RESUMO
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.
Assuntos
Inativação Gênica , Microscopia de Fluorescência/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Biocatálise , Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Cinética , Lisina/genética , Lisina/metabolismo , Metilação , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismoRESUMO
The establishment of silencing in Saccharomyces cerevisiae is similar to heterochromatin formation in multicellular eukaryotes. Previous batch culture studies determined that the de novo establishment of silencing initiates during S phase and continues for up to five cell divisions for completion. To track silencing phenotypically, we developed an assay that introduces Sir3 protein into individual sir3Delta mutant cells synchronously and then detects the onset of silencing with single-cell resolution. Silencing was completed within the first one to two cell divisions in most cells queried. Moreover, we uncovered 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. Our findings showed that removal of methyl modifications at H3K4 and H3K79 were important steps in silent chromatin formation and that Jhd2 and Set1 had competing roles in the process.