Chromatin Regulation by Deacetylation and SUMO-Targeted Ubiquitin Ligation

项目摘要(摘要) 染色体的功能是通过其结构的动态改变来调节的 蛋白质。乙酰化和去乙酰化是公认的修饰，它们影响 组蛋白和其他染色体蛋白影响转录、重组、 复制和修复损伤。一个独特的、新定义的活动是以相扑为目标的 泛素连接(STUbL)，由先前确定其作用的蛋白质催化。 基因组的稳定性和对DNA的响应。这项研究的目标是定义 染色质脱乙酰化和STUbL共同产生最佳效果的机制 转录沉默、基因组稳定性和生长调节。拟议的研究 建立在实验室最近发现的Sir2脱乙酰酶是物理和 在功能上与SLX5-SLX8复合体催化的STUbL活性相关联。该项目 将通过三个目标来实现。在第一个目标中，遗传和生化 研究将检验这一假设，即第二个预测的STUbL蛋白在 与SLX5-SLX8平行，通过STUbL活性促进结构和功能的优化。 突变体的转录沉默缺陷将通过分子和 遗传方法，包括遗传分析和染色质免疫沉淀 (芯片)。在第二个目标中，STUbL的基因组和亚核定位 将对组件进行评估。基因组和细胞生物学实验将测试 STUbL占据沉默的染色质，并定义了一种独特的基因组结合模式 亚核室(S)。第三个目标将定义特定于染色质的底物 通过生化方法检测STUbL活性。候选底物的组合 并将使用蛋白质组学分析。潜在的底物将被独立验证 通过额外的生物化学和分子遗传学方法。这将是确定的 Sir2脱乙酰酶活性是否影响STUbL活性或底物特异性。一起， 这三个目标的结果将建立关键的机制、基础和 对染色质功能至关重要的STUbL的基因组靶点。

Project Summary (ABSTRACT) Chromosomal functions are regulated by dynamic modifications of their structural proteins. Acetylation and deacetylation are well-established modifications that affect how histones and other chromosomal proteins influence transcription, recombination, replication and repair of damage. A distinct, newly defined activity is SUMO-targeted ubiquitin ligation (STUbL), catalyzed by proteins previously identified for their roles in genome stability and in response to DNA. The goals of the research are to define the mechanisms by which chromatin deacetylation and STUbL together yield optimal transcriptional silencing, genome stability and growth regulation. The proposed research builds on the lab's recent discovery that the Sir2 deacetylase is physically and functionally linked to a STUbL activity catalyzed by the Slx5-Slx8 complex. The project will be accomplished through three aims. In the first aim, genetic and biochemical studies will test the hypothesis that a second predicted STUbL protein functions in parallel to SLX5-SLX8 to promote optimal structure and function through STUbL activity. Transcriptional silencing defects of mutants will be characterized through molecular and genetic approaches, including genetic analysis and chromatin immunoprecipitation (ChIP). In the second aim, the genomic and subnuclear localization of STUbL components will be evaluated. Genomic and cell biological experiments will test if STUbLs occupy silent chromatin and define a distinct genomic binding pattern and subnuclear compartment(s). The third aim will define chromatin-specific substrates of STUbL activity through biochemical approaches. A combination of candidate substrate and proteomic analyses will be used. Potential substrates will be independently validated through additional biochemical and molecular genetic approaches. It will be determined if Sir2 deacetylase activity influences STUbL activity or substrate specificity. Together, the results from these three aims will establish the key mechanisms, substrates, and genomic targets of STUbL that are critical for chromatin function.