A balancing act: Interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans. A balancing act: Interactions within NuA4/TIP60 regulate picNuA4 function in Saccharomyces cerevisiae and humans

The NuA4 lysine acetyltransferase complex acetylates histone and non-histone proteins and functions in transcription regulation, cell cycle progression, and DNA repair. NuA4 harbors an interesting duality in that its catalytic module can function independently and distinctly as picNuA4. At the molecular level, picNuA4 anchors to its bigger brother via physical interactions between the C-terminus of Epl1 and the HSA domain of Eaf1, the NuA4 central scaffolding subunit. This is reflected at the regulatory level, as picNuA4 can be liberated genetically from NuA4 by disrupting the Epl1-Eaf1 interaction. As such, removal of either Eaf1 or the Epl1 C-terminus offers a unique opportunity to elucidate the contributions of Eaf1 and Epl1 to NuA4 biology and in turn their roles in balancing picNuA4 and NuA4 activities. Using high-throughput genetic and gene expression profiling, and targeted functional assays to compare eaf1∆ and epl1-C∆ mutants, we found that EAF1 and EPL1 had both overlapping and distinct roles. Strikingly, loss of EAF1 or its HSA domain led to a significant decrease in the amount of picNuA4, while loss of the Epl1 C-terminus increased picNuA4 levels, suggesting starkly opposing effects on picNuA4 regulation. The eaf1∆ epl1-C∆ double mutants resembled the epl1-C∆ single mutants, indicating that Eaf1’s role in picNuA4 regulation depended on the Epl1 C-terminus. Key aspects of this regulation were evolutionary conserved, as truncating an Epl1 homolog in human cells increased the levels of other picNuA4 subunits. Our findings suggested a model in which distinct aspects of the Epl1-Eaf1 interaction regulated picNuA4 amount and activity. Overall design: Microarray expression profiling was performed in duplicate. Cultures were grown in a 24-well plate incubator/reader to mid-log phase, with spiked-in RNA controls to monitor global changes in mRNA levels. Since no global changes in mRNA levels were detected, expression values were normalized to total mRNA levels. Fold changes were determined by comparing mutant to wild type profiles, with differentially expressed genes having a p-value 1.7 . Please note that the protocol_details.txt contains all protocol description.

NuA4赖氨酸乙酰转移酶复合物（NuA4 lysine acetyltransferase complex）可乙酰化组蛋白与非组蛋白，参与转录调控、细胞周期进程与DNA修复。该复合物存在有趣的双重特性：其催化模块可作为picNuA4独立且独特地发挥功能。在分子层面，picNuA4通过Epl1的C端与NuA4中央支架亚基Eaf1的HSA结构域之间的物理相互作用，锚定在其更大的同源复合物上。这一特性在调控层面也有所体现：通过破坏Epl1与Eaf1的相互作用，可从遗传层面将picNuA4从NuA4中解离出来。因此，敲除Eaf1或Epl1的C端，为阐明Eaf1与Epl1对NuA4生物学功能的贡献，以及它们在平衡picNuA4与NuA4活性中的作用提供了独特契机。本研究通过高通量遗传与基因表达谱分析、靶向功能实验，对比分析eaf1∆与epl1-C∆突变体，发现EAF1与EPL1既存在功能重叠，也具有独特的调控角色。值得注意的是，敲除EAF1或其HSA结构域会导致picNuA4的含量显著降低，而敲除Epl1的C端则会提升picNuA4的水平，这表明二者对picNuA4的调控存在截然相反的效应。eaf1∆ epl1-C∆双突变体的表型与epl1-C∆单突变体相似，这说明Eaf1对picNuA4的调控作用依赖于Epl1的C端。该调控机制的关键特征具有进化保守性：在人类细胞中截短Epl1的同源蛋白，同样会提升其他picNuA4亚基的水平。本研究的结果提出了一个模型：Epl1-Eaf1相互作用的不同方面，分别调控picNuA4的含量与活性。整体实验设计：采用重复实验进行微阵列表达谱分析。将细胞培养于24孔板培养箱/读数仪中，至对数中期，并加入掺入的RNA对照以监测mRNA水平的整体变化。由于未检测到mRNA水平的全局变化，因此将表达值标准化至总mRNA水平。通过对比突变体与野生型的表达谱计算倍数变化，差异表达基因的筛选阈值为p值1.7。请注意，protocol_details.txt中包含完整的实验方案描述。