GENOME INTEGRITY AND PROGRAMMED GENOME REARRANGEMENT IN THE CILIATE OXYTRICHA

Abstract

Ciliates are unicellular eukaryotes that undergo complex programmed genome rearrangements as part of their post-zygotic development and, as such, are long-standing models for studying genome integrity. Oxytricha trifallax has many unique features among ciliate model organisms. It exhibits the greatest number of programmed DNA elimination and translocation events among ciliates, and contains tens of thousands of high copy number nanochromosomes with extremely short telomeres. Similar DNA elimination and translocation events that drive genomic instability and copy number alterations are common occurrences in various types of cancers.

The first chapter of this thesis is a comprehensive overview of ciliate programmed genome rearrangements, with a focus on Oxytricha.

The second chapter focuses on a common hallmark of genomic instability across eukaryotic genomes, extrachromosomal circular DNAs (eccDNAs). Our work demonstrates that rearrangements in Oxytricha involve the production of copious amounts of eccDNA that originate from repetitive as well as nonrepetitive loci, and are a source of noncoding RNAs. Using an established eccDNA deep-sequencing pipeline, we perform the first genome-wide study of circular DNA in programmed genome rearrangements and identify thousands of eccDNAs in Oxytricha. We also demonstrate eccDNA transcription, suggesting that the circularly eliminated DNA is a source of novel ncRNAs. Our findings yield mechanistic insight into the process of DNA elimination during Oxytricha’s elaborate genome rearrangement. Together with recent findings that implicate a role for eccDNA transcription in humans, our findings also set the stage for a new understanding of extrachromosomal circular DNA, not just as a consequence of genome instability, but as biologically active molecules with important regulatory capacity.

The third chapter focuses on another hallmark of genomic instability across eukaryotic genomes, dysfunctional telomeres. Defective telomeres may contribute to senescence and catastrophic chromosomal rearrangements. In this chapter, we uncover a new role for the DNA repair protein complex Ku70/80 at the telomeres in ciliates. This complex has previously been shown to be involved in ciliate programmed genome rearrangements. We show that Ku70/80 is an important component for capping the naturally short telomeres of the ciliate Oxytricha and is required to prevent the loss of the telomeric G-rich tail and telomere-to-telomere fusions. We propose that the Ku heterodimer is an important telomere capping protein that is required for maintaining telomere integrity in Oxytricha likely by preventing the shortening of the G-rich tail.