Studies on the Establishment of Transcriptional Silencing in Yeast
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Epigenetic silencing refers to a transcriptionally inactive state, which involves the formation, maintenance and heritable transmission of heterochromatin, in concert with cell-cycle progression. It plays an important role in growth and development in eukaryotes ranging from yeast to human. My dissertation research concentrated on this process with budding yeast as the model system.I have studied how silencing is established in concert with cell-cycle progression. In S. cerevisiae, the two cryptic mating type loci, HML and HMR, are transcriptionally silent. Previous studies on one locus (HMR) identified an S-phase requirement for establishment of silencing, but couldn't explain the underlying mechanism. Although the other locus (HML) was assumed to be the same, I found it didn't impose such a requirement. That is, silencing could be partially established at HML without passage through S phase. Further analysis identified the promoters at these loci as the cause of this difference. Experiments with modified HM loci containing transcription units of different promoter strength demonstrated the competition between transcription and silencing: the stronger the promoter, the more resistant it is to silencing. This competition can be overcome by passage through S phase.Another part of my study focused on how the silent information regulator 1 (Sir1) facilitates the establishment of silencing. Current understanding suggests that the origin recognition complex (ORC) recruits Sir1, and Sir1, in turn, recruits other Sir proteins to the silencers, the cis-regulatory sites flanking the silent region. I have analyzed the distribution of Sir1 on silent chromatin and its interaction with other proteins both in vitro and in vivo. Sir1 contains an N-terminal domain which is homologous to its well-characterized C-terminal domain. My studies on the Sir1 N-terminal domain showed that, although it is not necessary for complementation of sir1 null mutants, it is important for protein stability and suppression of certain silencing defects. Using the known structure of the Sir1 C-terminal domain, I predicted the structure of the Sir1 N-terminal domain and tested its interaction with the Sir3 BAH domain.As an independent project of my dissertation research, I have investigated the cyclin-dependent kinase Bur1 and its interacting partners. A two-hybrid screen for Bur1 interacting proteins was carried out. Various assays were used to characterize the function of such interactions in the regulation of transcription, mRNA maturation and export.