Grech, L; (2018) Exploring the functional landscape of the fission yeast genome via Hermes transposon mutagenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In general, the non-protein-coding dark matter of eukaryotic genomes remains poorly understood. Neither diversity studies, comparative genomics, nor the biochemical outputs allow fine-scale descriptions of the genomic elements that are required for an organism to grow. Transposon mutagenesis offers an alternative approach to locate functional regions. In principle, insertion mutants are created in large pools, and mutants harbouring harmful insertions are quantitatively removed from the population. Subsequent sequencing of mutant libraries should reveal functionality in regions with fewer insertions. Transposon mutagenesis works well in bacteria. We applied the Hermes transposon system to locate functional regions in the Schizosaccharomyces pombe, or fission yeast genome. We created multiple dense insertion libraries, during log phase growth and chronological ageing, achieving a saturating (or near-saturating) insertion density of 1 insertion per 13 nucleotides of the genome for log phase samples. To account for the complexity and stochastic nature of the data, we applied a five-state hidden Markov model (HMM) that includes generalised linear models to account for systematic insertion biases (e.g. nucleosomes). The HMM state provides a semi-quantitative estimate of the functional significance of the genome at single nucleotide-level resolution. The HMM state values are strikingly consistent (but more detailed than) genome annotations. Here, we show that transposon insertions have functional consequences in 90% of the genome, including 80% of the non-protein-coding regions. Specifically, we discover 85 essential ncRNAs during vegetative growth, and 218 during ageing. We also find 54 pro-ageing and 136 anti-ageing genes. Overall, this functional annotation map distinguishes sub gene-level genomic segments that have differential effects on cell survival, and so will have extensive utility to the community.

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