Department of Horticultural Sciences, University of Florida, 2550 Hull Road, Fifield Hall, 32611, Gainesville, FL, USA.
Interdisciplinary Center for Biotechnology Research, University of Florida, 2033 Mowry Road, 32610, Gainesville, FL, USA.
Biol Direct. 2024 Apr 30;19(1):33. doi: 10.1186/s13062-024-00476-z.
The Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes.
To provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate.
FENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.
国际空间站上的高级植物实验-04-表观遗传表达（APEX-04-EpEx）实验研究了两种拟南芥遗传系（野生型 Col-0 和突变体 elp2-5）在空间飞行中改变的胞嘧啶甲基化，突变体 elp2-5 缺乏表观遗传调控因子延伸复合物亚基 2（ELP2）。全基因组亚硫酸氢盐测序（WGBS）显示出明显的与空间飞行相关的甲基化差异，这需要探索特定的空间改变的甲基化，以在单个分子分辨率下将特定的变化与与空间飞行相关基因的大片段联系起来。迄今为止，用于植物基因组的多重靶向 DNA 甲基化测序的工具仍然有限。
为了提供单个分子分辨率的甲基化数据，应用了 Flap-enabled 下一代捕获（FENGC），这是一种新型的靶向多重 DNA 捕获和富集技术，允许在任何指定的位点进行切割，用于调查空间飞行改变的基因区域内的 DNA 甲基化。FENGC 捕获面板包含 108 个靶标，范围从启动子或基因体区域的 509 到 704 个核苷酸，这些靶标来自空间飞行全基因组数据集的基因靶标。除了在空间飞行和地面控制之间具有表达和平均甲基化水平显著变化的基因外，还确定了具有空间改变的每个分子中甲基化胞嘧啶比例分布的靶标。此外，在同一 DNA 分子中显示了不同胞嘧啶环境的共甲基化趋势。我们进一步鉴定了三个以前未知生物学过程的基因的显著 DNA 甲基化变化，并且这两个基因中的两个功能丧失突变体（命名为 EMO1 和 EMO2，用于轨道 1 和 2 的 ELP2 调节甲基化）表现出增强的根生长速率。
FENGC 简化并降低了植物中靶向、单个分子甲基化的多重、靶向、单个分子甲基化分析的成本，提供了沿每个 DNA 分子的额外分辨率，这在基于群体的短读数据（如 WGBS）中是看不到的。这项案例研究揭示了在细胞亚群的单个 DNA 分子中发生的空间飞行改变的胞嘧啶甲基化的区域性修饰，而这些修饰在 WGBS 中无法识别。FENGC 的单个分子调查可能导致新的功能基因的鉴定。新鉴定的 EMO1 和 EMO2 是根生长调节剂，它们可能在植物适应空间飞行中涉及表观遗传。
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