Department of Physiological Chemistry, Butenandt Institute and LMU Biomedical Center, Faculty of Medicine, Ludwig Maximilians University of Munich, Butenandtstrasse 5, 81377 Munich, Germany.
Nature. 2013 Jul 4;499(7456):111-4. doi: 10.1038/nature12242. Epub 2013 May 22.
Facilitates chromatin transcription (FACT) is a conserved histone chaperone that reorganizes nucleosomes and ensures chromatin integrity during DNA transcription, replication and repair. Key to the broad functions of FACT is its recognition of histones H2A-H2B (ref. 2). However, the structural basis for how histones H2A-H2B are recognized and how this integrates with the other functions of FACT, including the recognition of histones H3-H4 and other nuclear factors, is unknown. Here we reveal the crystal structure of the evolutionarily conserved FACT chaperone domain Spt16M from Chaetomium thermophilum, in complex with the H2A-H2B heterodimer. A novel 'U-turn' motif scaffolded onto a Rtt106-like module embraces the α1 helix of H2B. Biochemical and in vivo assays validate the structure and dissect the contribution of histone tails and H3-H4 towards Spt16M binding. Furthermore, we report the structure of the FACT heterodimerization domain that connects FACT to replicative polymerases. Our results show that Spt16M makes several interactions with histones, which we suggest allow the module to invade the nucleosome gradually and block the strongest interaction of H2B with DNA. FACT would thus enhance 'nucleosome breathing' by re-organizing the first 30 base pairs of nucleosomal histone-DNA contacts. Our snapshot of the engagement of the chaperone with H2A-H2B and the structures of all globular FACT domains enable the high-resolution analysis of the vital chaperoning functions of FACT, shedding light on how the complex promotes the activity of enzymes that require nucleosome reorganization.
染色质转录辅助因子（FACT）是一种保守的组蛋白伴侣，它在 DNA 转录、复制和修复过程中重新组织核小体并确保染色质的完整性。FACT 的广泛功能的关键是其对组蛋白 H2A-H2B 的识别（参考文献 2）。然而，组蛋白 H2A-H2B 如何被识别以及这如何与 FACT 的其他功能（包括对组蛋白 H3-H4 和其他核因子的识别）整合在一起的结构基础尚不清楚。在这里，我们揭示了来自嗜热毛壳菌的进化上保守的 FACT 伴侣结构域 Spt16M 与 H2A-H2B 异二聚体复合物的晶体结构。一个新颖的“U 型转弯”基序架在 Rtt106 样模块上，包围 H2B 的α1 螺旋。生化和体内测定验证了结构，并剖析了组蛋白尾部和 H3-H4 对 Spt16M 结合的贡献。此外，我们报告了连接 FACT 和复制聚合酶的 FACT 异二聚化结构域的结构。我们的结果表明，Spt16M 与组蛋白发生了几种相互作用，我们认为这些相互作用允许该模块逐渐侵入核小体并阻断 H2B 与 DNA 的最强相互作用。FACT 会通过重新组织核小体组蛋白-DNA 接触的前 30 个碱基对来增强“核小体呼吸”。我们捕捉到的伴侣与 H2A-H2B 的结合以及所有球状 FACT 结构域的结构，使我们能够对 FACT 的重要伴侣功能进行高分辨率分析，揭示了该复合物如何促进需要核小体重排的酶的活性。
