De novo Assembly and Delivery of Synthetic Megabase-Scale Human DNA into Mouse Early Embryos

Epigenetic modifications on natural chromosomes are inherited and maintained in a default state, making it challenging to remove intrinsic marks to study the fundamental principles of their establishment and further influence on transcriptional regulation. Here, we developed SynNICE, a method for precisely assembling and transferring intact, naïve, synthetic Mb-scale human DNA into early mouse embryos, to study de novo epigenetic regulation. By synthesizing and transferring the 1.14 Mb human AZFa (hAZFa) locus, we observed the spontaneous incorporation of murine histones and the establishment of DNA methylation at the 1-cell stage. Notably, DNA methylation from scratch was strongly enriched for repeat sequences without H3K9me3 reinforcement. Furthermore, the transcription of hAZFa initiated at the 4-cell stage was regulated by newly established DNA methylation. This method provides a unique platform for exploring de novo epigenomic regulation mechanisms in higher animals. DNA-seq and RNA-seq of hAZFa in yeast, ChIP-seq, ATAC-seq and Hi-C of hAZFa in yeast and yeast nuclei, RNA-seq and WGBS of hAZFa in mouse early embryos.

天然染色体上的表观遗传修饰会以默认状态遗传并维持，这使得研究者难以去除其内在修饰标记，进而研究表观遗传修饰建立的基本原理及其对转录调控的后续影响。在此，我们开发了SynNICE技术，该方法可精准组装并将完整、初始未修饰的兆碱基级合成人类DNA转移至早期小鼠胚胎中，用于研究从头表观遗传调控机制。通过合成并转移1.14兆碱基的人类AZFa（hAZFa）基因座，我们观察到鼠源组蛋白的自发掺入以及1细胞期DNA甲基化的建立。尤为值得注意的是，从头形成的DNA甲基化显著富集于未受H3K9me3强化的重复序列区域。此外，hAZFa的转录在4细胞期启动，并受新建立的DNA甲基化调控。该技术为探究高等动物的从头表观基因组调控机制提供了独特的研究平台。本数据集包含酵母中hAZFa的DNA测序（DNA-seq）与RNA测序（RNA-seq）数据，酵母及酵母细胞核中hAZFa的染色质免疫共沉淀测序（ChIP-seq）、转座酶可及性测序（ATAC-seq）与Hi-C测序（Hi-C）数据，以及小鼠早期胚胎中hAZFa的RNA测序（RNA-seq）与全基因组亚硫酸氢盐测序（WGBS）数据。