RNA interference (RNAi)-related mechanisms participate in diverse epigenetic phenomena. Few are more extreme than the genome remodeling of the ciliate Tetrahymena thermophila. This organism eliminates nearly 15 megabases of its germline DNA from the somatic nucleus during its development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how ~6000 DNA segments, called internal eliminated sequences (IESs), are selectively excised is challenged by the fact that they share little common structure. The current model is built on the observations that bi-directional IES transcription leads to the generation of ~28 nucleotide RNA molecules (scan RNAs) that then target specific modification(s) to the homologous chromosomal location(s). The DNA rearrangement machinery recognizes this modified chromatin state and eliminates the targeted DNA segment. These studies will certainly provide fundamental insight into RNAi-related mechanisms that direct chromatin modifications that are critical for transcriptional gene silencing and heterochromatin formation in eukaryotes. The specific intellectual goals of this project are: 1) Identification of general cis-requirements for IES excision that should define basic constraints imposed on RNAi-directed chromatin modification. 2) Characterization of the specificity of germline, non-genic transcription that is postulated to be the initiating event in this remarkable genome reorganization. 3) Continued characterization of Dicer-like, RNAseIII homologues of Tetrahymena and their putative partner proteins. This will lead to elucidation of their roles in DNA rearrangement and/or other RNAi-related processes. The plan is to accomplish these goals through a combination of genetic and molecular biological approaches, taking specific advantage of tools available for studies in this model organism. Underlying this project is a goal to understand how RNA molecules can communicate genetic information between the parental and developing genomes, which has great potential to reveal novel roles for RNA in epigenetic programming. Additionally, because it is believed that many of the DNA segments targeted for elimination are important for germline chromosome structure, an increased understanding of how the cell specifically recognizes these sequences will contribute to the current knowledge base of mechanisms that ensure the chromosome stability that is essential to prevent aberrant rearrangements.
The ability of RNAs with sequences complementary to genomic DNA to regulate the activity of the eukaryotic genome has been revealed by recent studies of diverse biological processes, including genome rearrangements of Tetrahymena. The unique biology of this organism offers an excellent context which with to uncover the fundamental mechanisms by which such RNAs elicit their action on the DNA. In addition to providing such insights into this important genetic regulatory mechanism, this project will serve to train undergraduates, post-baccalaureate laboratory technicians, and PhD students in hypothesis-driven research and prepare them for future scientific careers. Underrepresented minorities in science have been trained using prior support and this continued support enables future mentoring. The research is directly linked to the further development of a laboratory course in which undergraduate students engage in original, sophisticated research. This course's problem-based approach teaches students how to use current technologies in a model organism to generate a thorough understanding of the biological process being explored. Gene silencing vectors that will be developed in this project will be incorporated into the design of future offerings of this course, providing a broader impact of the project's intellectual pursuits allowing direct carryover into its educational goals. The research tools and curriculum generated by this project, such as these vectors for gene inhibition studies, will be extremely valuable reagents that will be distributed to the broader Tetrahymena research and education community. Thus these efforts will help make this important model organism more accessible to biologists from other fields.
