Polycomb Group (PcG) proteins are part of an epigenetic cell memory system that plays essential roles in multicellular development, stem cell biology, X chromosome inactivation, and cancer. In animals, plants, and many fungi, Polycomb Repressive Complex 2 (PRC2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to assemble transcriptionally repressed facultative heterochromatin. PRC2 is structurally and functionally conserved in the model fungus Neurospora crassa, and recent work in this organism has generated insights into PRC2 control and function. To identify components of the facultative heterochromatin pathway, we performed a targeted screen of Neurospora deletion strains lacking individual ATP-dependent chromatin remodeling enzymes. We found the Neurospora homolog of IMITATION SWITCH (ISW) is critical for normal transcriptional repression, nucleosome organization, and establishment of typical histone methylation patterns in facultative heterochromatin domains. We also found that stable interaction between PRC2 and chromatin depends on ISW. A functional ISW ATPase domain is required for gene repression and normal H3K27 methylation. ISW homologs interact with accessory proteins to form multiple complexes with distinct functions. Using proteomics and molecular approaches, we identified three distinct Neurospora ISW-containing complexes. A triple mutant lacking three ISW accessory factors and disrupting multiple ISW complexes led to widespread up-regulation of PRC2 target genes and altered H3K27 methylation patterns, similar to an ISW-deficient strain. Taken together, our data show that ISW is a key component of the facultative heterochromatin pathway in Neurospora, and that distinct ISW complexes perform an apparently overlapping role to regulate chromatin structure and gene repression at PRC2 target domains.

Polycomb Group (PcG) 蛋白是表观遗传细胞记忆系统的一部分，在多细胞发育、干细胞生物学、X 染色体失活和癌症中发挥重要作用。在动物、植物和许多真菌中，Polycomb Repressive Complex 2 (PRC2) 催化组蛋白 H3 赖氨酸 27 (H3K27me3) 的三甲基化以组装转录抑制的兼性异染色质。PRC2 在模型真菌粗糙脉孢菌中的结构和功能上是保守的，最近在该生物体中的工作已经对 PRC2 的控制和功能产生了见解。为了识别兼性异染色质通路的成分，我们对缺乏单个 ATP 依赖性染色质重塑酶的脉孢菌缺失菌株进行了靶向筛选。我们发现了脉孢菌IMITATION SWITCH (ISW) 的同源物对于在兼性异染色质结构域中的正常转录抑制、核小体组织和典型组蛋白甲基化模式的建立至关重要。我们还发现PRC2和染色质之间的稳定相互作用取决于ISW。基因抑制和正常 H3K27 甲基化需要功能性 ISW ATPase 结构域。ISW 同源物与辅助蛋白相互作用形成具有不同功能的多种复合物。使用蛋白质组学和分子方法，我们确定了三种不同的脉孢菌属含 ISW 的复合物。缺乏三个 ISW 辅助因子并破坏多个 ISW 复合物的三重突变体导致 PRC2 靶基因的广泛上调并改变 H3K27 甲基化模式，类似于 ISW 缺陷菌株。总之，我们的数据表明，ISW 是脉孢菌中兼性异染色质途径的关键组成部分，并且不同的 ISW 复合物在调节 PRC2 靶域的染色质结构和基因抑制方面发挥着明显重叠的作用。