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Regulation of eukaryotic transcription by modification of chromatin structure


Modification of chromatin is a highly conserved and fundamental mode of transcriptional regulation in eukaryotes. Chromatin structure can be altered by covalent histone modification, variant histone incorporation and nucleosome remodeling. This thesis describes the study of these mechanisms in a model eukaryote, the budding yeast S. cerevisiae. Methylation of histone H3 on lysine 4 (K4) by Set1, the yeast homolog of human leukemia-related methyltransferases MLL1 and ALL1, is enriched along coding sequences and associated with active transcription. In contrast, the histone variant H2A.Z is found exclusively at the two nucleosomes flanking the transcription start site. H2A.Z regulates the spread of Sir-mediated silencing, which was believed to occur only in the vicinity of telomeres. Through the collaborative research detailed within, I identified members of the Set1 complex in a genetic screen for factors that cooperate with H2A.Z to antagonize Sir activity. I found that Set1 and H2A.Z share a genome-wide anti-silencing function that prevents ectopic binding and gene repression by the Sir2/3/4 complex across euchromatin. Exploring such functional redundancies between various chromatin modifiers will be important to fully understand their roles in transcription. Further collaborations led to the discovery of mechanisms that operate upstream of H2A.Z and Set1. H2A.Z interacts with a complex of 13 proteins, including the SWI/SNF-related ATPase Swr1. Swr1 is required for incorporation of H2A.Z into chromatin; loss of Swr1 or H2A.Z results in similar gene expression defects. H3 K4 methylation by Set1 requires histone H2B K123 mono-ubiquitination, which involves the E2 ubiquitin-conjugating enzyme Rad6. The RING-finger protein Bre1 is required for this modification. Rad6 acts on multiple protein targets; this putative E3 ubiquitin-ligase is likely to direct the mono-ubiquitination activity of Rad6 onto H2B. Finally, I have developed a software tool for the analysis of chromatin-profiling data from high-density tiling oligonucleotide microarrays. The use of this tool has shown that Sth1, an essential chromatin-remodeling enzyme and member of the RSC complex, slides nucleosomes around the 5' ends of coding sequences away from their predicted positions at nucleosome-positioning sequences (NPS). Thus nucleosome appear to be positioned by a combination of cis- and trans-acting factors.

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