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

Author(s): Ellahi, Aisha | Advisor(s): Rine, Jasper D. | Abstract: Regional promoter-independent gene silencing is critical in the establishment of cellularidentity in Saccharomyces. Domains of transcriptionally silent regions in the genome areassociated with certain heritable modifications made to chromatin, such as histonehypoacetylation and methylation. In Saccharomyces cerevisiae, this type of gene repressionoccurs through the activity of the four Silent Information Regulator, or SIR genes (SIR1-4). Froman evolutionary perspective, the SIR genes are unique: except for SIR2, all are specific tobudding yeasts. Many other organisms, from Schizosaccharomyces pombe to human, utilize theRNA interference (RNAi) pathway, whereas most budding yeasts lack this pathway entirely.Interestingly, SIR1, SIR3, and SIR4 are also rapidly evolving among Saccharomyces yeasts,providing a model by which to examine the essential principles governing successful silencingacross various species and the relationship between rapid sequence evolution and evolution offunction.To examine the relationship between gene duplication, extreme sequence divergence, andfunctional evolution, I studied the SIR1 gene in S. cerevisiae and its most ancestral paralog,KOS3, in the pre-whole-genome-duplication budding yeast, Torulaspora delbrueckii. T.delbrueckii also possesses genes for RNAi, AGO1 and DCR1, allowing us the possibility ofexploring how the evolutionary divergence of RNAi and SIR silencing occurred. In the process, Ideveloped genetic tools for T. delbrueckii. To fully characterize SIR1 function in S. cerevisiaeand SIR gene function in T. delbrueckii, I utilized chromatin immunoprecipitation followed bydeep-sequencing (ChIP-Seq) of tagged Sir proteins in both species. This strategy allowed for thediscovery of potential novel functions, as well, revealing functions that may have been gained orlost throughout SIR1’s evolution. To identify loci that were directly repressed by Sir proteins, Ialso generated whole-transcriptome data by performing mRNA-Seq on wild-type and sir mutantsin both species.Collectively, these data revealed that though SIR1 in both species is still involved insilencing, its role in that process has dramatically shifted. Previous data suggested that SIR1 isprimarily associated with the establishment or nucleation phase of silencing and not involved intelomeric silencing. The Sir1 ChIP data in S. cerevisiae corroborated this assessment. In T.delbrueckii, however, KOS3 was essential for silencing, and was also found at telomeres. Thus,Sir1 in its early evolution had a more essential role in silencing; this role may have changed dueto 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 complexmembers across budding yeasts, leading to different mutant phenotypes in each species. Assaysof silencer function in T. delbrueckii answered critical questions about when in the phylogenyimportant shifts in transcription factor binding sites took place. My work showed that the arrivalof the Rap1, ORC, and Abf1 binding sites in the silencers of budding yeasts took place prior tothe whole-genome duplication event. Analysis of silencer structure also revealed the diversity ofchromatin architecture in budding yeasts: S. cerevisiae silent mating type loci have two silencerson either side of each locus, whereas in T. delbrueckii, there appears to be a single silencer onone side of each mating type locus. Transcriptome analysis of RNAi mutants revealed that thispathway in T. delbrueckii does not function in heterochromatic gene silencing, suggesting thatthis pathway has already been repurposed for some other biological process.The examination of whole-transcriptome data in S. cerevisiae in conjunction with theenrichment patterns of the Sir proteins at telomeres allowed us to evaluate widely acceptedmodels regarding the molecular architecture of heterochromatin and expression at S. cerevisiaetelomeres. I established that repression of gene expression at native telomeres is not aswidespread as previously thought, and that many genes in proximity to regions of Sir proteinenrichment were, in fact, expressed just as equally in wild type as they were in sir mutant geneticbackgrounds. However, twenty-one genes were convincingly repressed by Sir proteins,highlighting the complex and individual nature of native telomeres and subtelomeric genes. Thesensitivity of RNA-Seq also uncovered a previously under-appreciated class of haploid-regulatedgenes: genes that were not fully repressed or de-repressed in the diploid a/α-cell type, but ratherweakly repressed or de-repressed. Thus, my work has expanded the set of known a/α-regulatedgenes in S. cerevisiae. In conclusion, this dissertation has broadened our understanding of thefunctional constraints dictating silencing gene evolution across species that diverged prior to andafter the whole-genome-duplication event. My data speaks to the actual chromatin architectureand expression state of native S. cerevisiae telomeres, leading to the refinement of existingmodels and an appreciation for how heterogeneous these regions of the genome can be.