Chromatin Regulation by Deacetylation and SUMO-Targeted Ubiquitin Ligation

描述（由申请人提供）：染色体功能由其结构蛋白的动态修饰调节。乙酰化和去乙酰化是公认的修饰，影响组蛋白和其他染色体蛋白如何影响转录、重组、复制和损伤修复。一个独特的，新定义的活性是sumo靶向泛素连接（STUbL），由蛋白质催化，先前发现它们在基因组稳定性和对DNA的响应中起作用。该研究的目标是确定染色质去乙酰化和STUbL共同产生最佳转录沉默、基因组稳定性和生长调节的机制。提出的研究建立在实验室最近发现的Sir2去乙酰化酶在物理和功能上与Slx5-Slx8复合物催化的STUbL活性相关联的基础上。该项目将通过三个目标来完成。在第一个目标中，遗传和生化研究将验证第二个预测的STUbL蛋白与SLX5-SLX8平行功能，通过STUbL活性促进最佳结构和功能的假设。突变体的转录沉默缺陷将通过分子和遗传学方法进行表征，包括遗传分析和染色质免疫沉淀（ChIP）。在第二个目标中，将评估STUbL成分的基因组和亚核定位。基因组学和细胞生物学实验将测试stubl是否占据沉默的染色质，并定义一个独特的基因组结合模式和亚核区室。第三个目标将通过生化方法定义STUbL活性的染色质特异性底物。将使用候选底物和蛋白质组学分析的组合。潜在的底物将通过额外的生化和分子遗传学方法独立验证。将确定Sir2脱乙酰酶活性是否影响STUbL活性或底物特异性。总之，这三个目标的结果将建立对染色质功能至关重要的STUbL的关键机制、底物和基因组靶点。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The STUbL enzymes that are the focus of this study affect critical processes such as cell growth, aging, metabolism, and response to DNA damage. In human cells, a STUbL was recently shown to be responsible for the therapeutic benefits in treatments of acute promyelocytic leukemia, yet many unanswered questions remain about this newly identified family of enzymes. Understanding their cellular roles, substrates and regulation will ultimately provide insight into basic processes that define human development and disease, and may be particularly relevant to mechanisms of healthy aging and cancer.