Analysis of MYST Family Acetyltransferase Functions

描述（由申请人提供）：本研究的长期目标是了解染色质修饰调节基因表达和染色体结构和功能的机制。该项目以MYST乙酰转移酶家族为中心，该家族与正常生长发育以及白血病、艾滋病毒感染和阿尔茨海默病等多种疾病有关。所提出的实验建立在MYST蛋白在转录激活和沉默中发挥作用的结果之上，并且在DNA损伤修复、细胞周期控制、固定期存活以及染色体和亚核结构中发挥关键作用。以前的研究主要集中在三个MYST基因，ESA1， SAS2和SAS3。计划中的研究将验证MYST酶的不同作用是通过基因组靶向，通过蛋白质相互作用和与其他染色质修饰蛋白的协调来指定的假设。转录分析、亲和分析、突变分析、染色质免疫沉淀和微阵列实验将用于这些测试。将确定导致SAS3和ESA1不同功能的遗传和物理相互作用。SAS3和ESA1的细胞周期控制和DNA损伤机制将被用于评估转录和结构效应。这些实验将监测检查点功能，并将验证和扩展微阵列数据。交叉互补实验将进行，以解决特异性和人类MYST蛋白功能保护的程度。酵母MYST酶的首选组蛋白底物已被确定，但非组蛋白底物可能是多种MYST功能的关键。蛋白质组学方法将识别非组蛋白底物，其意义将通过结合生化和遗传方法进行评估。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand mechanisms by which chromatin modifications regulate gene expression and chromosomal structure and function. The project centers on the MYST family of acetyltransferases that is implicated in normal growth and development and in conditions as diverse as leukemia, HIV-infection, and Alzheimer's disease. The proposed experiments build on results demonstrating that MYST proteins function in both transcriptional activation and silencing and have critical roles in DNA damage repair, cell cycle control, stationary phase survival, and chromosomal and subnuclear architecture. Previous studies focused on the three MYST genes, ESA1, SAS2 and SAS3. Planned studies will test the hypothesis that distinct roles for the MYST enzymes are specified through genomic targeting, mediated by interacting proteins and coordination with other chromatin modifying proteins. Transcriptional assays, affinity assays, mutational analysis, chromatin immunoprecipitation and microarray experiments will be used in these tests. Genetic and physical interactions that contribute to the distinct functions of SAS3 and ESA1 will be identified. Mechanisms of cell cycle control and DNA damage for SAS3 and ESA1 will be pursued to evaluate transcriptional vs. structural effects. These experiments will monitor checkpoint functions and will validate and extend microarray data. Cross-complementation experiments will be performed to address the specificity and extent of functional conservation of human MYST proteins. Preferred histone substrates have been identified for the yeast MYST enzymes, yet non-histone substrates are likely to be key to diverse MYST functions. Proteomic approaches will identify non-histone substrates, the significance of which will be evaluated through combined biochemical and genetic approaches.