Proteasome-dependent truncation of the negative heterochromatin regulator Epe1 mediates antifungal resistance

Epe1 histone demethylase restricts H3K9-methylation-dependent heterochromatin, preventing it from spreading over, and silencing, gene-containing regions in fission yeast. External stress induces an adaptive response allowing heterochromatin island formation that confers resistance on surviving wild-type lineages. Here we investigate the mechanism by which Epe1 is regulated in response to stress. Exposure to caffeine or antifungals results in Epe1 ubiquitylation and proteasome-dependent removal of the N-terminal 150 residues from Epe1, generating truncated tEpe1 which accumulates in the cytoplasm. Constitutive tEpe1 expression increases H3K9 methylation over several chromosomal regions, reducing expression of underlying genes and enhancing resistance. Reciprocally, constitutive non-cleavable Epe1 expression decreases resistance. tEpe1-mediated resistance requires a functional JmjC demethylase domain. Moreover, caffeine-induced Epe1-to-tEpe1 cleavage is dependent on an intact cell-integrity MAP kinase stress signalling pathway, mutations in which alter resistance. Thus, environmental changes provoke a mechanism that curtails the function of this key epigenetic modifier, allowing heterochromatin to reprogram gene expression, thereby bestowing resistance to some cells within a population. H3K9me-heterochromatin components are conserved in human and crop plant fungal pathogens for which a limited number of antifungals exist. Our findings reveal how transient heterochromatin-dependent antifungal resistant epimutations develop and thus inform on how they might be countered. Overall design: Comparison of H3K9me2 methylation and gene expression in three Spombe geneotypes: epe1D, Epe1-GFP, Epe1DNP150-GFP

Epe1组蛋白去甲基化酶（Epe1 histone demethylase）可限制依赖H3K9甲基化的异染色质（H3K9-methylation-dependent heterochromatin），防止其扩散并沉默裂殖酵母（fission yeast）中携带基因的区域。外界胁迫可诱导适应性应答，促成异染色质岛形成，使存活的野生型谱系获得抗性。

本研究探究了胁迫应答过程中Epe1的调控机制。当菌株暴露于咖啡因或抗真菌药物时，Epe1会发生泛素化（ubiquitylation），并通过蛋白酶体依赖途径切除其N端150个氨基酸残基，产生截短型tEpe1（truncated tEpe1），后者会在细胞质（cytoplasm）中积累。组成型表达tEpe1可提升多个染色体区域的H3K9甲基化水平，降低对应区域基因的表达量并增强抗性。反之，组成型表达不可剪切的Epe1突变体则会降低菌株的抗性。

tEpe1介导的抗性需要具有功能的JmjC去甲基化酶结构域（JmjC demethylase domain）。此外，咖啡因诱导的Epe1向tEpe1的剪切过程依赖于完整的细胞完整性丝裂原活化蛋白激酶（MAP kinase）应激信号通路，该通路的突变会改变菌株的抗性水平。

因此，环境变化可触发一种调控机制，削弱这一关键表观遗传修饰因子的功能，使异染色质重编程基因表达，从而赋予种群内部分细胞抗性。H3K9me-异染色质组分在人类和农作物真菌病原体中保守存在，而目前针对这些病原体的抗真菌药物种类有限。本研究揭示了瞬时依赖异染色质的抗真菌抗性表观突变（epimutation）的产生机制，可为此类抗性的干预策略提供理论依据。

实验整体设计：对比3株裂殖酵母（S. pombe）的H3K9me2甲基化水平与基因表达谱，涉及3种基因型：epe1Δ基因敲除株、带GFP标签的野生型Epe1（Epe1-GFP）以及N端150位氨基酸缺失的GFP标签Epe1突变体（原文标注为Epe1DNP150-GFP）。