Induction of DNA Methylation by Artificial piRNA Production in Male Germ Cells
Induction of DNA Methylation by Artificial piRNA Production in Male Germ Cells
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DOI：
10.1016/j.cub.2015.01.060
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发表时间：
2015-03-30
期刊：
影响因子：
9.2
通讯作者：
Nakano, Toru
中科院分区：
文献类型：
--
作者：
Itou, Daisuke;Shiromoto, Yusuke;Nakano, Toru
整体DNA去甲基化和随后的从头DNA甲基化发生在哺乳动物雄性胚胎生殖细胞中[1-3]。P元件诱导的懦弱睾丸（PIWI）相互作用RNA（piRNA）是生殖系特异性小RNA，已被假定对逆转录转座子基因的从头DNA甲基化至关重要，许多蛋白质（包括PIWI家族蛋白）在此过程中发挥关键作用[4-6]。在胚胎小鼠睾丸中，两种小鼠PIWI蛋白，小鼠PIWI样（MILI）和小鼠PIWI 2（MIWI 2），通过所谓的乒乓扩增循环参与piRNA的生物发生[7-10]，并且已经提出从皮尔纳簇的基因区域转录的长单链RNA是初始材料[11-16]。然而，仍然不清楚piRNA的生物发生是否需要来自piRNA簇的转录。为了回答这个问题，我们开发了一种新的人工皮尔纳生产系统，通过在皮尔纳生物发生阶段的胚胎雄性生殖细胞中简单表达正义和反义EGFP mRNA。EGFP表达被piRNA依赖性DNA甲基化沉默，表明正义和反义RNA转录物的伴随表达对于皮尔纳产生和随后的piRNA依赖性基因沉默是必要和足够的。此外，我们证明了这种人工皮尔纳诱导范例可以应用于精子发生所必需的内源性基因DNM T3 L [3，17，18]。这项研究不仅为皮尔纳产生的分子机制提供了新的见解，而且还提出了在生殖细胞中诱导表观遗传修饰的创新策略。
Global DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1-3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4-6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7-10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11-16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNM T3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells.