Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice.
Switching of dominant retrotransposon silencing strategies from posttranscriptional to transcriptional mechanisms during male germ-cell development in mice.
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DOI：
10.1371/journal.pgen.1006926
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发表时间：
2017-07
期刊：
影响因子：
4.5
通讯作者：
Sasaki H
中科院分区：
文献类型：
--
作者：
Inoue K;Ichiyanagi K;Fukuda K;Glinka M;Sasaki H
哺乳动物基因组中有数百万个反转录转座子拷贝，其中一些是转座活性的。在小鼠原核细胞中，PIWI相互作用小RNA（piRNA）对抗逆转录转座子活性以维持基因组完整性。皮尔纳系统破坏逆转录转座子衍生的RNA并引导一些逆转录转座子启动子处的从头DNA甲基化。然而，目前还不清楚DNA甲基化是否有助于逆转录转座子沉默在拟南芥。我们已经对Pld 6/Mitopld和Dnmt 3l敲除小鼠发育中的雄性生殖细胞中的DNA甲基化和polyA（+）RNA（转录组）进行了全面的研究，这两种小鼠分别在皮尔纳生物发生和从头DNA甲基化方面有缺陷。Dnmt 3l突变大大降低了大多数逆转录转座子的DNA甲基化水平，但其对RNA丰度的影响在拟南芥中是有限的。在Pld 6突变体生殖细胞中，虽然只有少数逆转录转座子表现出DNA甲基化降低，但许多逆转录转座子在RNA水平上表现出表达增加。更详细的分析RNA测序，新生RNA定量，切割RNA末端的分析，以及从双敲除小鼠获得的结果表明，PLD 6主要在转录后水平工作。Pld 6突变体中逆转录转座子表达的增加大于Dnmt 3l突变体，表明皮尔纳系统的RNA降解在拟南芥中比DNA甲基化起更重要的作用。然而，DNA甲基化有一个长期的影响：由Pld 6或Dnmt 3l突变引起的低甲基化导致减数分裂精母细胞中反转录转座子表达增加。因此，转录后沉默在生殖细胞发育的早期阶段起重要作用，然后转录沉默在后期阶段变得重要。此外，基因间和内含子反转录转座子序列，特别是那些含有反义L1启动子，驱动异位表达附近的基因在两个突变精母细胞，这表明反转录转座子沉默是重要的维护不仅基因组的完整性，但也转录组的完整性。反转录转座子是一类转座因子，其移动性具有致突变潜力。因此，调控反转录转座子的表达对维持基因组的完整性具有重要意义。在雄性生殖细胞中，DNA甲基化和皮尔纳系统被认为在逆转录转座子沉默中发挥作用。然而，在发育过程中，全基因组DNA甲基化一旦被删除（在原始生殖细胞中）并重新建立（在生殖细胞中）。在原核生物中，piRNA在一些逆转录转座子处引导从头DNA甲基化。为了阐明DNA甲基化和皮尔纳系统在雄性生殖细胞发育过程中对逆转录转座子沉默的贡献，我们分析了Dnmt 3l和Pld 6敲除小鼠中的DNA甲基化和RNA表达，这两种小鼠分别在从头DNA甲基化和皮尔纳生物合成中有缺陷。我们的研究结果表明，皮尔纳系统主要在转录后水平上起作用，并且在逆转录转座子沉默中比DNA甲基化起更重要的作用。然而，DNA甲基化在生殖细胞进入减数分裂（精母细胞）的后期变得更加重要。我们还发现，低甲基化的反转录转座子可以驱动附近基因的异位表达;因此，它们通过DNA甲基化的转录沉默对于维持转录组的完整性也很重要。
Mammalian genomes harbor millions of retrotransposon copies, some of which are transpositionally active. In mouse prospermatogonia, PIWI-interacting small RNAs (piRNAs) combat retrotransposon activity to maintain the genomic integrity. The piRNA system destroys retrotransposon-derived RNAs and guides de novo DNA methylation at some retrotransposon promoters. However, it remains unclear whether DNA methylation contributes to retrotransposon silencing in prospermatogonia. We have performed comprehensive studies of DNA methylation and polyA(+) RNAs (transcriptome) in developing male germ cells from Pld6/Mitopld and Dnmt3l knockout mice, which are defective in piRNA biogenesis and de novo DNA methylation, respectively. The Dnmt3l mutation greatly reduced DNA methylation levels at most retrotransposons, but its impact on their RNA abundance was limited in prospermatogonia. In Pld6 mutant germ cells, although only a few retrotransposons exhibited reduced DNA methylation, many showed increased expression at the RNA level. More detailed analysis of RNA sequencing, nascent RNA quantification, profiling of cleaved RNA ends, and the results obtained from double knockout mice suggest that PLD6 works mainly at the posttranscriptional level. The increase in retrotransposon expression was larger in Pld6 mutants than it was in Dnmt3l mutants, suggesting that RNA degradation by the piRNA system plays a more important role than does DNA methylation in prospermatogonia. However, DNA methylation had a long-term effect: hypomethylation caused by the Pld6 or Dnmt3l mutation resulted in increased retrotransposon expression in meiotic spermatocytes. Thus, posttranscriptional silencing plays an important role in the early stage of germ cell development, then transcriptional silencing becomes important in later stages. In addition, intergenic and intronic retrotransposon sequences, in particular those containing the antisense L1 promoters, drove ectopic expression of nearby genes in both mutant spermatocytes, suggesting that retrotransposon silencing is important for the maintenance of not only genomic integrity but also transcriptomic integrity. Retrotransposons are a class of transposable elements, of which mobility has mutagenic potential. Therefore, it is important to regulate the expression of retrotransposons for maintaining the genomic integrity. In male germ cells, DNA methylation and the piRNA system are thought to play roles in retrotransposon silencing. However, genome-wide DNA methylation is once erased (in primordial germ cells) and reestablished (in prospermatogonia) during development. In prospermatogonia, piRNAs guide de novo DNA methylation at some retrotransposons. To clarify the contribution of DNA methylation and the piRNA system to retrotransposon silencing in the course of male germ cell development, we analyzed DNA methylation and RNA expression in Dnmt3l and Pld6 knockout mice, which are defective in de novo DNA methylation and piRNA biogenesis, respectively. Our results reveal that, in prospermatogonia, the piRNA system works mainly at the posttranscriptional level, and plays a more important role than does DNA methylation in retrotransposon silencing. However, DNA methylation becomes much more important in later stages when germ cells enter meiosis (in spermatocytes). We also found that hypomethylated retrotransposons can drive ectopic expression of nearby genes; therefore, their transcriptional silencing by DNA methylation is important for maintaining the transcriptomic integrity as well.
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影响因子：
10.5
作者：
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