The complex architecture and epigenomic impact of plant T-DNA insertions
The complex architecture and epigenomic impact of plant T-DNA insertions
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DOI：
10.1371/journal.pgen.1007819
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发表时间：
2019-01-01
期刊：
影响因子：
4.5
通讯作者：
Ecker, Joseph R.
中科院分区：
文献类型：
--
作者：
Jupe, Florian;Rivkin, Angeline C.;Ecker, Joseph R.
根癌农杆菌（Agrobacterium tumefaciens）一直是植物基因组工程的主力。天然肿瘤诱导（Ti）质粒元件的定制替换使得能够将称为转移DNA（T-DNA）的感兴趣序列插入任何植物基因组中。虽然这些转移机制是很好的理解，插入事件的结构和表观基因组状态的详细了解是有限的，目前的技术。在这里，我们应用了两种单分子技术，并分析了拟南芥品系从三个广泛使用的T-DNA插入收集（SALK，SAIL和WISC）。四个随机选择的T-DNA系的光学图谱显示了1至7个插入/重排，单个插入的长度为27至236个碱基。两个分离系的基于从头纳米孔测序的组装部分解析了T-DNA结构，并揭示了染色体臂末端的多次易位和交换。对于目前的TAIR 10参考基因组，纳米孔重叠群校正了83%的非着丝粒错误组装。前所未有的连续核苷酸水平分辨率使T-DNA插入位点的表观基因组的深入研究成为可能。SALK_059379系T-DNA插入富集24 nt小干扰RNA（siRNA）和密集胞嘧啶DNA甲基化，导致通过RNA指导的DNA甲基化途径的转基因沉默。相比之下，SAIL_232系T-DNA插入主要由21/22 nt siRNA靶向，DNA甲基化和沉默限于报告基因，而不是抗性基因。此外，我们使用ChIP-seq在SALK_059379、SAIL_232和另外五个T-DNA系中分析了T-DNA插入周围的H3 K4 me 3、H3 K27 me 3和H2A.Z染色质环境。我们发现了各种效应，从染色质标记的完全丢失到H2A.Z的从头掺入以及T-DNA整合位点周围H3 K4和H3 K27的三甲基化。这项研究为将外源片段插入植物基因组的结构影响提供了新的见解，并展示了最先进的远程测序技术在快速识别意料之外的基因组变化方面的实用性。作者总结我们使用转基因技术在植物基因组中添加或改变基因的常规能力已被证明是植物科学的游戏规则改变者。转基因技术不仅使基因功能的研究成为可能，而且使现代作物的发展没有来自自然杂交的不必要的遗传包袱。一个主要的工具，以创造转基因是农杆菌系统，自然穿梭和整合片段的外来DNA到其宿主基因组。虽然整合的位置和数量相对容易追踪，但分子工具从未允许在单个图片中看到整合的DNA片段。在这里，我们利用最先进的DNA测序技术来捕获植物基因组中多个DNA插入事件的大小和结构。我们发现，预期的DNA片段的插入发生多个连接的完整和部分片段，导致在某些情况下，在染色体内和染色体间重排。我们对表观遗传景观的分析表明，从整合的外源DNA沉默到染色质标记的改变，从而改变染色质结构和功能，都有不同的影响。
The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes.Author summary Our routine ability to add or alter genes in plant genomes using transgenesis has proven to be a game changer to plant sciences. Transgenics not only enables the study of gene function but also allows the development of modern crop plants without the unwanted genetic baggage coming from natural crossing. A major tool to create transgenics is the Agrobacterium system which naturally shuttles and integrates pieces of foreign DNA into its host genome. While the position and number of integrations was relatively easy to track, molecular tools never allowed to see the integrated piece of DNA within a single picture. Here we have utilized state-of-the-art DNA sequencing technology to capture the size and structure of multiple DNA insertion events in a plant genome. We discovered that insertion of the anticipated DNA fragment occurred as multiple concatenated full and partial fragments that led in some cases to intra- and interchromosomal rearrangements. Our analysis of the epigenetic landscapes showed variable effects from silencing of the integrated foreign DNA to alterations of chromatin marks and thus chromatin structure and functionality.