ABSTRACT
In Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating loci HML and HMR is established by the combined actions of cis-acting silencers and trans-acting proteins, including Sir2p, Sir3p and Sir4p. The Sir proteins serve as an integral part of a special silent chromatin at the HM loci. Deletion of any of the SIR2-SIR4 genes leads to a complete loss of silencing. However, the SUM1-1 mutation can restore silencing at the HM loci. Recently, it has been shown that Sum1-1p is directed to the silencers and internal regions of the HM loci, and interacts with the Hst1p histone deacetylase that is a paralog of the Sir2p histone deacetylase. Like Sir-dependent silent chromatin, Sum1-1p-dependent chromatin is hypoacetylated. These suggest that Sum1-1p and Hst1p play roles similar to those of the Sir proteins in promoting transcriptional silencing. Here, we examine whether Sum1-1p-dependent chromatin is similar to Sir-dependent silent chromatin, which is characterized by densely and precisely positioned nucleosomes. We demonstrate that Sum1-1p-dependent primary chromatin structure at HMR largely resembles, but is not identical with, Sir-dependent silent chromatin, whereas Sum1-1p-dependent HML chromatin largely resembles, but is not identical with, derepressed chromatin found in a sir- background. This correlates with the previous finding that SUM1-1 restores silencing more efficiently at HMR than at HML. We show also that DNA in Sum1-1p-dependent silent chromatin assumes a distinct topology. Moreover, we present evidence indicating that Sum1-1p can increase the stability of Sir-dependent silent chromatin, thereby providing an explanation for the finding that SUM1-1 enhances HML/HMR silencing in a SIR+ background.
Subject(s)
Chromatin , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Transcription, Genetic , DNA/chemistry , Gene Silencing , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Mutation , Nuclear Proteins/genetics , Repressor Proteins , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Sirtuin 2 , Sirtuins/genetics , Sirtuins/metabolismABSTRACT
Increasing evidence indicates that transcriptionally silent chromatin structure is dynamic and may change its conformation in response to external or internal stimuli. We show that growth temperature affects all three forms of transcriptional silencing in Saccharomyces cerevisiae. In general, increasing the temperature within the range of 23-37 degrees C strengthens HM and telomeric silencing but reduces rDNA silencing. High temperature (37 degrees C) can suppress the silencing defects of histone H4 mutants. We demonstrate that DNA at the silent HML locus becomes more and more negatively supercoiled as temperature increases in a Sir-dependent manner, which is indicative of enhanced silent chromatin. This enhancement of silent chromatin is not dependent on silencers and therefore does not require de novo assembly of silent chromatin. We also present evidence suggesting that MAP kinase-mediated Sir3p hyperphosphorylation, which plays a role in regulating silencing in response to certain stress conditions, is not involved in high temperature-induced strengthening of silencing. In addition, Pnc1p, a positive regulator of Sir2p activity, plays no role in thermal regulation of silencing. Therefore, growth temperature regulates transcriptional silencing by a novel mechanism.
