Chromatin Regulation by Deacetylation and SUMO-Targeted Ubiquitin Ligation

描述(申请人提供)：染色体的功能由其结构蛋白的动态修改来调节。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。一种独特的、新定义的活性是相扑靶向泛素连接(STUbL)，它由先前被认为在基因组稳定和对DNA的反应中起作用的蛋白质催化。这项研究的目的是确定染色质脱乙酰化和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。这项拟议的研究建立在实验室最近的发现基础上，即Sir2脱乙酰酶在物理上和功能上与SLX5-SLX8复合体催化的STUbL活性有关。该项目将通过三个目标完成。在第一个目标中，遗传和生化研究将检验这样一个假设，即第二个预测的STUbL蛋白与SLX5-SLX8平行发挥功能，通过STUbL活性促进最佳结构和功能。突变体的转录沉默缺陷将通过分子和遗传方法来表征，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组分的基因组和亚核定位。基因组和细胞生物学实验将测试STUbL是否占据沉默的染色质，并定义独特的基因组结合模式和亚核区(S)。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分析相结合的方法。潜在的底物将通过其他生化和分子遗传学方法进行独立验证。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STUbL的关键机制、底物和基因组靶标，这些机制、底物和基因组靶标对染色质功能至关重要。 公共卫生相关性：STUbL酶是本研究的重点，影响细胞生长、衰老、新陈代谢和对DNA损伤的反应等关键过程。在人类细胞中，STUbL最近被证明在治疗急性早幼粒细胞白血病中起到了治疗作用，然而关于这个新发现的酶家族仍然有许多悬而未决的问题。了解它们的细胞作用、底物和调控最终将提供对定义人类发育和疾病的基本过程的洞察，并可能与健康衰老和癌症的机制特别相关。

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.