The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis [RNA-seq_med]

ARABIDOPSIS THRITHORAX-RELATED PROTEINS 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements, chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, upregulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6 null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels. Overall design: Total RNA was extracted from four-week-old rosette leaves grown on soil under long day conditions. Two to three replicates from seperate plants were collected for each genotype. RNA was extracted using Direct-zol RNA Miniprep kit (Zymo).

拟南芥Trithorax相关蛋白5（ATXR5）与ATXR6是拟南芥中组蛋白H3第27位赖氨酸单甲基化（H3K27me1）沉积以及基因组稳定性维持所必需的蛋白。降低ATXR5/6的活性会激活DNA损伤应答基因，同时伴随转座元件的组织特异性去抑制、染色质中心解聚，以及以异染色质过量DNA积累为特征的基因组不稳定。目前尚不清楚ATXR5/6缺失与H3K27me1水平下降如何导致上述表型。
本研究通过全面分析该突变体的基因表达与表观遗传变化，对atxr5/6功能减弱型突变体进行了系统表征。研究发现，该atxr5/6突变体中转座元件去抑制与过量DNA积累的组织特异性表型，与功能减弱型ATXR6等位基因残留的ATXR6表达水平相关。然而，DNA损伤基因的上调并不依赖ATXR6的表达水平，因此这一过程似乎是独立的。
本研究还分离得到了atxr6无效等位基因，实验结果表明ATXR5与ATXR6对于雌性生殖系发育是必需的。最后，本研究对此前报道的3种atxr5/6功能减弱型突变体的抑制子进行了表征，发现这些抑制子通过不同机制挽救atxr5/6突变表型，其中两种机制涉及提升H3K27me1的水平。
实验设计：从长日照条件下培养于土壤中的4周龄莲座叶中提取总RNA。每个基因型设置2~3次生物学重复，样本取自不同植株。RNA提取采用Direct-zol RNA迷你制备试剂盒（Zymo公司）。