Studies on the Evolution of Silencing in Budding Yeasts Using Comparative Genomics
- Author(s): Ellahi, Aisha
- Advisor(s): Rine, Jasper D.
- et al.
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
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/α-cell type, but rather
weakly repressed or de-repressed. Thus, my work has expanded the set of known a/α-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.