Collaborative Research: Discrete and Topological Models for Template-Guided Genome Rearrangements

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One of the breakthroughs of 21st century science is the precise genome editing that is enabled through RNA templates. The societal impact of understanding template-guided genome rearrangement is potentially enormous, both as a natural phenomenon that, when mis-guided, can lead to disease, and that if harnessed could lead to the next generation of tools for genome editing. This project seeks to learn from both theory and experiments how a mechanism for RNA-templated DNA recombination sculpts the assembly of genetic information in certain species of ciliates, the organisms with the greatest levels of natural genome editing. Through use of high-throughput experimental tools and novel mathematical concepts based on knot theory and discrete mathematics, the project will gain temporal and structural insight into the process of programmed genome reorganization, including genome-wide surveys of DNA-- DNA and DNA--RNA contacts during somatic development in ciliates. The proposed research will impact postdoctoral, graduate and undergraduate education in mathematics, biology, and chemistry, with at least two postdocs, three PhDs and several senior theses expected to result from this project. Because the ciliate Oxytricha undergoes hundreds of thousands of programmed DNA rearrangements during development, more than any other known organism, we use Oxytricha and its close relatives as a tractable lab model to study template-guided genome rearrangements. This project increases our understanding of template-guided chromosomal DNA rearrangements, both during the process of nuclear development and across the evolutionary steps that gave rise to scrambled genomes. Previous studies have shown that the general mechanism that orchestrates this rearrangement process is guided by maternal RNA templates that can be mathematically modeled by spatial graphs, while the rearrangement pathways can be modeled as paths in a directed acyclic graph whose vertices are the spatial graphs. The project approaches the problem through the lens of the intermediate molecules, as well as the higher level interactions that arise during rearrangement, and it will examine both experimentally and theoretically the global rearrangement pathways. Specific aims include: 1. Experimental dissection of the temporal order of events and the formation of DNA-- DNA and DNA--RNA interactions during genome rearrangement. 2. Build mathematical methods for genome rearrangements using graph based descriptions of the changes in the intermediates. 3. Develop mathematical models to measure differences in underlying scrambling patterns, as well as differences in possible rearrangement pathways between different species. 4. Initiate mathematical techniques based on graph theory, knot theory and algebraic topology that provide understanding of the structural patterns of the genome unscrambling pathways.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.