Measuring the buffering capacity of gene silencing in

Significance Gene silencing, once established, is stably maintained for several generations. Despite the high fidelity of the inheritance of the silent state, individual components of silenced chromatin are in constant flux. Models suggest that silent loci can tolerate fluctuations in Sir proteins and histone acetylation levels, but the level of tolerance is unknown. To understand the quantitative relationships between H4K16 acetylation, Sir proteins, and silencing, we developed assays to quantitatively alter a H4K16 acetylation mimic allele and Sir protein levels and measure the effects of these changes on silencing. Our data suggest that a two- to threefold change in levels of histone marks and specific Sir proteins affects the stability of the silent state of a large chromatin domain.
Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.