Genome rearrangements occur on an evolutionary scale, as well as on a developmental scale, in a wide range of eukaryotes. Even in cancer cells, dramatic DNA rearrangements are frequently observed in somatic cell lineages. This project uses certain species of ciliates as a model system to study gene rearrangements. These organisms undergo massive recombination during differentiation of an archival germline micronucleus into a somatic macronucleus capable of gene expression. The objective of this project is to increase the understanding of this process of assembly, starting from the basic principles of molecular DNA - RNA - enzyme interactions, through general knowledge of the possible pathways and intermediate steps that lead to the final product. Through mutual reinforcement, the project interlaces biological experiments with mathematical findings. The theoretical model utilizes spatial graphs with 4-valent vertices as a physical representation of the DNA at the time of recombination, and smoothing of the vertices models the actual recombination. Specific aims include: (a) examine the interactions of the template RNA and the involvement of possible cutting and splicing enzymes at the moment of recombination, (b) identify steps in the process by pinpointing putative intermediate molecules during unscrambling a variety of genes, and theoretically analyze sets of pointers leading to these intermediates (c) characterize theoretically and confirm experimentally all possible gene unscrambling pathways for a subset of genes, and (d) develop mathematical techniques based on methods from knot theory and formal languages to study the theoretical model.
Our understanding of genome-wide DNA rearrangements is still very limited. These processes appear on an evolutionary scale, where they can lead to species-specific differences and even the creation of species boundaries. On a developmental scale, genome rearrangements appear in a wide range of differentiating eukaryotic cells. The findings of this project will offer insight into the process of gene descrambling in ciliates and may also extend more generally to other model systems. 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 ciliates. This mechanism of template-mediated DNA rearrangement can be visualized as a scaffold for controlling and programming certain types of genetic information within a cell. Such control of information processing could facilitate biomolecular computing in vivo, as well as methods for epigenetic control of development.
In a wide range of species, from unicellular eukaryotes to mammals, genome rearrangements have been observed to occur on both an evolutionary scale, as well as on a developmental scale. Even in cancer cells, dramatic DNA rearrangements frequently occur in somatic cell lineages. Some of the most elaborate genome rearrangements and DNA recombination events appear in a lineage of ciliates, a group of eukaryotic unicellular organisms. This project combined theory and experiments to improve our understanding of the mechanism for RNA-template guided DNA recombination in the assembly of genetic information. An interdisciplinary team of biologists at Princeton and mathematicians at the University of South Florida developed and experimentally tested a theoretical model for RNA-templates guided DNA rearrangement in the single-celled ciliate, Oxytricha trifallax. The team developed a mathematical model based on spatial graphs and knot theory to study these genome rearrangements, which can be highly complex. Experimental sequencing of the germline genome (Chen et al., in review) and comparison to the somatic genome have allowed a detailed mapping of the precursor-product relationships between thousands of scrambled gene loci. This revealed thousands of new scrambled DNA rearrangement patterns, as well as many more discoveries, such as the presence of hundreds of shared or overlapping gene segments. Examination of partially processed molecules during the conversion from precursor to product DNA provided a window into the intricate process of DNA rearrangement Broader impacts include a book edited by Jonoska et al. and published by Springer, review articles, and public lectures given by the PI's, and the broadly cross-disciplinary training and mentorship of several postdoctoral fellows and graduate and undergraduate students at Princeton and USF, as well as a visiting mathematics professor who spent a summer learning hands-on molecular biology experiments in the Princeton laboratory. The USF-Princeton team also developed a variety of computational resources for the community, including STAGR (Software to Annotate Gene Rearrangements; Dolzhenko et al. submitted) and students at USF developed 3-dimentional plastic and wire models to visualize the process of rearrangement, both conceptually and as scientific art (see image).
