Sir2 Represses Endogenous Polymerase II Transcription Units in the Ribosomal DNA Nontranscribed Spacer

Abstract

Silencing at the rDNA, HM loci, and telomeres in Saccharomyces cerevisiae requires histone-modifying enzymes to create chromatin domains that are refractory to recombination and RNA polymerase II transcription machineries. To explore how the silencing factor Sir2 regulates the composition and function of chromatin at the rDNA, the association of histones and RNA polymerase II with the rDNA was measured by chromatin immunoprecipitation. We found that Sir2 regulates not only the levels of K4-methylated histone H3 at the rDNA but also the levels of total histone H3 and RNA polymerase II. Furthermore, our results demonstrate that the ability of Sir2 to limit methylated histones at the rDNA requires its deacetylase activity. In sir2⌬ cells, high levels of K4-trimethylated H3 at the rDNA nontranscribed spacer are associated with the expression of transcription units in the nontranscribed spacer by RNA polymerase II and with previously undetected alterations in chromatin structure. Together, these data suggest a model where the deacetylase activity of Sir2 prevents euchromatinization of the rDNA and silences naturally occurring intergenic transcription units whose expression has been associated with disruption of cohesion complexes and repeat amplification at the rDNA. INTRODUCTIONModified histones at silent genomic domains contribute to a chromatin environment that is refractory to gene expression and genetic recombination (reviewed in Strahl and Allis, 2000;Turner, 2000;Jenuwein and Allis, 2001). In Saccharomyces cerevisiae, chromatin at the homothallic mating-type loci HML and HMR, telomeres, and the ribosomal DNA locus (rDNA) silences genetic recombination and expression of native and ectopic genes transcribed by RNA polymerase (Pol) II. Silencing at the HM loci and telomeres has been studied extensively, whereas the mechanisms of Pol II silencing at the rDNA are not well characterized (reviewed in Moazed, 2001;Rusche et al., 2003). Increasing our understanding of the factors and mechanisms that regulate silencing at the rDNA will provide insight into the pathways that regulate gene expression and genome stability.In S. cerevisiae, the rDNA contains ϳ150 -200 tandem copies of a 9.1-kilobase (kb) repeat, with each repeat containing a Pol I-transcribed 35S rRNA gene and a nontranscribed spacer (NTS) that is subdivided into NTS1 and NTS2 by the Pol III-transcribed 5S rRNA gene (reviewed in Warner, 1999; Figure 1). Despite high levels of transcription by Pol I and Pol III in the rDNA locus, Pol II-transcribed genes integrated into the rDNA are silenced (referred to as rDNA silencing) (Bryk et al., 1997;Fritze et al., 1997;Smith and Boeke, 1997). Additionally, silent chromatin at the rDNA is essential for repression of genetic recombination (Gottlieb and Esposito, 1989;Davis et al., 2000;Kobayashi et al., 2004) and extension of replicative life span (reviewed in Guarente, 2000).Chromatin-associated proteins and modified histones regulate silencing of Pol II transcription at the rDNA (Bryk et al., 1997;Fritze et al., 199...

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…rDNA stability and the silencing of Pol II transcription in the rDNA While rDNA silencing was initially discovered fortuitously with reporter genes, study of the phenomenon ultimately led to the realization that Pol II naturally transcribes the NTS1 and NTS2 elements that are repressed by Sir2 ( Figure 12C) Li et al 2006;Vasiljeva et al 2008;Cesarini et al 2010). Such noncoding RNAs are derived from NTS1 and NTS2, and range in size from 1000 to 1700 nt.…”

A Genetic Screen for Increased Loss of Heterozygosity inSaccharomyces cerevisiae

“…The repetitive nature of the rDNA makes it inherently prone to recombination, however, mechanisms are in place that normally prevent this recombination (Burkhalter and Sogo 2004;Kobayashi et al 2004;Johzuka et al 2006;Li et al 2006). It is easy to imagine that mutations that affect recombination at the rDNA array may result in increased LOH at distal loci on chromosome XII, including MET15, but would not have an effect upon loci on other chromosomes.…”

Proliferating Cell Nuclear Antigen and ASF1 Modulate Silent Chromatin inSaccharomyces cerevisiaevia Lysine 56 on Histone H3

“…In Saccharomyces cerevisiae, the strength and stability of silent chromatin depends on the characteristics of histones present not only at silenced loci but also elsewhere throughout the genome. In yeast, transcribed loci are enriched in both acetylated and methylated histones whereas histones in silenced loci are hypoacetylated and hypomethylated (Braunstein et al 1996;Suka et al 2001;Bernstein et al 2002;Bryk et al 2002;Hoppe et al 2002;Rusché et al 2002;Ng et al 2003;SantosRosa et al 2004;Katan-Khaykovich and Struhl 2005;Rudner et al 2005;Li et al 2006). Loss of acetylation in silent chromatin is mediated by the NAD 1 -dependent histone deacetylase Sir2p (Rusché et al 2003 and references therein).…”