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dc.contributor.advisorCraig Peterson, PhD
dc.contributor.authorSwygert, Sarah G.
dc.date2022-08-11T08:08:45.000
dc.date.accessioned2022-08-23T16:06:59Z
dc.date.available2022-08-23T16:06:59Z
dc.date.issued2015-08-20
dc.date.submitted2015-09-28
dc.identifier.doi10.13028/M22C76
dc.identifier.urihttp://hdl.handle.net/20.500.14038/32157
dc.description.abstractHeterochromatin is a silenced chromatin region essential for maintaining genomic stability in eukaryotes and for driving developmental processes in higher organisms. A hallmark of heterochromatin is the presence of specialized architectural proteins that alter chromatin structure to inhibit transcription and recombination. Although it is generally assumed that heterochromatin is highly condensed, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, heterochromatin assembly at telomeres and the HM silent mating type loci requires the Sir proteins: Sir3, believed to be the major structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for SIR recruitment to silencing regions and deacetylation of lysine 16 of the histone H4 tail, a mark associated with active chromatin. A combination of sedimentation velocity, atomic force microscopy, and nucleosomal array capture was used to characterize the stoichiometry and conformation of SIR nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome, and that Sir2/4 may additionally bind at a ratio of one per nucleosome. Despite Sir3’s ability to repress transcription in vivo and homologous recombination in vitro in the absence of Sir2/4, Sir3 fibers were found to be significantly less compact than canonical magnesium-induced 30 nanometer fibers. However, heterochromatin fibers composed of all three Sir proteins did adopt a more condensed, globular structure. These results suggest that heterochromatic silencing is mediated both by the creation of more stable nucleosomes and by the steric exclusion of external factors.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectDissertations, UMMS
dc.subjectChromatin
dc.subjectChromatin Assembly and Disassembly
dc.subjectHeterochromatin
dc.subjectSilent Information Regulator Proteins, Saccharomyces cerevisiae
dc.subjectChromatin
dc.subjectChromatin Assembly and Disassembly
dc.subjectHeterochromatin
dc.subjectSaccharomyces cerevisiae
dc.subjectSilent Information Regulator Proteins
dc.subjectBiochemistry
dc.subjectGenetics and Genomics
dc.subjectStructural Biology
dc.titleThe Shape of Silence: The Solution-State Conformation of Sir Heterochromatin: A Dissertation
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1786&context=gsbs_diss&unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/790
dc.legacy.embargo2016-08-26T00:00:00-07:00
dc.identifier.contextkey7651816
refterms.dateFOA2022-08-24T04:18:21Z
html.description.abstract<p>Heterochromatin is a silenced chromatin region essential for maintaining genomic stability in eukaryotes and for driving developmental processes in higher organisms. A hallmark of heterochromatin is the presence of specialized architectural proteins that alter chromatin structure to inhibit transcription and recombination. Although it is generally assumed that heterochromatin is highly condensed, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, heterochromatin assembly at telomeres and the HM silent mating type loci requires the Sir proteins: Sir3, believed to be the major structural component of SIR heterochromatin, and the Sir2/4 complex, responsible for SIR recruitment to silencing regions and deacetylation of lysine 16 of the histone H4 tail, a mark associated with active chromatin. A combination of sedimentation velocity, atomic force microscopy, and nucleosomal array capture was used to characterize the stoichiometry and conformation of SIR nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome, and that Sir2/4 may additionally bind at a ratio of one per nucleosome. Despite Sir3’s ability to repress transcription in vivo and homologous recombination in vitro in the absence of Sir2/4, Sir3 fibers were found to be significantly less compact than canonical magnesium-induced 30 nanometer fibers. However, heterochromatin fibers composed of all three Sir proteins did adopt a more condensed, globular structure. These results suggest that heterochromatic silencing is mediated both by the creation of more stable nucleosomes and by the steric exclusion of external factors.</p>
dc.identifier.submissionpathgsbs_diss/790
dc.contributor.departmentProgram in Molecular Medicine
dc.description.thesisprogramBiochemistry and Molecular Pharmacology


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