SAGA催化的Ada3乙酰化调控基因表达的机制研究

The SAGA (Spt-Ada-Gcn5 acetyltransferase) is a group of transcriptional coactivators that facilitate gene expression, making them essential in cell growth and development. One important mechanism that SAGA regulates gene expression is catalyzing histone modifications. Nonetheless, because of the structural and functional complexity of both SAGA and chromatin, a detailed understanding of SAGA-mediated histone modifications and gene activation is lacking. In an effort to understand how SAGA promotes gene transcription, we have performed preliminary biochemical and molecular genetic studies on the SAGA complex from budding yeast. These studies have revealed a number of new aspects of SAGA action, including the discovery that 1) SAGA can acetylate its subunit Ada3 at three major lysine residues, 2) Ada3 acetylation promotes SAGA dimerization, 3) Ada3 acetylation is required for gene expression and cell growth under stress conditions. Based on these data, we proposed that SAGA could acetylate Ada3 to enhance its activity and facilitate gene transcription. To accomplish this goal, we propose the following three aims: 1) investigate how SAGA acetylates Ada3 and how Ada3 acetylation is regulated in budding yeast, 2) define the role that Ada3 acetylation plays in promoting SAGA dimerization and enzymatic activity, 3) elucidate how Ada3 acetylation and SAGA dimerization promote gene transcription. Given that SAGA is highly conserved from yeast to mammals, we will examine Ada3 acetylation in cancer cells and analyze its effect on SAGA activity, gene expression and cancer cell growth. We expect that these studies will not only reveal a novel mechanism for SAGA to regulate its activity and gene expression, but also lead to a better understanding of aberrant SAGA function in disease.

SAGA是一种高度保守的转录辅助复合体，通过调控基因表达在细胞生长发育中起重要作用。SAGA调控基因表达的机制之一是催化组蛋白修饰。由于SAGA和染色体结构的复杂性，关于 SAGA调控组蛋白修饰和基因表达的具体机制知之甚少。申请人前期发现SAGA可催化自身亚基 Ada3的乙酰化，乙酰化的Ada3可改变SAGA的结构以及调控基因表达，提示Ada3乙酰化可能通过影响SAGA的结构来调控基因表达。本项目拟在上述发现的基础上分别在酿酒酵母和肿瘤细胞里开展如下研究：（1）通过体外酶反应、蛋白质质谱、定点突变等技术研究SAGA催化Ada3乙酰化及其调控机制；（2）分析Ada3乙酰化对SAGA结构和活性的影响，通过RNA测序、荧光定量RT-PCR等技术分析Ada3乙酰化对基因表达和细胞生长的影响。上述研究的开展将揭示SAGA调控自身活性以及基因表达的新机制，为了解癌症中的基因异常表达提供重要线索。

SAGA是一种高度保守的转录辅助复合体，通过调控基因表达在细胞生长发育中起重要作用。SAGA调控基因表达的机制之一是催化组蛋白修饰。由于SAGA和染色体结构的复杂性，关于SAGA调控组蛋白修饰和基因表达的具体机制知之甚少。前期发现SAGA可催化自身亚基Ada3的乙酰化，乙酰化的Ada3可改变SAGA的结构以及调控基因表达，提示Ada3乙酰化可能通过影响SAGA的结构来调控基因表达。本项目利用体外酶反应、蛋白质质谱、定点突变等技术鉴定了SAGA的亚基Gcn5可催化Ada3乙酰化，且乙酰化位点是K8, K14和K182，进一步研究发现Ada3乙酰化受糖代谢压力调控。通过免疫共沉淀、密度梯度离心以及负染等实验手段，确定了Ada3乙酰化对SAGA二聚体的形成是必须的，增加Ada3乙酰化水平，SAGA二聚体的程度也会增加，其乙酰转移酶活性也高于单体。最后，Ada3乙酰化通过促进SAGA的二聚化来促进染色体上组蛋白的乙酰化水平，促进基因表达，促进细胞在以蔗糖作为碳源的培养基里生长。.上述研究工作揭示了SAGA通过自身乙酰化感应糖代谢压力，正协同催化核小体组蛋白乙酰化并调控基因表达，最终使细胞适应代谢压力的新机制。相关成果发表在Nature Structural & Molecular Biology、J Genet Genomics和BBA-Gene Regulatory Mechanisms上。该课题的顺利实施为今后进一步研究SAGA调控组蛋白修饰和基因表达奠定了良好的基础，为糖代谢偶联表观遗传调控的相关研究提供了重要的线索。