The overall goal of Project 1 is to use the human histone chaperone HIRA and the yeast post-translational modification enzyme, Sir2, as models to understand the structural basis for epigenetic control in aging and longevity; and to develop small molecule Sir2 effectors as reagents to study the linkage between epigenetic events and cell aging. HIRA forms a complex with ASFla to mediate transcriptional activation and formation of senescent heterochromatin, a process associated with tissue aging. Dr. Adams (Project 4) has also characterized new and related UBN proteins that interact with HIRA. These observations lead to the testable hypothesis that HIRA coordinates interactions with both ASFla and UBN proteins for chromatin regulation in senescence and gene activation. In preliminary studies, we have collaborated with Dr. 'Adams to characterize the structure and biochemistry of an ASFla/HIRA complex. The yeast Sir2 protein, and its orthologs in higher eukaryotes, are NAD+ dependent histone deacetylases. Yeast Sir2 promotes gene silencing and lifespan extension and its SIRT1 human ortholog has been implicated to counteract ageassociated diseases such as type-II diabetes, obesity and neurodegenerative disorders in mammals. In preliminary studies, we have determined crystal structures of the yeast Sir2 protein, Hst2, in various liganded forms and hypothesize that we can to use these structures as a molecular scaffold for the rational design of small molecule Sir2 effectors. In this proposal, we will combine biochemical, structural and small molecule screening methodologies to test our hypotheses with the following Specific Aims: (1) Determine the structural and biochemical basis for formation of a ASFla/HIRA/histone complex, (2) Determine the structural and biochemical basis for the role of UBN 1 and UBN2 in HIRA function, and (3) Develop small molecule activators and inhibitors of sirtuins as reagents to probe the involvement of sirtuins in epigenetic regulation and longevity in yeast. Our studies will be complemented by the largely biochemical, genetic, cell biology and organismal studies on the HIRA and sirtuin proteins that will be carried out by the other projects of the program, and the protein production capability of Core B, will contribute to the general understanding of the link between epigentics and aging and may lead to the development of small molecule compounds to treat age-associated disorders.