Role of Inositol Polyphosphates in Kinetochore Function and Transcription

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，这种酶在信号转导途径中起着至关重要的作用。PLC水解磷脂酰肌醇4,5-二磷酸[Ptdlns(4,5)P2]，产生两个重要的第二信使：肌醇1,4,5-三磷酸[lns(1,4,5)P3]和二酰基甘油（DAG）。由于Plc1p（由PLC1基因编码）是出芽酵母酿酒酵母中唯一的PLC酶，而Ptdlns(4,5)P2的水解是合成lns(1,4,5)P3和其他肌醇多磷酸（InsPs）的唯一途径，因此PLC1基因缺失的细胞（PLC1 -delta）完全缺乏所有的InsPs，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，如染色体分离/细胞周期进程和转录调控。我们的研究结果表明，plc1-delta细胞表现出核心着丝粒染色质结构的改变，染色体丢失的频率更高，有丝分裂延迟（Lin等，2000；DeLillo等，2003）。Plc1p参与着丝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体的活性（Shen等人，2003；Steger等人，2003），染色质重塑复合体之一RSC复合体与着丝粒位点相关，对高保真染色体分离很重要（Hsu等人，2003）。本提案的具体目标1将检验Plc1p和InsPs影响着丝点上RSC功能的募集、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，除了染色质重塑外，InsPs还通过一种独立于染色质重塑复合物的机制影响转录。Specific Aim 2将确定Plc1p和InsPs是否通过影响Msn2p/Msn4p激活因子、Tup1p/Ssn6p抑制因子复合体或Srb/介质复合体的Srb10p组分的募集来影响应激诱导基因的转录调控。Specific Aim 3将利用已被充分研究的GAL1启动子作为模型，确定plc1-delta细胞不能表达GAL1基因的原因。本提案的重点是阐明Plc1p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/细胞周期进程和转录调节，人类疾病（包括癌症）中被错误调节的过程。这项工作的结果将最终有助于鉴定癌症治疗的新分子靶点。

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.