Single-cell observations reveal intermediate transcriptional silencing states

Analysis of transcriptional silencing in Saccharomyces has provided valuable insights into heterochromatin formation and function. However, most of these studies revealed only the average behaviors of populations of cells. Here, we examined transcriptional silencing by monitoring individual yeast cells carrying distinguishable fluorescent reporter genes inserted at two different silent loci. These studies showed that two silent loci in a single cell behave independently, demonstrating that heterochromatin formation is locus autonomous. Furthermore, some silencing mutants with an intermediate phenotype, such as sir1, consist of two distinct populations, one repressed and one derepressed, while other mutants, such as those inactivating the SAS-I histone H4 K16 acetylase, consist of cells all with an intermediate level of expression. Finally, both establishment and decay of silencing can be influenced by specific gene activators, with establishment occurring stochastically over several generations. Thus, quantifying silencing in individual cells reveals aspects of silencing not evident from population-wide measurements.