Fine mapping and characterization of RLL6 locus required for anti-silencing of a transgene and DNA demethylation in Arabidopsis thaliana.
Fine mapping and characterization of RLL6 locus required for anti-silencing of a transgene and DNA demethylation in Arabidopsis thaliana.
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DOI：
10.3389/fgene.2022.1008700
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发表时间：
2022
影响因子：
3.7
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中科院分区：
文献类型：
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植物中DNA甲基化模式是由DNA甲基化和去甲基化途径的拮抗作用动态塑造的。虽然DNA甲基化途径已经得到了很好的研究，但DNA去甲基化途径迄今尚未完全了解。为了更深入地了解DNA去甲基化途径的机制，我们对参与防止表观遗传基因沉默的蛋白质进行了遗传学筛选，并对同样参与DNA去甲基化途径的蛋白质进行了进一步的研究。最终，回收了具有低荧光素酶发光（低LUC发光）的突变体，并将其命名为降低的LUC发光6-1（r116 -1）。图位克隆结果显示，rll 6 -1突变位于第4号染色体上，该区域内共有10个候选基因。对rll 6 -1突变体全基因组甲基化模式的分析表明，RLL 6位点突变导致拟南芥5条染色体上出现3,863个差异甲基化区域（hyper-DMR），并导致2 × 35 S启动子DNA甲基化水平升高，这与ros 1（repressor of silencing 1）突变体相似。进一步的分析表明，rll 6 -1和ros 1 -7突变体共有1,456个共同的超DMR，表明两种蛋白质以协同方式共同作用以去除DNA甲基化。进一步的研究表明，RLL 6位点的突变并不影响DNA糖基化酶/裂解酶家族的四个基因的表达。因此，我们的研究结果表明，RLL 6基因座编码的蛋白质不仅参与转录抗沉默的转基因，但也参与了DNA去甲基化途径。
DNA methylation patterns in plants are dynamically shaped by the antagonistic actions of DNA methylation and demethylation pathways. Although the DNA methylation pathway has been well studied, the DNA demethylation pathway, however, are not fully understood so far. To gain deeper insights into the mechanisms of DNA demethylation pathway, we conducted a genetic screening for proteins that were involved in preventing epigenetic gene silencing, and then the ones, which were also implicated in DNA demethylation pathway, were used for further studies. Eventually, a mutant with low luciferase luminescence (low LUC luminescence) was recovered, and named reduced LUC luminescence 6–1 (rll6-1). Map-based cloning revealed that rll6-1 mutation was located on chromosome 4, and there were a total of 10 candidate genes residing within such a region. Analyses of genome-wide methylation patterns of rll6-1 mutant showed that mutation of RLL6 locus led to 3,863 hyper-DMRs (DMRs for differentially methylated regions) throughout five Arabidopsis chromosomes, and elevated DNA methylation level of 2 × 35S promoter, which was similar to that found in the ros1 (repressor of silencing 1) mutant. Further analysis demonstrated that there were 1,456 common hyper-DMRs shared by rll6-1 and ros1-7 mutants, suggesting that both proteins acted together in a synergistic manner to remove DNA methylation. Further investigations demonstrated that mutation of RLL6 locus did not affect the expression of the four genes of the DNA glycosylase/lyase family. Thus, our results demonstrate that RLL6 locus-encoded protein not only participates in transcriptional anti-silencing of a transgene, but is also involved in DNA demethylation pathway.
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