Specific Tandem Repeats Are Sufficient for Paramutation-Induced Trans-Generational Silencing

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.

副突变（paramutation）是一种被广泛研究的表观遗传现象，指两种不同等位基因之间发生反式通讯，导致其中一个等位基因产生减数分裂可遗传的转录沉默。在b1基因座（b1 locus）上的副突变涉及RNA介导的转录沉默，且需要能够产生小干扰RNA（siRNA）的特定串联重复序列。本研究解答了三个关键问题：1）串联重复序列是否足以介导副突变？2）它们是否需要处于等位基因位置才能介导副突变？3）介导副突变的能力与重复序列的DNA甲基化水平之间是否存在关联？本研究通过使用多种携带b1串联重复序列的转基因株系实现了副突变，其中包括仅包含重复单元一半序列（413 bp）的串联重复序列的转基因事件，这证明这些序列足以介导副突变，且重复序列进行通讯并不需要处于等位基因位置。此外，转基因串联重复序列可提升玉米中报告基因的表达水平，表明该重复序列包含转录调控序列。转基因介导的副突变需要副突变介导因子1基因，该基因对于内源性副突变是必需的，这提示内源性副突变与转基因介导的副突变均依赖RNA介导的转录沉默通路。尽管所有受试的重复序列转基因均产生了小干扰RNA（siRNA），但并非所有转基因都能诱导副突变，这表明与内源性等位基因的情况一致，仅产生siRNA并不足以介导副突变。由转基因重复序列诱导的沉默传递效率低于内源性b1等位基因重复序列诱导的沉默，后者的传递效率始终为100%。转基因重复序列诱导的沉默强度存在差异，这使得我们能够检验重复序列内的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并不需要转基因序列内发生高水平的DNA甲基化。不过，在转基因诱导副突变后，内源性b1重复序列内的DNA甲基化水平升高，与内源性等位基因的更强沉默存在关联。