S-phase-independent silencing establishment in Saccharomyces cerevisiae

Abstract

The establishment of silent chromatin, a heterochromatin-like structure at HML and HMR in Saccharomyces cerevisiae, depends on progression through S phase of the cell cycle, but the molecular nature of this requirement has remained elusive despite intensive study. Using high-resolution chromatin immunoprecipitation and single-molecule RNA analysis, we found that silencing establishment proceeded via gradual repression of transcription in individual cells over several cell cycles, and that the cell-cycle-regulated step was downstream of Sir protein recruitment. In contrast to prior results, HML and HMR had identical cell-cycle requirements for silencing establishment, with no apparent contribution from a tRNA gene adjacent to HMR. We identified the cause of the S-phase requirement for silencing establishment: removal of transcription-favoring histone modifications deposited by Dot1, Sas2, and Rtt109. These results revealed that silencing establishment was absolutely dependent on the cell-cycle-regulated interplay between euchromatic and heterochromatic histone modifications.

An Inducible System for Silencing Establishment Reveals a Stepwise Mechanism in Which Anchoring at the Nuclear Periphery Precedes Heterochromatin Formation

“…In contrast to H2A-P, SIR recruitment and silencing establishment is a slow process, with Sir3 and Sir4 binding increasing significantly after 90 min and accumulating over several cell cycles, leading to gene silencing only after long-term induction (20 h). This slow establishment of silencing is in contrast with previous reports monitoring silencing establishment at HM loci [ 24 , 25 , 26 , 27 , 28 , 30 , 31 , 32 , 50 ]. This difference could be explained by several factors: (i) the chromosomal position of the reporter studied (euchromatic regions 339 kb away from Tel2R in our system versus subtelomeric HML loci); (ii) the strength of the gene promoter [ 30 ]; (iii) the method used to induce silencing establishment.…”

“…Binding and unbinding of the LacI protein could increase the turnover of nucleosomes independently of DNA replication, thus leading to the incorporation of unmodified histones that are more permissive to Sir3 binding. In particular, H3K79 tri-methylation, which is widespread in euchromatin, counteracts Sir3 spreading and prevents silencing establishment [ 5 , 32 , 52 ]. Given the lack of enzyme to erase this histone mark, its removal can be achieved only through turnover of the histones that bear it.…”

“…Given the lack of enzyme to erase this histone mark, its removal can be achieved only through turnover of the histones that bear it. Histone turnover occurs mainly during the S phase and was proposed to be a critical cell-cycle-regulated step in silencing establishment [ 32 ]. Our data suggest that the dynamics of LacI binding and unbinding could also stimulate histone turnover on the lacO array.…”

“…Heterochromatin establishment has been mainly studied using different strategies, most of them using conditional or inducible SIR3 alleles [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ], thus affecting all silent loci and possibly changing the stoichiometry of the SIR complex. Here, we developed and characterized an inducible system to study the establishment of silencing at a single ectopic locus, while silencing is already established at canonical loci, without affecting the levels of silencing factors.…”

A novel allele of SIR2 reveals a heritable intermediate state of gene silencing

“…A recent study found that Sir-based silencing establishment at both HML and HMR occurs through an intermediate silenced state, rather than an abrupt switch from the fully expressed to fully silenced state (Goodnight and Rine 2020). Furthermore, this intermediate state could be generated and stably maintained when certain histone modifying enzymes were absent in G1-arrested cells.…”