Department of Tropical Medicine and Parasitology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
Int J Mol Sci. 2021 Nov 2;22(21):11902. doi: 10.3390/ijms222111902.
persists in a dormant state with a protective cyst wall for transmission. It is incompletely known how three cyst wall proteins (CWPs) are coordinately synthesized during encystation. Meiotic recombination is required for sexual reproduction in animals, fungi, and plants. It is initiated by formation of double-stranded breaks by a topoisomerase-like Spo11. It has been shown that exchange of genetic material in the fused nuclei occurs during encystation, suggesting parasexual recombination processes of this protozoan. possesses an evolutionarily conserved Spo11 with typical domains for cleavage reaction and an upregulated expression pattern during encystation. In this study, we asked whether Spo11 can activate encystation process, like other topoisomerases we previously characterized. We found that Spo11 was capable of binding to both single-stranded and double-stranded DNA in vitro and that it could also bind to the promoters in vivo as accessed in chromatin immunoprecipitation assays. Spo11 interacted with WRKY and MYB2 (named from myeloblastosis), transcription factors that can activate gene expression during encystation. Interestingly, overexpression of Spo11 resulted in increased expression of - and genes and cyst formation. Mutation of the Tyr residue for the active site or two conserved residues corresponding to key DNA-binding residues for Spo11 reduced the levels of and gene expression and cyst formation. Targeted disruption of gene with CRISPR/Cas9 system led to a significant decrease in and gene expression and cyst number. Our results suggest that Spo11 acts as a positive regulator for differentiation into cyst.
它以具有保护囊壁的休眠状态存在，用于传播。目前还不完全清楚在囊形成过程中，三种囊壁蛋白 (CWP) 是如何协调合成的。
有丝分裂重组是动物、真菌和植物进行有性繁殖所必需的。它是由拓扑异构酶样 Spo11 形成双链断裂而引发的。已经表明，在囊形成过程中融合核发生遗传物质交换，这表明这种原生动物存在准性重组过程。
具有进化上保守的 Spo11，具有典型的切割反应结构域，并在囊形成过程中上调表达。在这项研究中，我们询问了 Spo11 是否可以像我们之前表征的其他拓扑异构酶一样激活囊形成过程。我们发现 Spo11 能够在体外结合单链和双链 DNA，并且可以在染色质免疫沉淀测定中在体内结合到 启动子。Spo11 与 WRKY 和 MYB2（分别来自髓样细胞瘤）相互作用，这两种转录因子可以在囊形成过程中激活 基因表达。有趣的是，过表达 Spo11 导致 和 基因的表达增加和囊形成。突变 Spo11 的 Tyr 残基（活性位点）或两个保守残基（对应于 Spo11 的关键 DNA 结合残基）会降低 和 基因表达和囊形成的水平。利用 CRISPR/Cas9 系统靶向敲除 基因会导致 和 基因表达和囊数量显著减少。我们的结果表明，Spo11 作为一种正调控因子，促进 分化为囊。
