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.
Highlights
In eukaryotic cells, discrete regions of the genome assume heritable chromatin structures that silence genes located in these regions
We previously showed a basal recruitment of Sir3 and Sir4 at the E silencer and at the ADE2 reporter gene flanked by the HML silencers, with or without an unbound lacO array located 1.9 kb upstream of the silencers (Dubarry et al, 2011)
We set up an inducible system to monitor silencing establishment at an ectopic locus without affecting silencing factors (SIRs) protein expression or activity
Summary
Discrete regions of the genome assume heritable chromatin structures that silence genes located in these regions. Most cell types, silent chromatin or heterochromatin is enriched at the nuclear periphery forming subnuclear compartments where general repressors of transcription concentrate, favoring silencing establishment at the nuclear periphery [1,2]. Telomeres and HM loci are preferentially located at the nuclear envelope, forming repressive domains that sequester the yeast silencing factors (SIRs) [3] in a manner comparable to heterochromatic chromocenters, which sequester HP1 in metazoans [1,4]. To HP1, the yeast silencing complex SIR2/3/4 is able to spread along the chromatin and to repress gene transcription owing to the histone deacetylase activity of. Sir that creates a binding surface for Sir3 [3] This spreading is limited by the amount of
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