Ordered Nucleation and Spreading of Silenced Chromatin inSaccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.

Long-Range Communication between the Silencers of HMR

“…Besides playing a role in tethering the HMR domain to telomeric foci, the role of the silencers would also be to nucleate the spread of Sir proteins (41,51). Our quantitative analysis demonstrating increased concentrations of Sir proteins at silencers with reduced levels of these proteins the further one traverses from the silencer would be consistent with this role for the silencers, though other models are equally possible.…”

Symmetry, asymmetry, and kinetics of silencing establishment in Saccharomyces cerevisiae revealed by single-cell optical assays

“…To turn off transcription, repressors and silencing proteins must override activating signals and vice versa. In the yeast Saccharomyces cerevisiae, transcriptional silencing of the cryptic mating genes at HML and HMR requires the localization of silent information regulatory (Sir) proteins across the silenced loci (1)(2)(3)(4). HML and HMR loci are constitutively repressed in wild-type cells.…”

“…Silencing establishment is thought to occur in multiple steps with Sir proteins initially recruited to the silencers flanking HML and HMR and subsequently enriched throughout the silenced loci (3,8,10). The apparent spreading of Sir proteins from silencers requires Sir2-dependent deacetylation of the critical histone residue H4 K16 and possibly other acetylated lysines as well (3,(11)(12)(13)(14).…”

Histone H3 Lysine 36 Methylation Antagonizes Silencing in Saccharomyces cerevisiae Independently of the Rpd3S Histone Deacetylase Complex

“…I N the budding yeast Saccharomyces cerevisiae, Sir proteins associate with DNA silencer sequences and spread to form silenced chromatin at telomeres and the silent mating-type loci HML and HMR (Jenuwein et al 2001;Moazed 2001;Rusche et al 2002). Unlike the initial nucleation event, the spreading of the Sir complex along chromatin appears to be independent of DNA sequence context (for review, see Moazed 2001).…”

“…Unlike the initial nucleation event, the spreading of the Sir complex along chromatin appears to be independent of DNA sequence context (for review, see Moazed 2001). The spread of a complex containing Sir2, Sir3, and Sir4 along DNA is dependent on the histone deacetylase activity of Sir2 (Hoppe et al 2002;Luo et al 2002;Rusche et al 2002). Sir3 and Sir4 have greater affinity for H3 and H4 that are hypoacetylated on their N-terminal tails (Carmen et al 2002).…”