Studies on the Evolution of Silencing in Budding Yeasts Using Comparative Genomics

Regional promoter-independent gene silencing is critical in the establishment of cellular/nidentity in Saccharomyces. Domains of transcriptionally silent regions in the genome are/nassociated with certain heritable modifications made to chromatin, such as histone/nhypoacetylation and methylation. In Saccharomyces cerevisiae, this type of gene repression/noccurs through the activity of the four Silent Information Regulator, or SIR genes (SIR1-4). From/nan evolutionary perspective, the SIR genes are unique: except for SIR2, all are specific to/nbudding yeasts. Many other organisms, from Schizosaccharomyces pombe to human, utilize the/nRNA interference (RNAi) pathway, whereas most budding yeasts lack this pathway entirely./nInterestingly, SIR1, SIR3, and SIR4 are also rapidly evolving among Saccharomyces yeasts,/nproviding a model by which to examine the essential principles governing successful silencing/nacross various species and the relationship between rapid sequence evolution and evolution of/nfunction./nTo examine the relationship between gene duplication, extreme sequence divergence, and/nfunctional evolution, I studied the SIR1 gene in S. cerevisiae and its most ancestral paralog,/nKOS3, in the pre-whole-genome-duplication budding yeast, Torulaspora delbrueckii. T./ndelbrueckii also possesses genes for RNAi, AGO1 and DCR1, allowing us the possibility of/nexploring how the evolutionary divergence of RNAi and SIR silencing occurred. In the process, I/ndeveloped genetic tools for T. delbrueckii. To fully characterize SIR1 function in S. cerevisiae/nand SIR gene function in T. delbrueckii, I utilized chromatin immunoprecipitation followed by/ndeep-sequencing (ChIP-Seq) of tagged Sir proteins in both species. This strategy allowed for the/ndiscovery of potential novel functions, as well, revealing functions that may have been gained or/nlost throughout SIR1’s evolution. To identify loci that were directly repressed by Sir proteins, I/nalso generated whole-transcriptome data by performing mRNA-Seq on wild-type and sir mutants/nin both species./nCollectively, these data revealed that though SIR1 in both species is still involved in/nsilencing, its role in that process has dramatically shifted. Previous data suggested that SIR1 is/nprimarily associated with the establishment or nucleation phase of silencing and not involved in/ntelomeric silencing. The Sir1 ChIP data in S. cerevisiae corroborated this assessment. In T./ndelbrueckii, however, KOS3 was essential for silencing, and was also found at telomeres. Thus,/nSir1 in its early evolution had a more essential role in silencing; this role may have changed due/nto the duplication and diversification of the other Sir complex members. This diversification may be contributing to the continual change in interactions between Sir1 and other Sir complex/nmembers across budding yeasts, leading to different mutant phenotypes in each species. Assays/nof silencer function in T. delbrueckii answered critical questions about when in the phylogeny/nimportant shifts in transcription factor binding sites took place. My work showed that the arrival/nof the Rap1, ORC, and Abf1 binding sites in the silencers of budding yeasts took place prior to/nthe whole-genome duplication event. Analysis of silencer structure also revealed the diversity of/nchromatin architecture in budding yeasts: S. cerevisiae silent mating type loci have two silencers/non either side of each locus, whereas in T. delbrueckii, there appears to be a single silencer on/none side of each mating type locus. Transcriptome analysis of RNAi mutants revealed that this/npathway in T. delbrueckii does not function in heterochromatic gene silencing, suggesting that/nthis pathway has already been repurposed for some other biological process./nThe examination of whole-transcriptome data in S. cerevisiae in conjunction with the/nenrichment patterns of the Sir proteins at telomeres allowed us to evaluate widely accepted/nmodels regarding the molecular architecture of heterochromatin and expression at S. cerevisiae/ntelomeres. I established that repression of gene expression at native telomeres is not as/nwidespread as previously thought, and that many genes in proximity to regions of Sir protein/nenrichment were, in fact, expressed just as equally in wild type as they were in sir mutant genetic/nbackgrounds. However, twenty-one genes were convincingly repressed by Sir proteins,/nhighlighting the complex and individual nature of native telomeres and subtelomeric genes. The/nsensitivity of RNA-Seq also uncovered a previously under-appreciated class of haploid-regulated/ngenes: genes that were not fully repressed or de-repressed in the diploid a/α-cell type, but rather/nweakly repressed or de-repressed. Thus, my work has expanded the set of known a/α-regulated/ngenes in S. cerevisiae. In conclusion, this dissertation has broadened our understanding of the/nfunctional constraints dictating silencing gene evolution across species that diverged prior to and/nafter the whole-genome-duplication event. My data speaks to the actual chromatin architecture/nand expression state of native S. cerevisiae telomeres, leading to the refinement of existing/nmodels and an appreciation for how heterogeneous these regions of the genome can be.

Advisor: Rine, Jasper D.