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

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.