Mechanism of Action of Yeast Silencing Complexes

描述（由申请人提供）：这项工作的长期目标是了解可遗传的沉默染色质结构域是如何组装和遗传的。沉默结构域是真核生物染色体的一个保守结构，在基因表达调控和染色体稳定性维持中起着重要作用。芽殖酵母酿酒酵母包含一个表观遗传学上可遗传的沉默染色质结构域的例子，这些结构域适合于遗传和生化分析。我们纯化了作用于芽殖酵母不同染色体区域的沉默复合物，并研究了它们的活性和定位于染色质的机制。在介导端粒和交配型沉默的SIR（沉默信息调节器）复合物中，NAD依赖性脱乙酰基酶Sir 2与组蛋白结合蛋白Sir 3和Sir 4缔合，并且所有三种蛋白质以脱乙酰基依赖性方式沿染色质纤维沿着分布。在抑制核糖体DNA（rDNA）重复内外源启动子的不适当重组和转录的核仁沉默和终期调节因子（Regulator of Nucleolar silencing and Telopase）复合物中，Sir2与Net1和Cdc14以及推定的rDNA姐妹染色单体钳复合物（称为RSC，rDNA姐妹染色单体钳）相关联。在这个提议中，我们将研究SIR复合物如何去乙酰化并与其染色质靶结合，以及去乙酰化和其他相互作用如何驱动SIR复合物组装并沿染色质纤维沿着。此外，我们将研究新发现的rSC复合物如何调节rDNA结构。这些研究旨在通过体外重组沉默染色质的分子解剖，对基因沉默进行完整的分子理解。 沉默的染色质结构域和Sir2样脱乙酰基酶的保守性表明，这里为芽殖酵母复合物开发的原则将适用于其他环境。对基因沉默机制的基本理解不仅可以为理解该过程如何失败提供框架，还可以为设计基于干预的治疗策略提供基础和知识。

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how epigetically heritable silent chromatin domains are assembled and inherited. Silent domains are a conserved feature of eukaryotic chromosmes and play important roles in regulation of gene expression and maintenance of chromosome stability. The budding yeast Saccharomyces cerevisiae contains an example of epigenetically heritable silent chromatin domains that are amenable to both genetic and biochemical analysis. We have purified the silencing complexes that act at different chromosome regions in budding yeast and have investigated their activities and mechanism of localization to chromatin. In the SIR (Silent Information Regulator) complex, which mediates telomeric and mating-type silencing, the NAD-dependent deacetylase Sir2 associates with histone binding proteins Sir3 and Sir4, and all three proteins spread along the chromatin fiber in a deacetylation-dependent manner. In the RENT (Regulator of Nucleolar silencing and Telophase) complex, which inhibits inappropriate recombination and transcription from foreign promoters within the ribosomal DMA (rDNA) repeats, Sir2 associates with Net1 and Cdc14, as well as a putative rDNA sister chromatid clamp complex (termed rSC, rDNA Sister chromatid Clamp). In this proposal, we will investigate how the SIR complex deacetylates and binds to its chromatin target and how deacetylation and other interactions drive SIR complex assembly and spreading along the chromatin fiber. In addition, we will investigate how the newly identified rSC complex regulate rDNA structure. These studies are aimed at a complete molecular understanding of gene silencing through the molecular dissection of in vitro reconstituted silent chromatin. The conservation of silent chromatin domains and Sir2-like deacetylases suggests that the principles developed here for the budding yeast complexes will apply in other settings. A basic understanding of the mechanism of gene silencing will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.