PROJECT SUMMARY Histone proteins package and organize DNA into chromatin, which regulates every DNA-dependent process. Alterations to chromatin structure is a hallmark of aging and is characterized by global changes in gene expression, histone abundance, histone PTM landscape, and chromatin accessibility. I propose that tight control over histone abundance and histone type, which we term histone homeostasis, is essential for normal cellular aging. Cells contain two histone types: canonical histones that are expressed during S phase of the cell cycle, and variant histones that are expressed throughout the cell cycle and in non-dividing cells. Variant histones are of particular interest to aging because they are the only source of new histones in non-dividing cells and accumulate with age. Additionally, variant histone misregulation results in chromatin defects and reduced lifespan. Knowing how canonical and variant histones regulate genome function is integral to understanding normal and pathological chromatin-based aging processes. Canonical histone H3.2 and variant histone H3.3 are some of the most highly conserved proteins across eukaryotes. The high conservation of amino acid differences between canonical H3.2 and variant H3.3 suggests that they may perform unique functions in the genome, yet it is not understood if variant H3.3 function is mediated by its cell-cycle independent expression or unique protein sequence. In Drosophila, I have discovered that variant H3.3 is essential for development when canonical histone gene copy number is reduced, suggesting a previously unknown requirement for coordination between canonical H3.2 and variant H3.3 expression. However, the mechanisms underlying this coordination are unknown. The proposed project tests the hypotheses that variant H3.3 is required for normal chromatin-based aging processes and that cells possess a homeostatic mechanism to maintain the correct relative expression of canonical H3.2 and variant H3.3 throughout development. The aims of this project are to (1) determine the contributions of H3.3 expression versus protein sequence to chromatin-based aging processes and, (2) elucidate the mechanisms that achieve proper H3 abundance through the coordination of canonical and variant histone expression. This work will expand the fundamental understanding of how canonical and variant histones cooperate to regulate genome function during aging.