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

Abstract

In Saccharomyces cerevisiae, silent chromatin inhibits the expression of genes at the HML, HMR, and telomeric loci. When silent chromatin forms de novo, the rate of its establishment is influenced by different chromatin states. In particular, loss of the enzyme Dot1, an H3 K79 methyltransferase, leads to rapid silencing establishment. We tested whether silencing establishment was antagonized by H3 K79 methylation or by the Dot1 protein itself competing with Sir3 for binding sites on nucleosomes. To do so, we monitored fluorescence activity in cells containing a GFP gene within the HML locus during silencing establishment in a series of dot1 and histone mutant backgrounds. Silencing establishment rate was correlated with Dot1's enzymatic function rather than with the Dot1 protein itself. In addition, histone mutants that mimicked the conformation of unmethylated H3 K79 increased the rate of silencing establishment, indicating that the H3 K79 residue affected silencing independently of Dot1 abundance. Using fluorophore-based reporters, we confirmed that mother and daughter cells often silence in concert, but in instances where asymmetric silencing occurs, daughter cells established silencing earlier than their mothers. This noninvasive technique enabled us to demonstrate an asymmetry in silencing establishment of a key regulatory locus controlling cell fate.

The Nuts and Bolts of Transcriptionally Silent Chromatin in Saccharomyces cerevisiae

“…In kinetic studies of de novo silencing establishment, loss of H3K4 and H3K79 methylation were among the last events observed in silent chromatin assembly (KatanKhaykovich and Struhl 2005). Moreover, elimination of the enzymes that methylate these residues accelerated the rate of silencing onset (Katan-Khaykovich and Struhl 2005; Osborne et al 2009;Osborne et al 2011). Additionally, in a wild-type population of cells with variegated expression of a telomeric reporter gene, histone methylation was found to be the only chromatin feature to distinguish the cells that still permitted transcription of the reporter from those that did not (Kitada et al 2012).…”

Mating-Type Genes andMATSwitching inSaccharomyces cerevisiae

“…This clever assay demonstrated that silencing was accomplished in 1-2 generations. In a later assay, Rine's group used GFP embedded in HML in a similar assay to ask how rapidly GFP intensity decreased (the assay relies on GFP turning over rapidly) (Osborne et al 2011). These assays revealed "unexpected complexity" in the contributions of a histone acetyltransferase (Sas2), two histone methytransferases (Dot1 and Set1), and one histone demethylase (Jhd2) to the dynamics of silencing and suggested that removal of methyl modifications at histone H3-K4 and -K79 were important steps in silent chromatin formation and that Jhd2 and Set1 had competing roles in the process.…”

Propagating Epigenetic States During DNA Replication

“…When mother and daughter cells switch epigenetic states simultaneously upon cell division, the daughter cell usually silences more rapidly than the mother. In instances where the switching event does not occur in both cells of a mother/daughter pair, the daughter cell is more likely to switch to a silenced state than the mother (Osborne et al, 2011). The mechanism behind this difference is unknown but could be linked to asymmetric inheritance of soluble proteins (e.g.…”

“…Sirs) or the sister chromatids during cell division. Alternatively, asymmetric expression of proteins that inhibit (in the mother cell) or promote (in the daughter cell) silent chromatin formation could also contribute to this process (Osborne et al, 2011).…”

“…In the absence of the H4 K16 acetyltransferase Sas2p, the probability of establishing silencing in a given cell cycle decreases, whereas loss of the H3 K4 and H3 K79 methyltransferases encoded by SET1 and DOT1 increases the probability of establishment (Osborne et al, 2009). In the case of DOT1, H3 K79 methylation status has been demonstrated to influence this switching event (Osborne et al, 2011). The rate and mechanism of removal of different histone modifications can also vary during establishment.…”