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表观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特征，在调节基因表达和维持染色体稳定性方面发挥着重要作用。出芽酵母酿酒酵母含有表观遗传的沉默染色质结构域，可用于遗传和生化分析。这些结构域是由结合DNA的核蛋白和称为SIR（沉默信息调节器）复合物的沉默复合物的组合活性组装和维持的。SIR复合体由nadd依赖的去乙酰化酶sirr2、组蛋白结合蛋白Sir3和适配蛋白Sir4组成。核蛋白将SIR复合体招募到DNA中。然后，SIR复合体以去乙酰化依赖的方式沿着染色质纤维远离成核位点扩散，形成一个染色质结构域，使转录和其他DNA交易沉默。尽管我们对SIR复合体如何被招募到DNA并与染色质结合的知识有所增加，但仍不清楚它是如何沉默与其相关的染色质区域的，以及在染色体复制和细胞分裂过程中产生的沉默区域是如何表观遗传的。在这个提议中，我们将在一个依赖于沉默染色质体外重构的系统中使用生化分析的组合来研究(1)sir介导的基因沉默的分子机制和(2)沉默染色质的表观遗传机制。此外，我们将研究SIR复合体如何改变底层染色质的结构，以了解染色质压实如何可能导致沉默。这些研究旨在通过体外重组沉默染色质的分子解剖，对基因沉默有一个完整的分子认识。沉默染色质结构域和sir2样去乙酰化酶的保存表明，这里为出芽酵母复合体开发的原则将适用于其他环境。对基因沉默机制的基本了解不仅可以为理解该过程如何失败提供框架，还可以为基于干预设计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 epigenetically heritable silent chromatin domains that are amenable to both genetic and biochemical analysis. These domains are assembled and maintained by the combined activities of nucleator proteins that bind to DNA and a silencing complex called the SIR (Silent Information Regulator) complex. The SIR complex is composed of the NAD-dependent deacetylase Sir2, the histone binding protein Sir3, and an adaptor protein Sir4. The nucleator proteins recruit the SIR complex to DNA. The SIR complex then spread along the chromatin fiber away from the nucleation sites in a deacetylation- dependent manner, creating a chromatin domain that silences transcription and other DNA transactions. Despite an increase in our knowledge of how the SIR complex is recruited to DNA and binds to chromatin, it has remained unclear how it silences the chromatin regions with which it is associated and how the resulting silent domains are epigenetically inherited during chromosome duplication and cell division. In this proposal we will use a combination of biochemical assays in a system that relies on in vitro reconstitution of silent chromatin to investigate (1) the molecular mechanism of SIR-mediates gene silencing and (2) the mechanism of epigenetic inheritance of silent chromatin. In addition, we will investigate how the SIR complex changes the structure of the underlying chromatin in order to understand how possible chromatin compaction contributes to silencing. 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.