The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N⁶-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

中文翻译：

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动态 DNA N6-腺嘌呤甲基化 (6mA) 控制着单细胞真核生物 Pseudocohnilembus persalinus 中的包囊过程

单细胞真核生物中常见的休眠囊肿的形成是一种复杂且高度调控的生存策略，以对抗环境压力，涉及剧烈的生理和生化变化。尽管大多数研究都集中在囊肿的形态和结构上，但人们对控制这一过程的分子机制知之甚少。最近的研究表明，DNA N ⁶ -腺嘌呤甲基化（6mA）可以响应外部刺激而动态变化；然而，其在调节囊肿形成中的潜在作用仍然未知。我们使用纤毛虫Pseudocohnilembus persalinus来研究 6mA 在滋养体和囊肿中的动态模式，这种纤毛虫很容易诱导形成囊肿。单分子实时 (SMRT) 测序揭示了营养体中高水平的 6mA，在包囊中减少，同时对称 6mA 转变为不对称 6mA。进一步分析表明，6mA作为转录活跃的标志，通过6mA水平的变化和6mA对称到不对称的转换，参与改变成膜相关基因的表达。最重要的是，我们发现通过敲低 DNA 6mA 甲基转移酶 PpAMT1 来降低 6mA 水平可加速囊肿形成。总之，我们描述了P. persalinus的全基因组 6mA 景观，并深入了解 6mA 在真核生物环境应激下基因调控中的作用。我们提出6mA作为活性转录的标志来调节包囊过程以及对称到不对称的转换，为从6mA修饰的角度理解分子对环境信号的反应提供了重要信息。