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.

出芽酵母中的基因沉默是由 Sir 蛋白与未乙酰化核小体结合形成抑制转录的染色质结构介导的。转录沉默的特点是沉默状态的高保真传输。尽管其相对稳定，但沉默状态的组成部分一直在不断变化，从而产生了一个模型，即沉默基因座可以容忍这种波动而不会产生功能性后果。然而，耐受水平是未知的，我们开发了测量组蛋白乙酰化阈值的方法，该阈值导致沉默的染色质状态切换到活跃状态，以及测量沉默所需的酶和结构蛋白的水平。我们表明，沉默的丧失需要 50% 到 75% 的乙酰模拟组蛋白，尽管精确水平受消音器强度和上游激活序列 (UAS) 增强子/启动子强度的影响。对沉默所需的阻遏蛋白水平的测量表明，减少SIR4基因剂量两到三倍显着削弱了沉默，尽管将 Sir2 或 Sir3 的基因拷贝数减少到相同程度并没有显着影响沉默，这表明 Sir4 是基因沉默中的限制成分。计算表明，乙酰转移酶在大量核小体中乙酰化核小体的能力仅降低两倍可能足以产生转录沉默结构域。