PROJECT SUMMARY Robust daily biological rhythms are key hallmarks of animal healthspan and are strongly regulated by circadian clocks. These cell-autonomous molecular timers enable animals to adapt to predictable daily changes in their environment. Clock-controlled outputs are all-encompassing and clock disruption is associated with a wide range of pathologies and chronic diseases. In the natural world, environmental signals, e.g. light and temperature, enable animal circadian clocks to control timing of food intake. Nutrient influx can therefore provide metabolic signals to reinforce environmental signals, promoting synchrony in cellular physiology to balance metabolism and energy use. Efforts to understand the underpinnings of circadian clocks and their control over daily biological rhythms have long focused on regulation at the transcriptional level, as core oscillator proteins are transcription factors that collaborate to govern rhythmic expression of genes involved in diverse cellular processes. More recent studies have uncovered complementary non- transcriptional mechanisms, including protein post-translational modifications (PTMs), that are critical for robust daily rhythms. The overall goal of this project is to advance our understanding of the role of nutrient-dependent PTMs in mediating metabolic regulation of time-of-day-specific protein functions to orchestrate daily biological rhythms. We will use the diurnal Drosophila model to test the central hypothesis that metabolic signals from clock-controlled feeding activity and cellular metabolism regulate rhythmic S-palmitoylation of cellular proteins, and S-palmitoylation is necessary for maintenance of robust daily biological rhythms. S-palmitoylation is the only reversible lipid PTM; it is the attachment of palmitate, a saturated fatty acid, to cysteines. S- palmitoylation targets a wide range of proteins from transcription factors to membrane receptors, and is known to alter their stability, activity, localization, and protein-protein interactions. The specific aims of this project are to investigate the mechanisms by which clock-dependent metabolic signals regulate S-palmitoylation rhythms and how these rhythms are impacted by nutritional stress (Aim 1); to identify proteins that exhibit daily rhythms in S-palmitoylation (Aim 2); and to determine if palmitoylation regulates daily biology rhythms (Aim 3). This project will advance our long-term goal to integrate post-translational regulatory pathways and obtain a comprehensive understanding of how daily biological rhythms are regulated by diet, nutrition, and timing of metabolic input. This project will have broad significance as misregulation in S- palmitoylation has been linked to a plethora of human diseases including cancer, metabolic, neurological, and immunological disorders.