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theses

The mechanisms by which epigenetic markings are generated and maintained are crucial to ensuring genome stability and appropriate gene expression levels, therefore understanding how chromatin states are faithfully maintained and propagated is critical. We performed a forward genetic screen in Schizosaccharomyces pombe using integration density profiling of transposable element insertions to identify modifiers of position effect variegation (PEV), an epigenetic phenomenon characterized by heritable yet stochastic heterochromatin spreading over a reporter gene. Interestingly, a number of essential genes including DNA replication and repair factors were enriched within the screen. Transposable element integration sites were subsequently used to guide the generation of new mutant alleles in the essential candidate PEV genes top2, top3, and tel2 using CRISPR-Cas9 mediated mutagenesis; with this approach we were able to isolate mutant alleles in all three genes affecting transcriptional silencing at heterochromatic regions. Finally, we demonstrate that dense transposon integrations may permit bypass of essentiality (BOE) by leveraging complex genetic interactions which suppress lethality in bypassable essential gene mutants. These interactions may facilitate the detection of novel phenotypes occurring as a consequence of essential gene inactivation. Essential genes have long been under-represented using traditional genetic screening approaches; however, a systematic approach utilizing these phenomena could potentially increase their representation in such screens to allow for future discovery and investigation of novel roles for essential genes in a number of vital cellular processes.

ETD_10210

Ph.D.

Includes bibliographical references

doi:10.7282/t3-rshj-8809

ETD doctoral

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