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. ? ?
