical advances, particularly low-input sequencing techniques, have enabled analysis of transcriptome and epigenetic changes during SCNT reprogramming. Despite high resolution and robustness of the traditional chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), millions of cells are needed, thereby limiting its application in rare biological samples. Therefore, how key transcriptional factors serve as critical barriers in SCNT-mediated reprogramming is unknown. In the present study, we will make full use of our advanced small-scale cleavage under targets and release using nuclease sequencing (CUT&RUN-seq) technology and bioinformatics method to generate, for the first time ever to our knowledge, a genome-wide CTCF-binding roadmap from mouse pre-implantation SCNT embryos. In addition, we will identify and rescue the widespread CTCF-binding defects by using targeted gene editing and/or epigenetic editing systems. Moreover, we will explore the synergistic effect of CTCF and epigenetic modifications in SCNT-mediated reprogramming process. Our study will significantly advance the understanding of genetic and epigenetic remodeling mechanism during nuclear transfer embryo development, and eventually facilitate in achieving higher cloning efficiency.

体细胞核移植可以将终末分化的体细胞重编程为具有全能性的胚胎，但是核移植胚胎往往存在明显的体内外发育缺陷。近年来，随着微量组学技术的发展，核移植胚胎中表观修饰的不完全重编程机制已经逐渐被揭示。但是受限于材料及技术瓶颈等问题，核移植胚胎中重要转录因子的调控机制，迄今尚未有研究。本研究将充分利用课题组已优化完善的少量细胞CUT&RUN-seq技术和相关分析方法，系统揭示核移植胚胎发育过程中转录因子CTCF在全基因组水平上的分布特征和动态变化规律。结合多种组学数据，对具有不同发育命运的核移植胚胎进行CTCF结合情况的比较，筛选出其中与胚胎发育命运密切相关的CTCF结合缺陷位点。并运用基因及表观修饰编辑技术，探讨CTCF结合缺陷的靶向修复方案对核移植胚胎发育的影响。本研究将有助于更深入的了解体细胞核移植介导的遗传和表观遗传重塑的分子机制，为进一步优化现有核移植技术提供重要的理论基础。

转录因子和表观调控因子驱动了哺乳动物胚胎发育过程中细胞潜能的剧烈变化。尽管早期胚胎发育中的表观调控机制已经逐渐被揭示，但是受限于材料及技术瓶颈等问题，这一关键发育阶段的转录因子调控机制一直得不到准确的解析。在本项目的资助下，我们利用微量细胞CUT&RUN技术，揭示了小鼠早期胚胎和核移植胚胎发育过程中CTCF在全基因组上的分布特征和动态变化规律。我们证实了大量的CTCF在小鼠胚胎植入前发育过程中会稳定地结合在基因组上，而仅有少部分染色质区域在合子中的Minor ZGA过程中新获得CTCF的结合，这一事件同时伴随着组蛋白H3K4me3修饰的富集；并且发现了CTCF介导的染色质环在早期胚胎中通过招募H3K4甲基转移酶KMT2B来调节转录活动；我们还证明了H3K9me3介导的异染色质建立造成了胚胎第一次细胞命运决定过程中CTCF占位和功能性的减弱。此外，我们还解析了核移植胚胎滋养层干细胞建系过程中的DNA甲基化修饰动态变化；并且揭示了人类胚胎期生殖细胞发育过程中组蛋白修饰的重编程调控机制。在项目执行期间，项目负责人发表SCI论文3篇，其中2篇标注基金号，并且获得2项国家发明专利的授权。