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.

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）中的含基因区域。外界应激可诱导适应性应答，促成异染色质岛形成，使存活的野生型谱系获得抗性。本研究探究了应激条件下Epe1的调控机制。咖啡因或抗真菌药物处理会导致Epe1发生泛素化（ubiquitylation），并经蛋白酶体（proteasome）依赖途径切除其N端150个残基，生成截短型tEpe1并使其在细胞质中积累。组成型表达tEpe1会提升多个染色体区域的H3K9甲基化水平，下调下游基因的表达并增强抗性。反之，组成型表达不可切割型Epe1则会降低细胞抗性。tEpe1介导的抗性依赖于具有功能活性的JmjC去甲基化酶结构域（JmjC demethylase domain）。此外，咖啡因诱导的Epe1向tEpe1的切割过程依赖于完整的细胞完整性MAP激酶应激信号通路（cell-integrity MAP kinase stress signalling pathway），该通路的突变会改变细胞的抗性水平。据此，环境变化可触发一种机制，削弱这一关键表观遗传修饰因子（epigenetic modifier）的功能，使异染色质得以重编程基因表达，从而赋予种群内部分细胞抗性。H3K9me异染色质组分在人类以及现有抗真菌药物种类有限的农作物致病真菌中保守存在。本研究揭示了瞬时依赖异染色质的抗真菌抗性表观突变（epimutations）的形成机制，为对抗此类抗性提供了理论依据。