Specific tandem repeats are sufficient for paramutation-induced trans-generational silencing.
Specific tandem repeats are sufficient for paramutation-induced trans-generational silencing.
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DOI：
10.1371/journal.pgen.1003773
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Chandler VL
中科院分区：
文献类型：
--
作者：
Belele CL;Sidorenko L;Stam M;Bader R;Arteaga-Vazquez MA;Chandler VL
异突变是一种广泛研究的表观遗传现象，其中两个不同等位基因之间的跨式通讯导致其中一个等位基因的减数分裂可遗传的转录沉默。在b1位点的平行突变涉及RNA介导的转录沉默，并需要产生siRNA的特定串联重复序列。本研究解决了三个重要问题：1）串联重复序列是否足以进行副突变，2）它们是否需要处于等位基因位置以介导副突变，以及3）介导副突变的能力与重复DNA甲基化水平之间是否存在关联？使用含有b1串联重复序列的多个转基因实现了副突变，包括具有仅一半重复单元（413 bp）的串联重复序列的事件，表明这些序列足以用于副突变，并且重复序列不需要等位基因位置来进行通信。此外，转基因串联重复序列增加了报告基因在玉米中的表达，表明重复序列含有转录调控序列。转基因介导的副突变需要paramutation1基因的介导，而paramutation1基因是内源副突变所必需的，这表明内源和转基因介导的副突变都需要RNA介导的转录沉默途径。虽然所有测试的重复转基因产生小干扰RNA（siRNA），但并非所有转基因诱导副突变，这表明与内源等位基因一样，siRNA产生不足以副突变。重复转基因诱导的沉默是不太有效地传递比沉默诱导的内源性b1等位基因的重复，这总是100%的效率。重复转基因诱导沉默的强度的可变性使得能够测试重复内的DNA甲基化程度是否与副突变效率的差异相关。转基因诱导的副突变不需要在转基因内广泛的DNA甲基化。然而，在转基因诱导的副突变后，内源性b1重复序列内的DNA甲基化增加与内源性等位基因的更强沉默相关。Paramutation是一个迷人的过程，在这个过程中，基因进行交流，以有效地建立其表达的变化，这些变化可以稳定地传递给后代，而不会改变DNA序列。虽然副突变在20世纪50年代首次被描述，并在20世纪60年代得到了广泛的研究，但其潜在机制多年来一直是个谜。在过去的十年中，玉米b1基因座的副突变被证明需要位于b1上游100 kb的转录的非编码串联重复序列。这些重复序列产生小RNA，并且在转录水平介导小RNA沉默的多个基因中的突变防止副突变。虽然潜在的机制是共享的，目前的RNA介导的转录沉默模型是基于S。粟酒裂殖酵母和拟南芥不能解释副突变的许多方面。在这份手稿中，我们使用了转基因的方法来证明，b1非编码串联重复序列足以发送和响应副突变信号，即使重复序列不在其正常的染色体位置，这种情况也会发生。
Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele. Paramutation is a fascinating process in which genes communicate to efficiently establish changes in their expression that are stably transmitted to future generations without any changes in DNA sequences. While paramutation was first described in the 1950s and extensively studied through the 1960s, its underlying mechanism remained mysterious for many years. Over the past ten years paramutation at the b1 locus in maize was shown to require transcribed, non-coding tandem repeats located 100 kb upstream of b1. These repeats generate small RNAs, and mutations in multiple genes mediating small RNA silencing at the transcriptional level prevent paramutation. While underlying mechanisms are shared, current models for RNA-mediated transcriptional silencing that are based on experiments with S. pombe and Arabidopsis do not explain many aspects of paramutation. In this manuscript we used a transgenic approach to demonstrate that the b1 non-coding tandem repeats are sufficient to send and respond to the paramutation signals and that this occurs even when the repeats are not at their normal chromosomal location.
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将RNA定向DNA甲基化的两步募集到串联重复序列。
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