Studies On Silencing

基因组结构域的沉默需要被称为沉默子的失活中心和许多抑制蛋白之间一系列复杂的相互作用。沉默者招募由Sir蛋白组成的抑制蛋白复合物，与核小体中的组蛋白相互作用，形成无法进入的染色质结构域。我们的研究将集中在(1)沉默结构域的建立机制和组蛋白变异在沉默中的作用(2)沉默结构域结构的生化和分子特征及其体外重建。(3)沉默结构域局限于基因组特定区域的机制。通过分析Sir1p中即使在Sir1被募集到沉默子上也无法建立沉默状态的突变，来检验Sir1p在交配型位点上的沉默建立。这些突变体被用来分离抑制因子。确定了这些抑制因子与Sir1p突变体的关系。组蛋白H2A变体的分离表明该蛋白在沉默中起作用，目前正在研究中。被沉默的染色质结构域被限制在基因组的特定区域的机制正在被研究。寻找介导边界功能的特定DNA序列元件。一旦确定是一个特定的t-RNA基因。这一结果以及其他结果表明，沉默功能的障碍可能是通过形成一个稳定的复合体来重塑染色质，并阻断沉默染色质的传播。遗传学研究表明，多个位点在转录上受到Sir蛋白复合体不同组合的抑制。进行了从酵母细胞中纯化Sir蛋白复合物的研究，随后对单个成分进行了鉴定和表征。进一步的研究将旨在确定相关的酶活性以及确定复合物的分子结构。我们目前对转录沉默的理解的一个重要指标是是否有可能在体外重建沉默状态。将开发将Sir蛋白沉积到重组核小体上的方法，并使用大量生化分析来测试组装的染色质模拟沉默状态的能力。这样一个系统将允许对沉默过程进行生化解剖，并将对基因沉默的机制产生新的见解。

Silencing of genomic domains requires a complex series of interactions between inactivation centers called silencers and numerous repressor proteins. The silencers recruit repressor protein complexes composed of the Sir proteins that interact with histones in nucleosomes to form a chromatin domain that is inaccessible Our studies will focus on (1) the mechanisms by which silent domains are established and the role of histone variants in silencing (2) the biochemical and molecular characterization of the structure of the silent domain and its reconstitution in vitro. (3) The mechanism by which a silenced domain is restricted to a specific region of the genome. The establishment of silencing by Sir1p at the mating type loci was examined by analyzing mutations in Sir1p that are unable to establish silent state even after recruitment of Sir1 to the silencer. These mutants were employed to isolate suppressors . The relationship of such suppressors with Sir1p mutants were determined. The isolation of a histone H2A variant suggests a role for this protein in silencing and is being investigated. The mechanism by which the silenced chromatin domain is restricted to a specific region of the genome is being investigated. Specific DNA sequence elements that mediate boundary function were sought. Once such identified was a specific t-RNA gene. This as well as other results suggest that barriers to silencing function by forming a stable complex that probably remodels chromatin and blocks the spread of the silenced chromatin Genetic studies have revealed that multiple loci are transcriptionally repressed by distinct combinations of the Sir protein complexes. Studies were performed to purify Sir protein complexes from yeast cells followed by the identification and characterization of the individual components. Further studies will aim at identifying associated enzymatic activities as well as determining the molecular structure of the complexes. An important index of our current understanding of transcriptional silencing is whether or not it is possible to reconstitute the silenced state in vitro. Methods will be developed to deposit the Sir proteins onto reconstituted nucleosomes and the assembled chromatin will be tested for its ability to mimic the silenced state using numerous biochemical assays. Such a system will allow a biochemical dissection of the process of silencing and will yield new insights into the mechanisms by which genes are silenced.