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