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。芽殖酵母酿酒酵母含有表观遗传学上可遗传的沉默染色质结构域，其适于遗传和生化分析。这些结构域通过与DNA结合的成核蛋白和称为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。SIR复合物由NAD依赖性脱乙酰酶Sir2、组蛋白结合蛋白Sir3和衔接蛋白Sir4组成。成核蛋白将SIR复合物募集到DNA中。然后SIR复合物以脱乙酰化依赖性方式沿着沿着染色质纤维远离成核位点扩散，产生沉默转录和其他DNA交易的染色质结构域。尽管我们对SIR复合物如何被招募到DNA并结合到染色质的知识有所增加，但仍然不清楚它如何沉默与其相关的染色质区域，以及所得沉默结构域如何在染色体复制和细胞分裂期间表观遗传地遗传。在本提案中，我们将在依赖于沉默染色质体外重建的系统中使用生物化学测定的组合来研究（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遗传机制。此外，我们将研究SIR复合物如何改变基础染色质的结构，以了解可能的染色质压缩如何有助于沉默。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 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.