Chromatin Dynamics and Transcription Regulation in the Animal Model Eukaryote Tetrahymena

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The unicellular ciliate protozoan Tetrahymena thermophila is an animal model organism that has proven very useful for cell and molecular studies addressing basic biological questions. Nobel Prize winning studies on telomeres and telomerase and on catalytic RNA and ribozymes were done with Tetrahymena. Additional fundamental studies include the initial description of a histone modifying enzyme as a transcription factor opening molecular analysis of epigenetics, establishing the role of the linker histone H1, and much more. Both the somatic and germinal genome sequences are available and have facilitated molecular and genetic analysis of fundamental biological processes. ***A unique biological feature of Tetrahymena, is nuclear dimorphism with both a transcriptionally active polyploid somatic nucleus (MAC) and a mostly transcriptionally silent diploid germinal nucleus (MIC) in a common cytoplasm. This provides a powerful model for addressing questions about regulation of gene expression focusing on the different chromatin configuration in the two structurally and functionally distinct nuclei. Extensive RNA-directed genome reorganization occurs during the development of the somatic nucleus involving irreversible genome silencing and is a model for RNA involved gene silencing. ***Research in my lab in 50 years of continuous NRC/NSERC funding has contributed to addressing fundamental biological questions using molecular, cell, and genetic approaches with Tetrahymena, the development of technologies important for these studies, and for the training of HQP. The long term goal of my research program is to address questions about epigenetic regulation of gene expression and RNA-mediated irreversible gene silencing and developmental genome reorganization by exploiting the different chromatin states of the two nuclei in a common cytoplasm. Short term goals will focus on three aims in order of priority; 1) chromatin dynamics and epigenetics with a tChd chromatin remodeler protein family member (chromodomain helicase-DNA-binding; ATP-dependent helicases of the SNF2 superfamily), candidate DNA and RNA binding proteins (DRBP), involved in the developmental RNAi pathway of irreversible gene silencing and genome reorganization. We will use gene knockouts, RNAi gene knockdowns, and CRISPR/Cas and RNAi screens of Chd encoding genes, structural analysis of Chd proteins, analysis of Chd proteins binding to histone post translational modifications, and RNA binding properties of Chd proteins; 2) an additional aim will be comparative proteomic analysis of MAC and MIC proteins; and 3) interactome studies of histones, chromatin remodeling proteins, and proteins involved in transcription and its regulation. Addressing these defined aims will provide significant insights and impact the important fundamental question of how RNAs and epigenetics orchestrate chromatin dynamics in complex eukaryotic regulatory mechanisms and provide an excellent training environment for HQP. **