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

Abstract

In eukaryotic cells, silent chromatin is mainly found at the nuclear periphery forming subnuclear compartments that favor silencing establishment. Here, we set up an inducible system to monitor silencing establishment at an ectopic locus in relation with its subnuclear localization in budding yeast. We previously showed that introducing LacI bound lacO arrays in proximity to gene flanked by HML silencers favors the recruitment of the yeast silencing complex SIR at this locus, leading to its silencing and anchoring at the nuclear periphery. Using an inducible version of this system, we show that silencing establishment is a stepwise process occurring over several cell cycles, with the progressive recruitment of the SIR complex. In contrast, we observed a rapid, SIR-independent perinuclear anchoring, induced by the high amount of LacI binding at the lacO array leading to nucleosome eviction at this array and to the phosphorylation of H2A in the neighboring nucleosomes by Mec1 kinase. While the initial phosphorylation of H2A (H2A-P) and perinuclear anchoring are independent of the SIR complex, its latter recruitment stabilizes H2A-P and reinforces the perinuclear anchoring. Finally, we showed that Sir3 spreading stabilizes nucleosomes and limits the access of specific DNA-binding protein to DNA.

Anchoring of parasitic plasmids to inactive regions of eukaryotic chromosomes through nucleosome signal

“…These observations further support that a long (>9 kb) inactive region from a different organism but with a similar GC content than S. cerevisiae can be a contact hotspot for the 2µ plasmid. It has been shown that a high level of LacI binding results in nucleosome eviction (Loïodice et al, 2021). The observation that specific contact is lost when the LacI protein is attached to the region suggests that the resulting large nucleosome-free region could be responsible for the detachment of the 2µ plasmid.…”

Remodeling of perturbed chromatin can initiatede novotranscriptional and post-transcriptional silencing

“…First, in Neurospora , a synthetic tetO operator array was shown to activate RIP by the heterochromatin-related pathway, suggesting that such a construct could initiate transcriptional silencing de novo [29]. Second, in budding yeast, the association of such operator arrays with their corresponding repressor proteins (LacI or TetR) was also shown to induce transcriptional silencing of a nearby reporter [30,31]. This process started with phosphorylation and concomitant depletion of histone H2A, suggesting that the tight binding of repressor proteins led to chromatin stress and nucleosome loss; and it did so by a mechanism independent of DNA replication [30].…”

“…Second, in budding yeast, the association of such operator arrays with their corresponding repressor proteins (LacI or TetR) was also shown to induce transcriptional silencing of a nearby reporter [30,31]. This process started with phosphorylation and concomitant depletion of histone H2A, suggesting that the tight binding of repressor proteins led to chromatin stress and nucleosome loss; and it did so by a mechanism independent of DNA replication [30]. The perturbed array was directed to the perinuclear compartment, where it became gradually occupied by Silent Information Regulator (SIR) proteins, the critical effectors of transcriptional silencing in budding yeast [30].…”

“…This process started with phosphorylation and concomitant depletion of histone H2A, suggesting that the tight binding of repressor proteins led to chromatin stress and nucleosome loss; and it did so by a mechanism independent of DNA replication [30]. The perturbed array was directed to the perinuclear compartment, where it became gradually occupied by Silent Information Regulator (SIR) proteins, the critical effectors of transcriptional silencing in budding yeast [30]. At the end, the heterochromatinized array featured stabilized nucleosomes and had its overall accessibility reduced [30].…”

“…The perturbed array was directed to the perinuclear compartment, where it became gradually occupied by Silent Information Regulator (SIR) proteins, the critical effectors of transcriptional silencing in budding yeast [30]. At the end, the heterochromatinized array featured stabilized nucleosomes and had its overall accessibility reduced [30]. With respect to a possible mechanism, it was proposed that the tight yet dynamic binding of multiple repressor molecules could accelerate nucleosome turnover and promote the incorporation of new histones without certain modifications which would normally interfere with the recruitment of the SIR proteins [30].…”