体细胞核移植中的DNA修饰缺陷的产生与修正方法的研究

Differentiated somatic cells can be reprogrammed into totipotent embryos through somatic cell nuclear transfer (SCNT), and recent evidence has shown that mutations in the mitochondrial genome as well as aging-induced telomere shortness and mitochondrial dysfunction, which holds promise for future clinical applications of therapeutic cloning. However, most cloned embryos arrest at early stage, and impaired DNA methylation imprinting is observed in clonal animals and their placentas. And underlying molecular mechanism of these deficiencies remains unexplored. Until recently, our group and Yi Zhang’s group proved that unfaithful remove of H3K9me3 might be an important reason for the 2-cell arrest of mice and human SCNT embryos. Injection of the mRNA of H3K9me3 eraser will greatly improve the blastocyst development of SCNT embryos. However the improvement in the birth rate is not equally obvious. By combining SCNT embryo biopsies with single-cell RNA sequencing, we established a new analysis system to dissect the molecules responsible for the developmental arrest of cloned embryos, and found for the first time that Kdm5b, an H3K4me3 demethylase, serves as the key factor for 4-cell arrest. Here in this project we want to find DNA methylation defects which associated with developmental arrest of SCNT embryo, by single-cell DNA methylation sequencing. We will further develop a locus-editable repair system to rescue the crucial arrest-related DNA methylation disorders identified in our analysis, to further confirm the role of DNA methylation in SCNT embryo development. Also we will further confirm the DNA methylome of different group of SCNT embryos which are treated by published histone-modification-repair strategy like Kdm4d-mRNA injection, SCR treatment, and justify the efficiency and safety of these strategy in the view of DNA methylome rebuilding.

核移植技术作为一种体细胞重编程的方法，被认为具有修正线粒体缺陷以及改善端粒长度等优势。然而克隆胚胎容易发生植入前阻滞，克隆动物的胎儿和胎盘往往具有印记基因等方面的缺陷。但克隆胚胎发育异常和甲基化异常的分子机制之前并不清楚。我们和国外研究组在近期的研究成果都证明了核移植过程中H3K9me3的修饰擦除失败，是鼠和人的克隆胚胎发生2细胞阻滞的重要原因，但这一修饰的修正对克隆小鼠出生率的改善不明显。我们在研究中首创了胚胎活体取样结合单细胞测序的研究平台，并首次报道了4细胞阻滞发生因素并初步解析了克隆胚胎DNA甲基化的异常。本项目的研究希望通过单细胞DNA甲基化测序，寻找与克隆胚胎发育缺陷相关的DNA甲基化缺陷，并建立定点修复系统，阐释建立从DNA甲基化方面改善克隆胚胎表观缺陷的新方法。并通过这一技术平台从DNA甲基化修饰方面，探讨组蛋白修复方法在核移植中应用的安全性和有效性。

核移植技术作为一种体细胞重编程的方法，被认为具有修正线粒体缺陷以及改善端粒长度等优势。我们研究团队聚焦引发克隆胚胎的重编程与后续发育缺陷的表观调控机制，有针对性的将单细胞活体取样和单细胞测序相结合，绘制了不同发育命运的核移植胚胎，在植入前发育各个阶段的转录组和DNA甲基化组图谱。在甲基化组的分析中，我们发现克隆胚胎发育中CpG位点的DNA存在着再甲基化的现象，特别是4细胞时期较为明显。结合转录组数据，我们发现这一再甲基化过程是导致克隆胚胎中合子基因和部分逆转座子不完全激活的重要原因。通过干扰DNA甲基化酶（Dnmt3a+Dnmt3b）的表达，我们可以有效降低克隆胚胎中异常的DNA再甲基化，提高克隆胚胎的发育率和出生率，并改善胎盘的发育。上述工作发表于Cell Stem Cell 杂志，并引起业内关注。我们进一步发现克隆胚胎的DNA再甲基化和组蛋白修饰重编程缺陷是两个相对独立的壁垒，联合使用可以进一步提高克隆胚胎的发育能力。另外在项目资助下，我们也积极开展了新的DNA修饰检测方法的建立，以及逆转座子的RNA以及RNA修饰对于核内表观修饰调控的机制研究，进一步拓展了我们的研究方向。