Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells.
Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells.
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DOI：
10.1371/journal.pone.0046036
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Matsui Y
中科院分区：
文献类型：
--
作者：
Mochizuki K;Tachibana M;Saitou M;Tokitake Y;Matsui Y
原始生殖细胞(PGC)依次诱导发育所需的特定基因。我们专注于调控PGC特异性基因表达的表观遗传学变化。MIL-1、Blimp1和Stella在PGC中优先表达，在PGC分化过程中表达上调。在这里，我们首先检测了外胚层和原生殖细胞中mIL-1、Blimp1和Stella调控区的DNA甲基化状态，发现它们在E9.0以后的原生殖细胞分化过程中发生了低甲基化，在这些基因中这些基因都是高表达的。我们使用siRNA抑制胚胎干细胞中的维持DNA甲基转移酶Dnmt1，发现所有三个基因的侧翼区域都发生了低甲基化，每个基因的表达都增加了1.5-3倍。此外，我们还发现，通过敲除Dnmt1基因，在ES细胞诱导的PGCLCs(PGC样细胞)中，PGC基因的表达增加了1.5-5倍。我们还获得了证据表明，mIL-1调节区的甲基化导致报告试验中表达的2.5倍下降。综上所述，这些结果表明，DNA去甲基化在新生PGCs中PGC基因的初始激活中并不起主要作用，但在E9.0之后有助于增强其在PGCs中的表达。然而，我们也发现，在PGCs中，具有代表性的体细胞基因HoxA1和Hoxb1以及组织特异性基因GFAP的抑制不依赖于DNA甲基化；它们的侧翼区域是低甲基化的，但在E13.5的PGCs中没有观察到它们的表达。它们的启动子区域显示了PGCs中的二价组蛋白修饰，这可能参与了对其表达的抑制。我们的结果表明，PGC基因和体细胞基因的表观遗传状态是不同的，这表明表观遗传机制在调控PGC中特定基因的表达方面做出了贡献。
Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.
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