Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.

中文翻译：

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重要的组蛋白伴侣协作调节转录和染色质完整性

组蛋白伴侣对于转录、DNA 复制和 DNA 修复过程中控制染色质完整性至关重要。三个保守的必需伴侣 Spt6、Spn1/Iws1 和 FACT 与延伸 RNA 聚合酶 II 相关，并在物理和/或功能上相互作用；然而，人们对它们各自的功能或它们之间的关系知之甚少。在这项研究中，我们选择了温度敏感的spt6突变的抑制剂，该突变会破坏 Spt6-Spn1 的物理相互作用，并且还会导致转录和染色质缺陷。这一选择发现了 FACT 中的新突变。令人惊讶的是，基于免疫共沉淀、ChIP 和质谱实验，FACT 抑制并没有恢复 Spt6-Spn1 相互作用。此外，FACT 的抑制绕过了 Spn1 的完全丧失。有趣的是，FACT 抑制突变沿着 FACT-核小体界面聚集，表明它们改变了 FACT-核小体相互作用。与这一观察结果一致，我们发现破坏 Spt6-Spn1 相互作用的 spt6 突变导致 FACT 与染色质关联水平升高，而 FACT 抑制剂降低了 FACT-染色质关联水平，从而恢复正常的 Spt6-FACT平衡染色质上。总而言之，这些研究揭示了组蛋白伴侣之间以前未知的调节，这对于它们的基本体内功能至关重要。