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Role of Inositol Polyphosphates in Kinetochore Function and Transcription

肌醇多磷酸在着丝粒功能和转录中的作用

基本信息

批准号：
7011303
负责人：
Ales Vancura
金额：
$ 24.75万
依托单位：
ST. JOHN'S UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2009-12-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of our laboratory is to identify and characterize eukaryotic cellular processes regulated by phospholipase C (PLC), an enzyme which plays vital roles in signal transduction pathways. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] to produce two important second messengers: inositol 1,4,5-trisphosphate [lns(1,4,5)P3] and diacylglycerol (DAG). Since Plc1p (encoded by PLC1 gene) is the only PLC enzyme in the budding yeast Saccharomyces cerevisiae and hydrolysis of Ptdlns(4,5)P2 is the only pathway for synthesis of lns(1,4,5)P3 and other inositol polyphosphates (InsPs), cells with deletion of PLC1 gene (plc1-delta) are completely devoid of all InsPs and S. cerevisiae thus represents an ideal system in which to study metabolism and cellular roles of InsPs. The central hypothesis of this proposal is that InsPs play important role(s) in nuclear events, such as chromosome segregation/cell cycle progression and transcriptional regulation. Our results demonstrate that plc1-delta cells display alterations in the structure of core centromeric chromatin, higher frequency of chromosome loss, and mitotic delay (Lin et al., 2000; DeLillo et al., 2003). The mechanism of Plc1p's involvement in kinetochore function, not apparent at first, was suggested by recent work that showed that InsPs regulate the activity of chromatin remodeling complexes (Shen et al., 2003; Steger et al., 2003) and that the RSC complex, one of the chromatin remodelers, associates with centromeric loci and is important for high fidelity chromosome segregation (Hsu et al., 2003). The Specific Aim 1 of this proposal will test the hypothesis that Plc1p and InsPs affect recruitment, integrity, or other aspects of RSC function at the kinetochore. Genetic experiments and genome-wide expression analysis indicate that in addition to chromatin remodeling, InsPs affect transcription also by a mechanism independent of chromatin remodeling complexes. The Specific Aim 2 will determine whether Plc1p and InsPs affect transcriptional regulation of stress-induced genes by affecting recruitment of Msn2p/Msn4p activators, Tup1p/Ssn6p represser complex, or Srb10p component of the Srb/mediator complex. The Specific Aim 3 will utilize the well-studied GAL1 promoter as a model and determine, why plc1-delta cells fail to express GAL1 gene. The focus of this proposal is to elucidate the role of Plc1p and InsPs in two very important aspects of cell physiology: chromosome segregation/cell cycle progression and transcriptional regulation, processes that are misregulated in human disease, including cancer. The results of this work will ultimately contribute to identification of novel molecular targets for cancer therapy.

描述（由申请人提供）：我们实验室的长期目标是鉴定和表征由磷脂酶C（PLC）调节的真核细胞过程，磷脂酶C是一种在信号转导途径中起重要作用的酶。PLC水解磷脂酰肌醇4，5-二磷酸[Ptdlns（4，5）P2]产生两种重要的第二信使：肌醇1，4，5-三磷酸[lns（1，4，5）P3]和甘油二酯（DAG）。由于Plc 1 p（由PLC 1基因编码）是芽殖酵母Saccharomyces cerevisiae中唯一的PLC酶，Ptdlns（4，5）P2的水解是合成lns（1，4，5）P3和其它肌醇多磷酸（InsPs）的唯一途径，因此缺失PLC 1基因的细胞（plc 1-delta）完全缺乏InsPs和S.因此，酿酒酵母是研究InsPs代谢和细胞作用的理想系统。该建议的中心假设是InsPs在核事件中发挥重要作用，例如染色体分离/细胞周期进程和转录调控。我们的结果表明plc 1-δ细胞显示核心着丝粒染色质结构的改变、更高频率的染色体丢失和有丝分裂延迟（Lin等人，2000; DeLillo等人，2003年）。Plc 1 p参与动粒功能的机制最初并不明显，但最近的研究表明InsPs调节染色质重塑复合物的活性，从而提出了Plc 1 p参与动粒功能的机制（Shen等人，2003; Steger等人，2003），并且RSC复合物（染色质重塑剂之一）与着丝粒基因座相关并且对于高保真染色体分离是重要的（Hsu et al.，2003年）。本提案的具体目标1将检验Plc 1 p和InsPs影响动粒RSC功能的招募、完整性或其他方面的假设。遗传实验和全基因组表达分析表明，除了染色质重塑，InsPs影响转录也通过一种机制，独立于染色质重塑复合物。具体目标2将确定Plc 1 p和InsPs是否通过影响Msn 2 p/Msn 4p激活剂、Tup 1 p/Ssn 6p阻遏物复合物或Srb/介体复合物的Srb 10 p组分的募集来影响应激诱导基因的转录调控。具体目标3将利用充分研究的GAL 1启动子作为模型，并确定为什么plc 1-delta细胞不能表达GAL 1基因。该提案的重点是阐明Plc 1 p和InsPs在细胞生理学的两个非常重要的方面的作用：染色体分离/细胞周期进展和转录调控，这些过程在人类疾病（包括癌症）中被错误调节。这项工作的结果将最终有助于确定癌症治疗的新分子靶点。