Establishment and Regulation of Silenced Chromatin in Saccharomyces Cerevisiae
Heterochromatin, or condensed chromatin, is a transcriptionally repressive form of chromatin that occurs in many eukaryotic organisms. At its natural locations, heterochromatin is thought to play important roles in genome organization as well as gene expression. Just as important is the restriction of this repressive form of chromatin to appropriate regions of the genome. In the budding yeast <italic>Saccaromyces cerevisiae</italic>, domains of condensed, transcriptionally silenced chromatin are found at telomeres and at the silent-mating type cassettes, <italic>HML<italic/> and <italic>HMR</italic>. At these locations, a complex of Silent Information Regulator (SIR) proteins gets recruited to DNA through discrete silencer elements. Once recruited, the Sir protein complex then spreads along chromosomes in a step-wise manner. This process results in the silencing of gene expression. It is unclear whether silenced chromatin is established in the same manner at different genomic locations. Understanding how silenced chromatin is formed is important for determining how these chromatin structures are regulated.
To better understand how silenced chromatin is established in different genomic contexts, I used chromatin immuoprecipitation to follow the rate of silenced chromatin formation at different locations. The rates of Sir protein assembly were compared at two locations, telomere VI-R and <italic>HMR</italic>. I discovered that the silencers at these two locations were equally proficient at recruiting Sir proteins. However, the rate of Sir protein assembly onto nucleosomes was far more rapid at <italic>HMR</italic> than at the telomere VI-R. Furthermore, the rate of Sir protein assembly was more rapid on one side of the <italic>HMR-E</italic> silencer at <italic>HMR</italic> than the other. Moreover, insertion of the <italic>HMR-E</italic> silencer adjacent to the telomere VI-R significantly improved the rate of Sir protein assembly onto nucleosomes. Additionally, observations that the association of Sir protein occurs simultaneously across several kilobases at <italic>HMR</italic> and that silencing at <italic>HMR</italic> is insensitive to co-expression of wild-type and catalytically inactive Sir2 proteins suggest that <italic>HMR-E</italic> enables the assembly of silenced chromatin in a non-linear fashion. These results suggest that <italic>HMR-E</italic> functions to both recruit Sir proteins and promote their assembly across several kilobases.
In addition to the <italic>HMR-E</italic> silencer, <italic>HMR</italic> is also characterized by the presence of a second auxiliary <italic>HMR-I</italic> silencer and a tRNA gene that functions as a boundary element to restrict the spread of silenced chromatin. I used chromatin immunoprecipitation to determine how each of these regulatory elements contribute to the steady-state levels of Sir protein association with chromatin. Consistent with a role for <italic>HMR-E</italic> beyond recruitment, I discovered that the <italic>HMR-E</italic> silencer alone promoted higher levels of Sir proteins on nucleosomes compared to the telomere VI-R. The levels of Sir protein association with <italic>HMR</italic> were further elevated by the <italic>HMR-I</italic> silencer, even though this silencer does not recruit Sir proteins on its own and does not contribute to any of the known functions of silenced chromatin at <italic>HMR</italic>. Additionally, although the tRNA gene did block the spread Sir proteins, I discovered that the capacity for Sir proteins to spread beyond a few kilobases was severely limited even in the absence of the boundary.
The results of this thesis work provide new insights into the mechanisms of silenced chromatin establishment and regulation in budding yeast. I show here that the capacity of Sir proteins to assemble onto nucleosomes is inherently limited. Additionally, silencers vary in their ability to promote this assembly. I conclude that the silencer is a key factor in determining the relative size, efficiency, and location of silenced chromatin domains in the cell.
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