The Establishment of Gene Silencing at Single-Cell Resolution

The establishment of silencing in Saccharomyces cerevisiae is similar to heterochromatin formation in multi-cellular eukaryotes. Previous batch culture studies determined that the de novo establishment of silencing initiates during S phase and continues for up to 5 cell divisions for completion. To track silencing phenotypically, we developed an assay that introduces Sir3 protein into individual sir3Δ 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 revealed that removal of methyl modifications at H3 K4 and H3 K79 were important steps in silent chromatin formation, and that Jhd2 and Set1 played competing roles in the process.