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<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>
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