Chromatin dynamics in yeast

Chromatin, the physiological packaged state of eukaryotic DNA, plays a complex but generally inhibitory role in the control of gene expression. Nucleosomes, the fundamental unit of chromatin, can occlude transcription factor binding sites and are an obstacle to transcriptional elongation. Further, covalent modifications of histones, the protein components of nucleosomes, can serve as binding sites for transcription promoting or inhibiting proteins. These complex and interdependent interactions between chromatin and the proteins that regulate gene expression are the focus of my thesis research.;Several well studied promoters in yeast lose nucleosomes upon transcriptional activation and gain them upon repression, an observation that has prompted the model that transcriptional activation and repression requires nucleosome remodelling of regulated promoters. I examined global nucleosome positioning before and after glucose-induced transcriptional reprogramming, a condition under which over half of all yeast genes significantly change expression. The majority of induced and repressed genes exhibit no change in promoter nucleosome arrangement, although promoters that do undergo nucleosome remodelling tend to contain a TATA box. Rather, I found multiple examples where the pre-existing accessibility of putative transcription factor binding sites before glucose addition determined whether the corresponding gene would change expression in response to glucose addition. I also investigated the role of the histone chaperone ASF1 in nucleosome remodelling during the glucose response and found that in yeast lacking ASF1 nucleosome remodelling appears to occur with delayed kinetics. My results suggest that the primary role of most nucleosomes may be to occlude spurious instances of transcription factor motifs rather than the regulation of transcription per se.;Yeast contain multiple genomic regions subject to heterochromatic silencing by binding of the repressive SIR protein complex. I investigated the dynamics of silencing in single cells, using fluorescent reporters integrated at the silenced loci HML and HMR. I examined silencing in genetic backgrounds where silencing is weakened, and found that HML and HMR gain and lose silencing independently. In wild type cells, I found that the establishment of silencing takes multiple generations and is stochastic in nature. I also investigated multiple mutant backgrounds where the level of SIR proteins available to repress physiological loci is reduced, either directly or indirectly, and found that these all result in an intermediate silencing state, where transcription is partially but not fully repressed. These data suggest that SIR proteins at least partially act by mass action and demonstrate the silencing is not 'all or nothing'.