Browsing by Subject "chromatin structure"
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Item The Role of Chromatin Structure and Histone Modifications in Gene Silencing at the Ribosomal DNA Locus in Saccharomyces cerevisiae(2012-07-16) Williamson, Kelly M.One of the fundamental questions in science is how chromatin transitions from actively transcribed euchromatin to silent heterochromatin, and what factors affect this transition. One area of my research has focused on understanding the differences in the chromatin structure of active and silent regions in the ribosomal DNA locus (rDNA), a heterochromatin region in S. cerevisiae. Secondly, I have focused on understanding a histone methyltransferase Set1, which is involved in both euchromatin and heterochromatin regions. To distinguish actively transcribed open regions of chromatin from silent and closed regions of chromatin, we have expressed a DNA methyltransferase M.CviPI in vivo to utilize its accessibility to GpC sites. We have used this technique to study changes in nucleosome positioning within the NTS2 region of the rDNA in two cases: as a result of a silencing defect caused by the loss of Sir2, a histone deacetylase involved in silencing at the rDNA, and as an indicator of active transcription by RNA Pol I. Using this technique, we observed differences between open and closed chromatin structure by changes in nucleosome positioning within NTS2. Additionally, we have observed the presence of bound factors within the 35S rRNA gene promoter that are unique to actively transcribed genes. The second area of my research focused on the protein methyltransferase Set1 that mono-, di-, and trimethylates lysine 4 of histone H3 (H3K4) utilizing the methyl group from S-adenosyl methionine (SAM). Set1 is part of a multi protein complex called COMPASS (Complex associated with Set1), and is associated with both actively transcribed and silent regions. Thirty mutants of Set1 were made within the SET domain to learn more about the catalytic mechanism of Set1. The crystal structures of human SET domain proteins, as well as sequence alignments and a random mutagenesis of yeast Set1, were used to identify conserved amino acids in the SET domain of Set1. Mutants were analyzed for their effect on histone methylation in vivo, silencing of RNA Pol II transcription within the rDNA, suppression of ipl1-2, and COMPASS complex formation. Our results show that trimethylated H3K4 is required for silencing of RNA Pol II transcription at the rDNA. Overall, we have shown the importance of tyrosine residues in SET domain proteins. To summarize, my research has strived to understand chromatin structure and the factors that affect the transition between euchromatin and heterochromatin.Item The role of histones and histone modifying enzymes in ribosomal dna silencing in saccharomyces cerevisiae(2009-05-15) Li, ChonghuaIn S. cerevisiae, the ribosomal DNA locus is silent for RNA polymerase II (Pol II) transcription and recombination (rDNA silencing). Our goal is to understand how histones and histone-modifying enzymes regulate the silent chromatin at the rDNA locus. Sir2, a NAD+-dependent histone deacetylase, is required for rDNA silencing. To understand how Sir2 regulates rDNA silencing, we performed chromatin immunoprecipitation to measure the association of modified histones across the rDNA repeat in wild-type and sir2? cells. We found that in sir2? cells, histone H3 at the rDNA became hyperacetylated and hypermethylated. High levels of K4-methylated H3 correlate with Pol II transcription. Consistent with this, we found that the nontranscribed spacer (NTS) region was transcribed by Pol II in sir2? cells. To investigate if transcription of the NTS region regulates rDNA silencing, we overexpressed this region both in trans and in cis. Our data showed that overexpression of the NTS region in cis caused Pol II silencing defect and hyperrecombination at the rDNA. These data suggest that Sir2 contributes to maintain the silent chromatin at the rDNA by repressing Pol II transcription in the NTS region. We also found that the NTS transcripts could be translated in vitro and that they copurified with polysomes, suggesting that the transcripts may encode proteins or that the transcripts are somehow involved in the process of translation. Additionally, we examined the role of linker histone H1 in regulating rDNA silencing. We found that, unlike Sir2 that represses both Pol II transcription and recombination, histone H1 only represses recombination at the rDNA. The hyperrecombination defect at the rDNA is more severe in sir2? hho1? double mutant than in either single mutant, suggesting histone H1 and Sir2 act independently. Consistently, hho1? cells did not accumulate extrachromosomal rDNA circles (ERCs) or the Holliday junction intermediates, which accumulate in sir2? cells. These data suggest that histone H1 and Sir2 regulate different recombination pathways. In summary, my research has provided insight into the mechanism of how silent chromatin at the rDNA locus is regulated, which will help us understand how fundamental components of chromosomes affect gene expression and genome stability.