The role of histones and histone modifying enzymes in ribosomal dna silencing in saccharomyces cerevisiae

In 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.