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Thesis Advisor: Marc R. Gartenberg, Ph.D.
Graduate Program of Cellular and Molecular Pharmacology
RWJMS Research Tower
4th floor conference room
Wednesday, December 3, 2008
In Saccharomyces cerevisiae, transcription silencing of entire domains occurs at the HM mating-type loci, telomeres and the rDNA. While specific protein contribute to the process at each of the three sites, only Sir2 is required for silencing at all of them. Sir2 is the founding member of an evolutionarily conserved family of NAD-dependent protein deacetylases. At the mating-type loci where the actions of Sir2 are best characterized, silencing is thought to occur in two steps. In the first step, Sir2 is thought to nucleate with Sir3 and Sir4 on DNA by binding cis acting elements known as silencers. In the second step, deacetylation of adjacent histone tails by Sir2 is thought to create additional binding sites for Sir3 and Sir4. Through further cycles of histone deacetylation and histone binding, the Sir proteins are thought to spread to create a silenced chromosomal domain. A byproduct of the Sir2 reaction, O-acetyl-ADP-ribose (OAADPr), is thought to aid spreading by binding one of the Sir proteins.
I have investigated these models for Sir2 action by attempting to replace Sir2 with heterologous deacetylases. If histone deacetylation is the only function of Sir2 in silencing, then a heterologous deacetylase should be able to substitute if it is targeted appropriately. If OAADPr is essential for silencing, then only NAD-dependent deacetylases should be able to substitute for Sir2. I have used a non-conventional approach to deliver heterologous deacetylases by tethering them to Sir3. As a proof of principle, I began this study by creating fusions between Sir3 and a fragment of Sir2 that retains the histone deacetylase domain but lacks a targeting domain. I then made fusions between Sir3 and Hos3, an NAD-independent deacetylase that has no known functions in silencing. Both chimeras restored silencing to the mating-type loci and telomeres in sir2 null strains. I found this form of silencing to be quantitatively comparable to native silencing, to rely on typical cofactors of silenced chromatin, and to be accompanied by the histone modifications associated with silencing. Remarkably, the Sir3-Hos3 chimera functioned in cells lack all of the known OAADPr producing enzymes, indicating that silencing can be achieved in the absence of the metabolite. I conclude that OAADPr is not required for silencing, and that the only function of Sir2 in silencing is to generate deacetylated histone tails.
I extended my studies with protein chimeras to test the importance of deacetylation by Sir2 in other situations where Sir2 is known to act. I used a Sir2 chimera, in which the core deacetylase domain was replaced with Hos3, to target a heterologous deacetylase to the rDNA. I found that histone deacetylation by Hos3 was sufficient for silencing at the NTS1 rDNA site but that it this wasn’t sufficient for rDNA stability. I concluded that NTS1 silencing can be uncoupled from rDNA stability. I also used the Sir3-Hos3 chimera to study a relatively unexplored relationship between Sir2 and Cdc6, a regulator of DNA replication initiation. I found that Sir3-Hos3 genetically interacts with cdc6-1 just like Sir2. This suggests that histone deacetylation is the activity that underscores the interaction, and that Sir3 delivers Sir2 in the events that are genetically coupled to Cdc6.
Initially, I have used the chimera approach to probe the importance of motifs in other Sir proteins. Specifically, the C-terminal region of Sir3 contains an AAA ATPase-like domain. I show that this domain is dispensable in silencing when the Sir2 deacetylase domain is fused to the N-terminal portion of Sir3. This indicates that the N-terminal domain executes the critical functions of Sir3 in silencing, and that the function of Sir3 might be limited to Sir2 recruitment. Collectively, these experiments offer a new way to think about the contributions of Sir2 in transcriptional silencing.