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The impact of subtelomeric DNA on epigenetic switching in Saccharomyces cerevisiae

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Title: The impact of subtelomeric DNA on epigenetic switching in Saccharomyces cerevisiae
Author: Abbasi, Sanna
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Yankulov, Krassimir
Abstract: Gene expression is regulated through multiple mechanisms including epigenetic means, such as chromatin structure. Chromatin structure is heritable and ensures the proper transmission of gene expression programs to future daughter cells. However, switches in the expression state of a gene are observed in many organisms. While epigenetic switching is evident in various organisms, crucial for human cell differentiation, and implicated in multiple diseases, the underlying mechanism for such conversions is not fully understood. To study epigenetic switching in Saccharomyces cerevisiae, I specifically looked at the subtelomere of chromosome VII. Because these regions undergo random epigenetic switches about once per 20 generations, genes typically display both the active or silenced state. I used the Telomere Position Effect (TPE) variegation assay, previously established in our lab, to determine the frequency of switches of a reporter gene inserted at this subtelomere. Previously we have shown that two genes, CAC1 and RRM3, affect the frequency of such conversions. In these earlier studies, we used a reporter construct that removed the natural subtelomeric elements found at chromosome VII. In the current study, I attempted to identify the role these removed elements play in epigenetic conversions by using the TPE variegation assay with both the standard reporter construct and a modified reporter construct that includes the elements. Using the standard reporter, I confirmed the involvement of two additional genes in epigenetic switching, SAS2 and, very mildly, RTT106. Meanwhile, the addition of subtelomeric elements appeared to increase the frequency of conversions from the active to silent state in all tested strains and matched previous silencing data, however it appeared to have no impact on conversions from the silent to active state. I noted substantial concerns regarding this assay when using the modified reporter construct, particularly when selecting for the silenced state. Using PCR, I deduced that the incidence of false positives with the modified construct was enhanced compared to the standard construct. This enrichment was particularly evident when selecting for the initially silent state of the modified reporter construct. These false positives could be occurring due to the prolonged presence of the integrating construct in a non-integrated form, made possible by the origin of replication sequences within the subtelomeric elements. In the future, to understand the role subtelomeric DNA may play in epigenetic switching, the assay will need to be re-optimized to reduce the number of false positives.
Date: 2016-06
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