Mechanism of Action of Yeast Silencing Complexes

描述(申请人提供)：这项工作的长期目标是了解如何在表观可遗传的沉默染色质结构域组装和遗传。沉默结构域是真核生物染色体的一种保守特征，在基因表达调控和维持染色体稳定性方面发挥着重要作用。萌芽酵母酿酒酵母包含一个表观可遗传的沉默染色质结构域的例子，这些结构域同时适用于遗传和生化分析。我们纯化了芽期酵母中作用于不同染色体区域的沉默复合体，并研究了它们对染色质的活性和定位机制。在介导端粒和交配型沉默的SIR复合体中，依赖NAD的脱乙酰酶Sir2与组蛋白结合蛋白Sir3和Sir4结合，这三种蛋白都以脱乙酰化依赖的方式沿染色质纤维扩散。在核仁沉默和端粒酶调节因子(Rent)复合体中，Sir2与Net1和CDc14以及可能的rDNA姐妹染色单体钳复合体(RSC，rDNA姐妹染色单体钳)结合，抑制核糖体DMA(RDNA)重复序列内外来启动子的不适当重组和转录。在本提案中，我们将研究SIR复合体是如何脱乙酰化并结合到染色质靶标上的，以及脱乙酰化和其他相互作用是如何驱动SIR复合体组装和沿染色质纤维扩散的。此外，我们还将研究新发现的RSC复合体如何调节rDNA结构。这些研究的目的是通过对体外重组沉默染色质的分子解剖，从分子水平上理解基因沉默。 沉默的染色质结构域和类似Sir2的脱乙酰酶的保守表明，这里为萌芽酵母复合体开发的原理将适用于其他环境。对基因沉默机制的基本了解不仅将为理解这一过程如何失败提供一个框架，而且还将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.