Transcriptional factor acetylation in yeast

描述（由申请人提供）：特定基因集的时空表达对于复杂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减数分裂过程中瞬时转录调控的分子机制。Ume6p通过募集Sin3-Rpd3组蛋白去乙酰化酶到EMG启动子来抑制有丝分裂中的早期减数分裂基因（EMG）。肌电信号基因诱导需要组蛋白乙酰转移酶Gcn5p，这突出了蛋白乙酰化在肌电信号诱导中的重要性。本研究主要关注Ume6p乙酰化在肌电图表达调控中的作用。我们发现EMG诱导需要破坏Ume6p。Ume6p的破坏仅限于进入减数分裂的细胞，这表明有减数分裂特异性途径向Ume6p发出降解信号。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没有在酵母中被证明，并且只在哺乳动物系统中被描述为少数因素。为了解Ume6p乙酰化在调节肌电图表达中的作用，提出以下目标：测定有丝分裂和减数分裂细胞中Ume6p的乙酰化模式。阐明Ume6p乙酰化与肌电抑制作用的第一步是确定有丝分裂细胞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表征有丝分裂和减数分裂细胞中EMG启动子上Ume6p的乙酰化模式。Aim2。表征GcnSp和RpdSp在Ume6p乙酰化和肌电图转录中的作用。有丝分裂细胞需要Ume6p-Sin3p-Rpd3p相互作用，再加上减数分裂需要Gcn5p，表明乙酰化对EMG表达的调节至关重要。我们将确定RpdSp和Gcn5p的相反活性是否通过修饰Ume6p来调节肌电图表达。Aim3。确定乙酰化如何拮抗ume6p依赖性抑制。Ume6p乙酰化可能通过几种功能冗余机制调节肌电图表达。我们将确定Ume6p乙酰化的功能后果，并将其与肌电图转录联系起来。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，我们将了解控制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.