<p>Chromatin is composed of DNA, histones, and other proteins and contributes to DNA packaging, controlling gene expression and DNA replication. This work focuses on the contributions of histones H3 and H4 to gene regulation in the yeast <italic>Saccharomyces cerevisiae</italic>. I identified a region of the nucleosome that is critical for three types of long-range transcriptional silencing but not for local repression mediated by some of the same proteins. </p><p>In <italic>S. cerevisiae</italic>, the Sir complex performs long range silencing of the mating type loci, while the promoter specific Sum1 complex represses mid-sporulation genes. Interestingly, the <italic>SUM1-1</italic> mutation changes the Sum1 repression complex into a silencing complex capable of long range spreading. Sum1-1 provides a good model to distinguish between properties of nucleosomes important for long-range silencing (common to Sum1-1 and Sir silencing), and specific interactions nucleosomes might make with the Sum1 complex (common to Sum1 and Sum1-1 complexes). Interactions between nucleosomes and silencing proteins are critical to Sir silencing, and the spreading ability of Sum1-1p suggests that a component of the Sum1-1 complex may also interact with nucleosomes. Since the Sum1-1 and Sum1 complex components are shared, histone contacts may also contribute to wild type Sum1 repression.</p><p>I investigated the contributions of histones H3 and H4 to Sum1-1 silencing and Sum1 repression using a genetic screen. Interestingly, I found histone mutations that disrupt Sum1-1 silencing and cluster in the LRS/H4 region of the nucleosome, which was previously identified to disrupt silencing at the mating type loci, telomeres, and rDNA. Therefore, this region of the nucleosome is important to silencing mediated by three distinct complexes- Sir, RENT, and Sum1-1. The Sir3p bromo-adjacent homology (BAH) domain binds this region of the nucleosome to facilitate Sir spreading and silencing, and I tested Orc1p, a paralog of Sir3p, to determine if it makes similar contributions to Sum1-1 silencing. Using reporter mating assays and chromatin immunoprecipitation, I found that mutations and deletion of the BAH domain of Orc1p disrupt Sum1-1 silencing. These results suggest that Orc1p may interact with this region of the nucleosome and contribute to Sum1-1 silencing outside of recruitment.</p><p>Surprisingly, Sum1 repression was not disrupted by histone mutations. I conducted <italic>in vitro</italic> binding assays to identify a region in Sum1p that may interact with histones and account for the spreading ability of Sum1-1p. Consistent with results that histones do not contribute to Sum1 repression, I did not find evidence of Sum1p binding to histone peptides. Therefore, interactions with histones H3 and H4 are important to Sir and Sum1-1 silencing and not Sum1 repression. These interactions with histones may facilitate the formation of higher order chromatin structures necessary for long range silencing complexes. </p><p>I also identified mutations in the H3 tail that disrupt Sum1-1 silencing. Surprisingly, these mutations did not disrupt the enrichment of Sum1-1p. Similar observations have been made for Sir proteins in the absence of the H3 tail, and the H3 tail may contribute to chromatin compaction and silencing after the assembly of silencing proteins. Therefore, the Sir and Sum1-1 complexes may share several features that facilitate silencing. The use of the LRS/H4 region of the nucleosome may be a common interaction surface with silencing proteins, and the H3 tail may assist in the formation of a specialized chromatin structure. These interactions may also be utilized in the formation of heterochromatin in higher eukaryotes.</p> / Dissertation
Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/3842 |
Date | January 2011 |
Creators | Prescott, Eugenia Christine Tsamis |
Contributors | Rusche, Laura N |
Source Sets | Duke University |
Detected Language | English |
Type | Dissertation |
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