PROJECT SUMMARY: The major goal of this K99/R00 proposal is to determine mechanisms by which SIRT6 regulates and extends lifespan. Therapeutic modulation of SIRT6 activity in humans may be an effective way to treat many age- related diseases and disorders and extend healthy lifespan. The ability of SIRT6 to regulate lifespan in mice has been demonstrated, and our preliminary data show substantial lifespan extension by overexpressing the SIRT6 Drosophila melanogaster ortholog, dSirt6, in flies, but the extent of this ability and mechanisms by which SIRT6 operates are poorly understood. This proposal will use Drosophila melanogaster to clarify these issues through three Specific Aims. Aim 1 will determine which tissues dSirt6 acts through to extend lifespan, and the relative contributions of dSirt6 deactylase (associated with chromatin silencing) and ADP-ribosylase (associated with DNA repair) functions towards regulating lifespan. The results from these experiments should provide better confirmation of the ability of SIRT6 to extend lifespan, and mechanisms by which it operates to do so. Aim 2 will measure global gene expression changes (RNA-seq) and epigenomic alterations (CUT&RUN for dSirt6 substrates H3K9ac and H3K56ac) during aging, with and without dSirt6 overexpression. These results will provide information about the impact of dSirt6 overexpression on chromatin during aging and the relative impact on gene expression and lifespan, and also general insight into the relationship between aging, chromatin state, and lifespan. Aim 3 will use both targeted and unbiased screens to identify other genes that regulate dSirt6 function. The results of these experiments will provide better mechanistic insight into how dSirt6 extends lifespan, and potentially allow us to extend it even further than with dSirt6 overexpression alone. In addition, this aim is designed to provide the applicant with a wide array of data and directions to serve as the basis for future grant proposals. The proposed research complements the training goal of this proposal: to provide the applicant with advanced skills transgenic fly construction and transcriptomic and epigenomic analyses, with guidance from expert mentors. Transgenic organisms and genome-wide analyses are at the forefront of aging research, and can be applied together to gain deep insight into the molecular causes and consequences of aging. The training activities of this proposal include individualized hands-on training, attendance of training workshops, didactic coursework, and individual and team mentoring in professional development. The proposed research will advance our understanding and ability to treat age-related disease and - together with the proposed training activities - will provide me with the resources and tools to transition into an independent career dedicated to understanding and treating age-related diseases and disorders at the molecular level.