Department of Molecular Cell Developmental Biology, University of California, Santa Cruz, CA 95064.
Department of Molecular Cell Developmental Biology, University of California, Santa Cruz, CA 95064
Proc Natl Acad Sci U S A. 2021 Dec 7;118(49). doi: 10.1073/pnas.2111841118.
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 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.
酿酒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。转录沉默的特征是沉默状态的高保真传递。尽管它相对稳定，但沉默状态的组成部分在不断变化，这就产生了一个模型，即沉默基因座可以在没有功能后果的情况下容忍这种波动。然而，其耐受程度尚不清楚，我们开发了一些方法来测量导致沉默染色质状态向活性状态转变所需的组蛋白乙酰化阈值，以及测量沉默所需的酶和结构蛋白的水平。我们发现，沉默的丧失需要 50%至 75%的乙酰化模拟组蛋白，尽管确切水平受到沉默器强度和上游激活序列（UAS）增强子/启动子强度的影响。对沉默所需的抑制蛋白水平的测量表明，基因剂量减少两到三倍会显著削弱沉默，尽管将 Sir2 或 Sir3 的基因拷贝数减少到相同程度不会显著影响沉默，这表明 Sir4 是基因沉默的限制因素。计算表明，在一个大的核小体阵列中，乙酰转移酶乙酰化核小体的能力仅降低两倍，可能足以产生一个转录沉默的结构域。
