Competition between Heterochromatic Loci Allows the Abundance of the Silencing Protein, Sir4, to Regulate de novo Assembly of Heterochromatin

Abstract

Changes in the locations and boundaries of heterochromatin are critical during development, and de novo assembly of silent chromatin in budding yeast is a well-studied model for how new sites of heterochromatin assemble. De novo assembly cannot occur in the G1 phase of the cell cycle and one to two divisions are needed for complete silent chromatin assembly and transcriptional repression. Mutation of DOT1, the histone H3 lysine 79 (K79) methyltransferase, and SET1, the histone H3 lysine 4 (K4) methyltransferase, speed de novo assembly. These observations have led to the model that regulated demethylation of histones may be a mechanism for how cells control the establishment of heterochromatin. We find that the abundance of Sir4, a protein required for the assembly of silent chromatin, decreases dramatically during a G1 arrest and therefore tested if changing the levels of Sir4 would also alter the speed of de novo establishment. Halving the level of Sir4 slows heterochromatin establishment, while increasing Sir4 speeds establishment. yku70Δ and ubp10Δ cells also speed de novo assembly, and like dot1Δ cells have defects in subtelomeric silencing, suggesting that these mutants may indirectly speed de novo establishment by liberating Sir4 from telomeres. Deleting RIF1 and RIF2, which suppresses the subtelomeric silencing defects in these mutants, rescues the advanced de novo establishment in yku70Δ and ubp10Δ cells, but not in dot1Δ cells, suggesting that YKU70 and UBP10 regulate Sir4 availability by modulating subtelomeric silencing, while DOT1 functions directly to regulate establishment. Our data support a model whereby the demethylation of histone H3 K79 and changes in Sir4 abundance and availability define two rate-limiting steps that regulate de novo assembly of heterochromatin.

Recruitment and allosteric stimulation of a histone-deubiquitinating enzyme during heterochromatin assembly

“…7). The eventual dilution of the anti-silencing mark H3K79me and the prevention of new H3K79 methylation by removing H2B-Ub would facilitate heterochromatin establishment, as has been observed previously for de novo silencing kinetics (9,24,55). In conjunction with previous genetic analyses of Ubp10 in silencing, we present a more refined and mechanistically detailed model for how Ubp10 promotes assembly of SIR complexmediated heterochromatin.…”

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…The supply of Sir proteins is limiting for transcriptional silencing. Increased concentration of Sir proteins at one site diminishes the pool of available proteins for silencing at another Maillet et al 1996;Marcand et al 1996;Larin et al 2015). By sequestering individual silent chromatin domains at the periphery, the concentration of perinuclear Sir proteins is raised for all other silent chromatin domains anchored at the periphery.…”

“…Sir4 levels also change in response to environmental stimuli. Levels of the protein drop precipitously in cells subjected to extended arrest by the a-factor mating pheromone (Larin et al 2015).…”

Determinants of Sir2-Mediated, Silent Chromatin Cohesion

“…The subtle silencing defects associated with mutations in EKDK were reversed by mild increases in Sir4, a factor whose quantity is limiting for silencing (30). Thus, if cohesion promotes silencing in any way, it might be to increase the local concentration of Sir4.…”

“…To disentangle the roles of the EKDK motif in silencing and silent chromatin cohesion, we attempted to suppress the silencing defect of the AKAA mutant by providing additional Sir4. The protein is limiting for silencing, and even subtle increases in the SIR4 gene dosage can improve transcriptional repression (26,(28)(29)(30). In the case of the sectoring assay, extra SIR4 provided by a low-copy-number centromere plasmid in a wild-type strain increased the fraction of completely red colonies from 65% to nearly 100% (Fig.…”