From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132.
the Biology Faculty, Lomonosov, Moscow State University, Leninskie Gory 1, Moscow 119992, Russia.
J Biol Chem. 2018 Apr 20;293(16):6121-6133. doi: 10.1074/jbc.RA117.000199. Epub 2018 Mar 7.
The essential histone chaperone FACT (cilitates hromatin ranscription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization , indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A-H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACT's histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.
基本组蛋白伴侣 FACT（促进染色质转录）促进核小体的组装和拆卸。FACT 是 Spt16 与 SSRP1 或 Pob3 形成的异二聚体，主要区别在于 SSRP1 具有高迁移率族 B（HMGB）DNA 结合结构域。酵母 FACT 缺乏 SSRP1 同源物中存在的固有 HMGB 结构域，但酵母 FACT 活性由 Nhp6 支持，Nhp6 是一种独立的、具有单个 HMGB 结构域的蛋白质。FACT 结构域与组蛋白结合的重要性已经确立，但 DNA 结合活性的作用仍知之甚少。在这里，我们通过将单个或多个 HMGB 模块融合到 Pob3 上来模拟 SSRP1 或测试扩展 DNA 结合能力来研究这些作用。人类 FACT 和酵母模拟物都需要 Nhp6 来支持核小体重排，这表明单个固有 DNA 结合 HMGB 模块不足以完全发挥 FACT 活性。三个融合的 HMGB 模块在没有 Nhp6 协助的情况下支持活性，但这种 FACT 变体不能有效地从核小体中释放出来，并且具有毒性。值得注意的是，固有 DNA 结合 HMGB 模块降低了与核小体重排相关的 DNA 可及性和组蛋白 H2A-H2B 二聚体的丢失。我们提出，HMGB 结构域的 DNA 弯曲通过暴露 FACT 的组蛋白结合位点来促进核小体的解稳定和重排，但 DNA 弯曲也产生了容纳核小体组装所需的核小体曲率。因此，固有 DNA 弯曲活性有利于 FACT 组装核小体而不是核小体重排，但过度的活性会损害 FACT 的释放，这表明在核小体组装过程中有一个质量控制检查点。
