Abstract #40

Quantitative Study of Epigenetic Gene Expression in Live Cells. Eugenia Xu, Karl Zawadzki, James Broach. Department of Molecular Biology, Princeton University, Princeton, NJ.
   In Saccharomyces cerevisiae, silencing at the mating-type loci HML and HMR depends on the assembly of a repressive chromatin structure consisting of Sir2p, Sir3p, Sir4p, and the core histones. This repressive chromatin structure can be established, maintained and stably inherited in wild type yeast cells. To explore the mechanism of transcriptional silencing in more quantitative details, we have developed a dual reporter assay by inserting distinguishable fluorescent reporter genes at the two different silent loci and examined silencing either by fluorescent microscopy or by fluorescent activated cell sorting.
By studying the epigenetic mutants defective in silent chromatin formation, we observed that two different silent loci in a single cell behave independently, demonstrating that heterochromatin formation is locus autonomous. Furthermore, some silencing mutants with intermediate phenotypes, such as sir1 and asf1, consist of two distinct populations, one fully repressed and one derepressed, while other mutants, such as those in SAS genes encoding components of the histone H4 K16 acetylase, consist of a uniform population of cells all with an intermediate silencing phenotype. Finally, both establishment and decay of silencing can be influenced by specific gene activators, with establishment occurring stochastically over several generations. Thus, observations of silencing in individual cells have revealed aspects of silencing not evident from population-wide measurements. We are currently using our single-cell analysis tools to characterize the nature of the silencing defect in other reported epigenetic mutants.

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