Mechanisms of Heterochromatin Silencing in Saccharomyces cerevisiae
- Author(s): Steakley, David Lee
- Advisor(s): Rine, Jasper
- et al.
Sir proteins are responsible for maintaining stable transcriptional repression and silencing of telomeres, rDNA, and silent mating-type loci in S. cerevisiae. Decades of research identify a thorough list of the proteins required to establish and maintain silencing at HML and HMR, but despite many models being put forward, the mechanism by which Sir-proteins repress transcription has not been definitively determined. I sought to determine the mechanism of transcriptional silencing within heterochromatin in yeast.
I tested models of transcriptional silencing using a variety of orthogonal in vivo approaches to build a more comprehensive model of the mechanism of silencing by Sir proteins. Specifically, I assayed for RNA Pol II in an engaged, DNA-melted form at the promoter of HMLα1 to conclusively test predictions of the downstream-inhibition model. I then adapted the single-subunit RNA polymerase of T7 phage and its cognate promoter to test the ability of a silenced template to be transcribed by a heterologous transcription assay in vivo. Finally, I adapted the thoroughly studied GAL1 promoter and Gal4 activator to test whether the extent of repression by Sir-proteins at HMR is sensitive to the nature of the transcription factor or to the promoter being repressed.
These studies provided a framework for understanding the mechanism of Sir-protein repression. I propose a dynamic affinity-based competition model for transcriptional silencing where Sir proteins primarily restrict transcription before RNA Polymerase II has melted DNA at its target and competition between Sir proteins and gene-specific activators at promoters determines the efficiency of silencing.