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  • Thumbnail for Identifying Mutations That Affect the TFIIIC-Dependent Pathway of Heterochromatin Boundary Function in S. pombe
    Identifying Mutations That Affect the TFIIIC-Dependent Pathway of Heterochromatin Boundary Function in S. pombe by Roth, Robert Killion

    Heterochromatin boundary elements inhibit the spread of repressive histone methylation through gene coding regions to prevent the silencing of nearby genes. Two parallel and redundant pathways are responsible for the function of heterochromatin boundaries in the fission yeast, Schizosacchromyces pombe; A pathway that involves TFIIIC, a transcription factor that associates with specific DNA elements, and a pathway that involves Epe1, a Jmjc domain-containing protein enriched at heterochromatin boundaries. Although TFIIIC and Epe1 are known to regulate heterochromatin boundaries, their mechanisms of action are still relatively unknown. To elucidate the proteins involved in the TFIIIC-dependent pathway of boundary function, chemical mutagenesis was employed using a reporter strain that reads out boundary function and lacks the Epe1-dependent boundary pathway. Mutants that exhibited impaired TFIIIC-dependent boundary function were sequenced to identify individual point mutations in four unique genes, sda1, cog5, dpb2, and byr3. To test if these genes play a role in TFIIIC-dependent boundary function, a CRISPR/Cas9 system was engineered to target wildtype genes in the Epe1-deficient reporter strains and reintroduce the identified mutations. The CRISPR/Cas9 plasmids were successfully amplified with sgRNA inserts capable of targeting Cas9 to sda1+, cog5+, dpb2+, and byr3+. The plasmids and sgRNA sequences were confirmed by CspCI restriction enzyme digest and Sanger sequencing. These plasmids will be transformed into S. pombe to generate reporter strains harboring each mutation, which can be used to verify if these mutations impair TFIIIC-dependent boundary function.