Functional And Comparative Genomics Of Saccharomyces And Non Saccharomyces Yeasts Potential For Industrial And Food Biotechnology PDF Download

Regional promoter-independent gene silencing is critical in the establishment of cellular identity in Saccharomyces. Domains of transcriptionally silent regions in the genome are associated with certain heritable modifications made to chromatin, such as histone hypoacetylation and methylation. In Saccharomyces cerevisiae, this type of gene repression occurs through the activity of the four Silent Information Regulator, or SIR genes (SIR1-4). From an evolutionary perspective, the SIR genes are unique: except for SIR2, all are specific to budding yeasts. Many other organisms, from Schizosaccharomyces pombe to human, utilize the RNA 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 silencing across various species and the relationship between rapid sequence evolution and evolution of function. To examine the relationship between gene duplication, extreme sequence divergence, and functional 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 of exploring how the evolutionary divergence of RNAi and SIR silencing occurred. In the process, I developed genetic tools for T. delbrueckii. To fully characterize SIR1 function in S. cerevisiae and SIR gene function in T. delbrueckii, I utilized chromatin immunoprecipitation followed by deep-sequencing (ChIP-Seq) of tagged Sir proteins in both species. This strategy allowed for the discovery of potential novel functions, as well, revealing functions that may have been gained or lost throughout SIR1's evolution. To identify loci that were directly repressed by Sir proteins, I also generated whole-transcriptome data by performing mRNA-Seq on wild-type and sir mutants in both species. Collectively, these data revealed that though SIR1 in both species is still involved in silencing, its role in that process has dramatically shifted. Previous data suggested that SIR1 is primarily associated with the establishment or nucleation phase of silencing and not involved in telomeric 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 due to 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 members across budding yeasts, leading to different mutant phenotypes in each species. Assays of silencer function in T. delbrueckii answered critical questions about when in the phylogeny important shifts in transcription factor binding sites took place. My work showed that the arrival of the Rap1, ORC, and Abf1 binding sites in the silencers of budding yeasts took place prior to the whole-genome duplication event. Analysis of silencer structure also revealed the diversity of chromatin architecture in budding yeasts: S. cerevisiae silent mating type loci have two silencers on either side of each locus, whereas in T. delbrueckii, there appears to be a single silencer on one side of each mating type locus. Transcriptome analysis of RNAi mutants revealed that this pathway in T. delbrueckii does not function in heterochromatic gene silencing, suggesting that this pathway has already been repurposed for some other biological process. The examination of whole-transcriptome data in S. cerevisiae in conjunction with the enrichment patterns of the Sir proteins at telomeres allowed us to evaluate widely accepted models regarding the molecular architecture of heterochromatin and expression at S. cerevisiae telomeres. I established that repression of gene expression at native telomeres is not as widespread as previously thought, and that many genes in proximity to regions of Sir protein enrichment were, in fact, expressed just as equally in wild type as they were in sir mutant genetic backgrounds. However, twenty-one genes were convincingly repressed by Sir proteins, highlighting the complex and individual nature of native telomeres and subtelomeric genes. The sensitivity of RNA-Seq also uncovered a previously under-appreciated class of haploid-regulated genes: genes that were not fully repressed or de-repressed in the diploid a/[alpha]-cell type, but rather weakly repressed or de-repressed. Thus, my work has expanded the set of known a/[alpha]-regulated genes in S. cerevisiae. In conclusion, this dissertation has broadened our understanding of the functional constraints dictating silencing gene evolution across species that diverged prior to and after the whole-genome-duplication event. My data speaks to the actual chromatin architecture and expression state of native S. cerevisiae telomeres, leading to the refinement of existing models and an appreciation for how heterogeneous these regions of the genome can be.