Role of Inositol Polyphosphates in Kinetochore Function and Transcription

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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.