Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes

DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.

DNA甲基化模式（DNA methylation patterns）会在原始生殖细胞（primordial germ cells）以及着床前胚胎中通过去甲基化及后续的从头甲基化（de novo methylation）过程发生重编程。已有研究表明，表观遗传重编程（epigenetic reprogramming）或许是胚胎基因组恢复至多能状态所必需的过程。本研究对小鼠胚胎干细胞（embryonic stem, ES cells）、胚胎生殖细胞（embryonic germ, EG cells）、精子、滋养层干细胞（trophoblast stem, TS cells）以及原代胚胎成纤维细胞（primary embryonic fibroblasts, pMEFs）开展了全基因组启动子区域DNA甲基化分析。全局聚类分析（global clustering analysis）结果显示，ES细胞、EG细胞与精子的甲基化模式惊人相似，这表明尽管精子属于高度特化的细胞类型，但其启动子表观基因组（epigenome）已基本完成重编程，且与多能状态的细胞高度相似。通过对多能组织与原代胚胎成纤维细胞的比较分析，本研究发现多个多能性相关基因（包括Nanog、Lefty1、Tdgf1）以及核小体重塑因子Smarcd1（nucleosome remodeller Smarcd1）在干细胞中呈低甲基化状态，而在分化细胞中呈高甲基化状态。在超过60%的分析基因中，启动子甲基化差异与转录水平的显著变化显著相关。因此，本研究采用的启动子甲基化比较分析方法，筛选出了参与多能性调控与表观遗传重编程的候选基因。尽管整体而言精子基因组的甲基化水平与ES细胞及EG细胞相近，但仍存在若干关键例外，例如Nanog与Lefty1在精子中呈高度甲基化状态。在受精后至桑椹胚（morula）阶段开始表达前，Nanog启动子的甲基化会通过主动去甲基化（active demethylation）与被动去甲基化（passive demethylation）过程被清除。在ES细胞中，若对原本活跃的Nanog启动子进行从头甲基化修饰，其转录会被沉默。本研究表明，启动子甲基化重编程是调控多能性基因表观遗传的关键决定因素之一。因此，受精前生殖细胞系中的表观遗传重编程，以及早期胚胎中关键多能性基因的重编程，对于多能性的传递至关重要。