Promoter Strength Influences the S Phase Requirement for Establishment of Silencing at the Saccharomyces cerevisiae Silent Mating Type Loci

Abstract

In Saccharomyces cerevisiae, the two cryptic mating type loci, HML and HMR, are transcriptionally silent. Previous studies on the establishment of silencing at HMR identified a requirement for passage through S phase. However, the underlying mechanism for this requirement is still unknown. In contrast to HMR, we found that substantial silencing of HML could be established without passage through S phase. To understand this difference, we analyzed several chimeric HM loci and found that promoter strength determined the S phase requirement. To silence a locus with a strong promoter such as the a1/a2 promoter required passage through S phase while HM loci with weaker promoters such as the a1/a2 or TRP1 promoter did not show this requirement. Thus, transcriptional activity counteracts the establishment of silencing but can be overcome by passage through S phase. E PIGENETIC silencing refers to a transcriptionally inactive state and its heritable transmission. It involves the formation, maintenance, and inheritance of a specialized, constitutively compact chromatin structure, termed heterochromatin. This kind of transcriptional silencing plays an important role in establishing and maintaining distinct patterns of gene expression in genetically identical cells during growth and differentiation. Examples of transcriptional silencing include the inactive mammalian X chromosome, position effect variegation in Drosophila melanogaster, and the cryptic mating-type loci in fission and budding yeasts (Rusche et al. 2003;Probst et al. 2009).In the budding yeast Saccharomyces cerevisiae, the MAT locus encodes transcriptional regulatory proteins that are responsible for the differences between the two mating types. HML and HMR harbor cryptic copies of the mating type information genes, a and a, respectively. Transcriptional silencing at these loci relies on cisregulatory DNA elements called silencers and on a number of trans -acting gene products. Previous studies revealed that establishment of silencing involves a series of protein-DNA and protein-protein interactions (reviewed in Gasser and Cockell 2001;Rusche et al. 2003;Fox and Mcconnell 2005). The silencer elements flanking the HM loci recruit the DNA binding proteins Rap1, Abf1, and ORC, which in turn recruit the silent information regulator (Sir) proteins, Sir1, Sir2, Sir3, and Sir4. A Sir2-Sir3-Sir4 complex spreads from the silencers into nearby nucleosomes (Hoppe et al. 2002;Rusche et al. 2002;Liou et al. 2005;Rudner et al. 2005). This spreading requires Sir2, which deacetylates histone H4 K16, thereby creating a binding site for Sir3 and Sir4 and hence the Sir2/3/4 complex (Carmen et al. 2002;Liou et al. 2005). Multiple rounds of deacetylation by Sir2 allow the Sir complex to spread to adjacent nucleosomes, thus creating a stretch of silent chromatin.To investigate the establishment of silencing and its relationship to the cell cycle, previous studies utilized conditional or inducible alleles of the Sir proteins to create a transition from Sir À to Sir 1 and th...

A Cis-Acting tRNA Gene Imposes the Cell Cycle Progression Requirement for Establishing Silencing at the HMR Locus in Yeast

“…Independent experiments in which they exchanged the HML and HMR promoters, or introduced ectopic promoters at HML and HMR, convincingly demonstrated that promoter strength per se influences the dependence on cell cycle progression for the establishment of silencing (Ren et al 2010). Our result, indicating a decreased dependence on cell cycle progression at HML when we introduced an ectopic gene, is consistent with these results.…”

“…During the final review of this study, Ren et al (2010) published data also demonstrating that the HML locus is significantly less dependent on cell cycle progression for the establishment of silencing compared to HMR. Independent experiments in which they exchanged the HML and HMR promoters, or introduced ectopic promoters at HML and HMR, convincingly demonstrated that promoter strength per se influences the dependence on cell cycle progression for the establishment of silencing (Ren et al 2010).…”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…Similarly, silencing of a telomeric URA3 reporter occurred only during passage from M to G 1 phases (Martins-Taylor et al 2004;MartinsTaylor et al 2011). Finally, the a1 mating-type gene at HML was silenced in cells that were not cycling at all (Ren et al 2010;Lazarus and Holmes 2011). That the different reporter genes shut off in such different ways during establishment assays may indicate that the timing of silencing onset is regulated predominantly by the factors that perpetuate transcription of the genes, rather than the action of a silent chromatin-specific trigger.…”

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…As further discussed below, silencing of HMR requires passage through S phase (but not replication per se). However, "substantial silencing of HMLa" could be established without passage through S phase (Ren et al 2010). These authors attributed this difference in promoter strength-with the a1-a2 promoter being weaker than a1-a2-because HMRa is partially silenced without S phase, while HMLa requires S phase passage.…”