IMITATION SWITCH is required for normal chromatin structure and gene repression in PRC2 target domains

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 new 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 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 upregulation 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.

多梳蛋白家族（Polycomb Group, PcG）是表观遗传细胞记忆系统的组成部分，在多细胞发育、干细胞生物学、X染色体失活及癌症发生过程中发挥不可或缺的作用。在动物、植物与多数真菌中，多梳抑制复合体2（Polycomb Repressive Complex 2, PRC2）可催化组蛋白H3赖氨酸27三甲基化（H3K27me3），进而组装形成转录抑制型兼性异染色质。PRC2在模式真菌粗糙脉孢菌（Neurospora crassa）中结构与功能均保守，近期针对该模式生物的研究已为阐明PRC2的调控机制与功能提供了新见解。为鉴定兼性异染色质通路的新组分，我们对缺失单个ATP依赖型染色质重塑酶的粗糙脉孢菌缺失菌株开展了靶向筛选。研究发现，粗糙脉孢菌的IMITATION SWITCH（ISW）同源蛋白对于正常转录抑制、核小体组织以及兼性异染色质区域典型组蛋白甲基化模式的建立至关重要。我们同时发现，PRC2与染色质的稳定相互作用依赖于ISW蛋白。具有功能活性的ISW ATP酶结构域是实现基因抑制与H3K27甲基化所必需的。ISW同源蛋白可与辅助蛋白结合，形成多种具有不同功能的复合物。通过蛋白质组学与分子生物学手段，我们鉴定出三种不同的粗糙脉孢菌ISW复合物。缺失三种ISW辅助因子、破坏多种ISW复合物的三重突变体，会导致PRC2靶基因广泛上调，并改变H3K27甲基化模式，其表型与ISW缺陷菌株一致。综上，本研究数据表明，ISW是粗糙脉孢菌兼性异染色质通路的关键组分，且不同的ISW复合物发挥功能上的重叠作用，共同调控PRC2靶区域的染色质结构与基因抑制。