Grant: Discovery Grants Program - Individual

The unicellular eukaryotic ciliated protozoa Tetrahymena thermophila is characterized by nuclear dimorphism. The polyploid macronucleus (MAC) functions as the cell's somatic nucleus, divides amitotically and is the site of vegetative transcription. The diploid micronucleus (MIC) containing 5 metacentric chromosomes is the cell's germinal nucleus, transcriptionally silent during vegetative growth. This nucleus undergoes meiosis in the the sexual life cycle (conjugation) leading to programmed genome rearrangement and genome instability. The chromatin state of the two nuclei differ with the somatic MAC being mainly in an open euchromatic state and the MIC being mainly in a closed heterochromatic state. The presence of the two functionally distinct nuclei in the same cytoplasm provides a unique opportunity to address functional consequences of nuclear chromatin configurations. These include transcriptional activity, genome stability including DNA repair as well as the mechanism(s) of histone deposition and chromatin remodeling in the two nuclei. Tetrahymena's complex metazoan-like biology, the completely sequenced somatic and germinal nuclei, extensive proteomic analysis, and microarray and RNA seq analysis of gene expression throughout the life cycle make Tetrahymena a useful and much exploited model system for these basic and applied studies. This request for renewal of 40+ years of NSERC Discovery Grant support focuses on the chromatin state of the independent nuclei and on molecular studies of meiosis approached from the perspective of genome stability and genome rearrangements. Experiments proposed will depend heavily on my very significant recent contributions to Tetrahymena genomics, proteomics, and expression microarray studies with significant support from NSERC Discovery Grant funding. This focus of recent NSERC funding allows the exploitation of these tools in functional analysis to address important and interesting basic and applied questions in meiosis, genome stability, histone deposition and chromatin remodeling, as well as questions about signaling in early conjugation events. Areas of focus are: 1) Meiosis in Tetrahymena does not involve a synaptonemal complex (SC). I propose analysis of proteins as well as protein/protein interactions (the Interactome) of Tetrahymena orthologues of putative SC proteins or of proteins suggested to interact or complex with SCs. This will provide important information about the organization of meiotic chromosomes in the absence of an SC. 2) Studies of genome stability through double strand break (DSB) repair. GFP or other tagged proteins will be localized in MIC during meiosis and in response to treatment with inducers of DSB. Relationships between DSB repair, DSBs in meiosis, and chromosome fragmentation in developmentally programmed genome rearrangements will be addressed. 3) Histone deposition in the MIC focusing on DSB repair and on meiosis. These studies will involve the H3-H4 histone chaperone Asf1p. Affinity purification and mass spectrometry have been used to identify a number of proteins interacting with Asf1. A number of interesting proteins have been identified and functional analysis of some of these will be pursued. Focus will be on proteins that appear to be MIC-specific and may have particular roles in MIC function including meiosis. Computational network analysis of expression data will address questions about protein/protein interactions identified. 4) A novel signaling pathway involving tyrosine phosphorylation induced during cell pairing and early stages of conjugation. Co-immunoprecipitation with anti phosphtyrosine antibodies and mass spectrometry will allow isolation and identification of the phosphorylated molecule(s) involved.