Suppressor mutations that make the essential transcription factor Spn1/Iws1 dispensable in Saccharomyces cerevisiae

Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications as an integral member of the RNA polymerase II elongation complex. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated eight suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA co-activator complex, Sgf73. The identification of these distinct groups and their analysis suggests that there are multiple mechanisms by which Spn1 bypass can occur, including changes in histone acetylation and alterations of other histone chaperones. Thus, Spn1 may participate in multiple functions during transcription. Our results suggest that bypass of a subset of these functions allows viability in the absence of Spn1. Overall design: ChIP-seq of histone H4 and histone H4 acetylation, in Spn1-depletion yeast strains with wild-type and Set2 deletion backgrounds.

Spn1/Iws1是一种必需的真核转录延伸因子（transcription elongation factor），在从酵母到人类的所有物种中均保守存在。多项研究证实，作为RNA聚合酶II（RNA polymerase II）延伸复合物的核心组成成员，Spn1以组蛋白伴侣（histone chaperone）的功能调控转录、RNA剪接（RNA splicing）、基因组稳定性（genome stability）及组蛋白修饰（histone modifications）。然而，目前学界对Spn1的确切功能仍不明确，且对其为何是细胞存活所必需的机制知之甚少。
为解决上述问题，我们在酿酒酵母（Saccharomyces cerevisiae）中筛选得到8个可绕过Spn1必需性的抑制突变体（suppressor mutations）。出乎意料的是，这些抑制突变体关联了多种功能迥异的复合物与活性，包括组蛋白伴侣FACT、组蛋白甲基转移酶Set2、Rpd3S组蛋白去乙酰化复合物（Rpd3S histone deacetylase complex）、组蛋白乙酰转移酶Rtt109、核小体重塑因子Chd1，以及SAGA共激活复合物（SAGA co-activator complex）成员Sgf73。
对这些不同功能类群的鉴定与分析表明，存在多种可绕过Spn1必需性的机制，包括组蛋白乙酰化状态的改变以及其他组蛋白伴侣的功能调控。由此可见，Spn1可能在转录过程中参与多种功能。我们的研究结果提示，只要绕过其中部分功能，细胞即可在缺失Spn1的情况下存活。
整体实验设计：针对野生型与Set2缺失背景的Spn1敲降酵母菌株，开展组蛋白H4及乙酰化组蛋白H4的染色质免疫共沉淀测序（ChIP-seq）分析。