Transcriptional factor acetylation in yeast

描述(由申请人提供)：特定基因集合的空间和时间表达对于复杂的分化程序的执行至关重要。分化程序执行不当可能导致发育缺陷、胚胎死亡或癌症。本研究的长期目标是阐明酿酒酵母在减数分裂过程中调节瞬时转录的分子机制。Ume6p通过将Sin3-Rpd3组蛋白脱乙酰酶招募到EMG启动子来抑制有丝分裂中的早期减数分裂基因(EMG)。肌电基因的诱导需要组蛋白乙酰转移酶Gcn5p，强调了蛋白质乙酰化在肌电诱导中的重要性。本研究的重点是Ume6p乙酰化在调节肌电表达中的作用。我们发现，肌电诱导需要Ume6p的破坏。Ume6p的破坏被限制在进入减数分裂的细胞中，这表明了一条减数分裂特有的途径，该途径发出了Ume6p降解的信号。在寻找潜在的减数分裂特异性破坏信号时，我们发现Ume6p是GcnSp的底物。转录因子的乙酰化以前还没有在酵母中被证明，并且在哺乳动物系统中只被描述为少数几个因子。为了了解Ume6p乙酰化在调节肌电表达中的作用，我们提出了以下目标：Aim1。确定Ume6p在有丝分裂和减数分裂细胞中的乙酰化模式。要阐明Ume6p乙酰化与肌电抑制相关的作用，第一步是确定在有丝分裂和减数分裂细胞中哪些赖氨酸被乙酰化。我们将使用乙酰化特异性抗体来表征Ume6p在有丝分裂和减数分裂细胞中的肌电启动子处的乙酰化模式。AIM2.研究GcnSp和RpdSp在Ume6p乙酰化和肌电转录中的作用。有丝分裂细胞对Ume6p-Sin3p-Rpd3p相互作用的要求，以及Gcn5p对减数分裂的要求，表明乙酰化对EMG表达的调控至关重要。我们将确定RpdSp和Gcn5p的相反活性是否通过修饰Ume6p来调节EMG的表达。Aim3.确定乙酰化如何拮抗Ume6p依赖的抑制。Ume6p乙酰化可能通过多种功能冗余机制来调节肌电的表达。我们将确定Ume6p乙酰化的功能后果，并将它们与EMG转录联系起来。这项研究的长期目标是确定控制细胞分化的分子机制。使用Ume6p作为通过转录因子乙酰化进行基因调控的模型，我们将了解管理分化程序正确执行的过程。

DESCRIPTION (provided by applicant): The spatial and temporal expression of specific gene sets is critical for the execution of complex differentiation programs. Improper execution of differentiation programs can lead to developmental defects, embryonic death, or cancer. The long-term goal of this research is to elucidate the molecular mechanisms regulating transient transcription during meiosis in the budding yeast S. cerevisiae. Ume6p represses early meiotic genes (EMG) in mitosis by recruiting the Sin3-Rpd3 histone deacetylase to EMG promoters. EMG gene induction requires the histone acetyltransferase Gcn5p highlighting the importance of protein acetylation in EMG induction. This proposal focuses on the role of Ume6p acetylation in regulating EMG expression. We have discovered that Ume6p destruction is required for EMG induction. Ume6p destruction is restricted to cells entering meiosis suggesting a meiosis-specific pathway that signals Ume6p for degradation. While searching for potential meiosis-specific destruction signals, we found that Ume6p is a substrate for GcnSp. The acetylation of transcription factors has not been demonstrated previously in yeast and has only been described for a few factors in mammalian systems. To understand the role that Ume6p acetylation plays in regulating the EMG expression, the following aims are proposed: Aim1. Determine the acetylation pattern of Ume6p in mitotic and meiotic cells. The first step in elucidating the role of Ume6p acetylation as it relates to EMG repression is to determine which lysines are acetylated in mitotic and meiotic cells. We will characterize the acetylation pattern of Ume6p at EMG promoters in mitotic and meiotic cells using acetylation specific antibodies. Aim2. Characterize the role of GcnSp and RpdSp in Ume6p acetylation and EMG transcription. The requirement of the Ume6p-Sin3p-Rpd3p interaction in mitotic cells, coupled with the requirement of Gcn5p for meiosis, show that acetylation is critical for the regulation of EMG expression. We will identify if the opposing activities of RpdSp and Gcn5p regulate EMG expression by modifying Ume6p. Aim3. Determine how acetylation antagonizes Ume6p-dependent repression. Ume6p acetylation may modulate EMG expression using several functionally redundant mechanisms. We will determine the functional consequences of Ume6p acetylation and relate them to EMG transcription. The long term goal of this research is to determine the molecular mechanisms governing cellular differentiation. Using Ume6p as a model for gene regulation via transcription factor acetylation, we will learn about the processes that govern the proper execution of differentiation programs.