Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes.
Global mapping of DNA methylation in mouse promoters reveals epigenetic reprogramming of pluripotency genes.
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DOI：
10.1371/journal.pgen.1000116
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发表时间：
2008-06-27
期刊：
影响因子：
4.5
通讯作者：
Reik W
中科院分区：
文献类型：
--
作者：
Farthing CR;Ficz G;Ng RK;Chan CF;Andrews S;Dean W;Hemberger M;Reik W
在原始生殖细胞和植入前胚胎中，DNA甲基化模式通过去甲基化和随后的从头甲基化进行重编程。有人认为，胚胎基因组要恢复到多能状态，表观遗传重编程可能是必要的。我们已经进行了小鼠胚胎干细胞（ES），胚胎生殖细胞（EG），精子，滋养层干细胞（TS）和原代胚胎成纤维细胞（pMEFs）的DNA甲基化的全基因组启动子分析。全局聚类分析显示，ES细胞、EG细胞和精子的甲基化模式惊人地相似，这表明虽然精子是一种高度特化的细胞类型，但其启动子表观基因组已经在很大程度上被重编程，类似于多能状态。多能性组织和pMEFs之间的比较揭示了许多多能性相关基因，包括Nanog、Lefty1和Tdgf1，以及核小体重塑因子Smarcd 1，在干细胞中是低甲基化的，而在分化细胞中是高甲基化的。启动子甲基化的差异与超过60%的分析基因中转录水平的显著差异相关。因此，我们的启动子甲基化的比较方法确定了多能性和表观遗传重编程调控的候选基因。虽然精子基因组总体上与ES和EG细胞的甲基化相似，但也有一些关键的例外，包括Nanog和Lefty1，它们在精子中高度甲基化。Nanog启动子甲基化在受精后在桑椹胚中开始表达之前被主动和被动去甲基化消除。在ES细胞中，正常活性的Nanog启动子在被从头甲基化靶向时沉默。我们的研究表明，启动子甲基化的重编程是多能性基因表观遗传调控的关键决定因素之一。因此，受精前生殖系中的表观遗传重编程和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。大规模的表观遗传重编程发生在哺乳动物生殖细胞和早期胚胎中。这种重编程的生物学目的在很大程度上是未知的，尽管有人认为这可能是胚胎基因组恢复到多能状态所必需的。我们在小鼠中进行了基因组范围的启动子甲基化筛选，将生殖细胞与多能细胞、多能细胞和更分化的细胞类型进行了比较。我们发现启动子甲基化是发育潜能的表观遗传标记。与多能性相关的基因通常在干细胞中低甲基化，在更分化的细胞类型中高甲基化（和沉默），我们的全基因组筛选为多能性的调节提供了新的候选者。重要的是，生殖细胞类似于多能细胞类型，因为大多数启动子已经被重编程。然而，一小组关键的多能性调节因子（包括Nanog）在成熟的生殖细胞中被甲基化，大概是为了在生殖细胞分化的关键阶段抑制多能性。事实上，这些基因中的甲基化在受精后重新编程，以便胚胎能够恢复全能性。因此，这项工作首次表明，表观遗传重编程对于维持生殖细胞和胚胎干细胞的多能性至关重要。
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. Large scale epigenetic reprogramming occurs in mammalian germ cells and the early embryo. The biological purpose of this reprogramming is largely unknown, although it has been suggested that it may be required for the embryonic genome to return to a pluripotent state. We carried out a genome-wide screen of promoter methylation in the mouse, comparing germ cells with pluripotent cells, multipotent cells, and more differentiated cell types. We find that promoter methylation is an epigenetic signature of developmental potency. Genes linked to pluripotency are generally hypomethylated in stem cells and hypermethylated (and silenced) in more differentiated cell types, and our genome-wide screen provides new candidates for the regulation of pluripotency. Importantly, germ cells resemble pluripotent cell types in that most promoters have been reprogrammed. However, a small group of key pluripotency regulators (including Nanog), are methylated in mature germ cells, presumably in order to suppress pluripotency at critical stages of germ cell differentiation. Indeed, methylation in these genes becomes reprogrammed after fertilisation so that the embryo can regain totipotency. This work, therefore, shows for the first time that epigenetic reprogramming is crucial for maintaining the pluripotency of germ and embryonic stem cells.
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