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Exploring mechanisms of trinucleotide expansion and studying the normal function of the ataxin-7 yeast ortholog Sgf73

  • Author(s): Mason, Amanda Gayle
  • Advisor(s): La Spada, Albert R
  • et al.
Abstract

In this dissertation I explore trinucleotide repeat instability as well as the characterization of the ataxin-7 protein through the interrogation of its yeast ortholog Sgf73. Upon expansion of its CAG tract, ataxin-7 is known to cause the neurodegenerative disease, spinocerebellar ataxia type 7 (SCA7). Using microarray expression analysis, we sought to identify the genetic basis of regional instability differences between two different brain tissues, the striatum and the cerebellum. These tissues are known to have high and low levels of trinuclotide expansion, respectively. We identified eight candidate genes involved in DNA replication and repair enriched in cerebellum, and validated five - Pcna, Rpa1, Msh6, Fen1, and Lig1. Expression levels of these candidates are significantly higher in the cerebellum than in the striatum, but were not dependent on disease status in a line of transgenic Huntington disease mice. In Chapters 2 and 3, we studied the function of the ataxin-7 yeast ortholog, Sgf73, a member of the deubiquitinase module (DUBm) in the SAGA transcriptional co-activator complex. It is known that the yeast sgf73Δ mutant has an extended replicative life span (RLS). We tested the role of different SAGA complex members in RLS and found that other DUBm mutants have increased RLS, but not to the degree seen in sgf73Δ. Deleting other non-DUBm SAGA components did not extend RLS. Additionally we found that the sgf73Δ RLS extension is dependent on the presence of the histone deacetylase, Sir2, which physically interacts with Sgf73. Lastly, we performed ChIP-Seq to determine the DNA binding locations of Sgf73. We identified 389 genomic regions bound by Sgf73, with an enrichment in the region 5' to ribosomal protein encoding genes. Additionally, 31 of the 389 identified Sgf73 genomic targets had previously been described to be involved in RLS. Using these data and transcriptome data from SAGA-related mutants, we were able to pinpoint several high-confidence Sgf73 targets for further studies on the mechanism of RLS in yeast as well as expanded ataxin-7 mediated neurodegeneration in humans.

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