Project Summary/Abstract: Circadian rhythms are highly conserved, 24-hour, oscillations that tune human physiology to the day/night cycle, enhancing fitness by ensuring that appropriate activities occur at biologically advantageous times. Disruption of proper circadian synchronization negatively impacts the human long-term medical outlook, making revealing the mechanism underlying circadian regulation over cellular physiology critical to human health. Circadian rhythms are timed via a transcription-translation based negative feedback loop (TTFL), or clock. The current paradigm for circadian regulation over physiology, termed the clocks “output”, is that transcriptional programing generated by the TTFL drives temporally specific waves of gene expression. However, our research has revealed that transcriptional programing cannot wholly account for clock output, as we discovered little to no correlation between mRNAs and proteins that oscillate with a circadian periodicity in fungi and macrophages, revealing robust circadian post-transcriptional control over immunometabolism. While the mechanisms that time this circadian post-transcriptional control are unknown, we have demonstrated the proteins involved in the transcriptionally repressive arm of the clock act as hub proteins for the formation of large macromolecular complexes. Many of the proteins bound to these complexes are circadianly controlled at the post-transcriptional level. Moreover, we established the clock repressive arm proteins have conserved regions of intrinsic protein disorder that correlate with the predicted binding sights of the identified interactors. Our research goal is to validate the hypothesis that circadian post-transcriptional regulation is tuned via the formation of time-of-day defined macromolecular protein complexes centered around the repressive arm proteins. We posit that the formation of these complexes is enabled by conserved intrinsic disorder in the repressive arm proteins, allowing flexibility to widely impart circadian post-transcriptional regulation over immunometabolism. To test our hypothesis, we will create a Conformational/Temporal Interactome (CiTI) map of clock repressive complex proteins in mammals and fungi. We will generate these CiTI maps in a sex and immune-specific manner to define the effect of post-transcriptional circadian control over physiology. As a mechanism for keeping time, circadian feedback loops are highly conserved and much of the definition of clock mechanisms comes from the investigation of model systems. We will therefore exploit cost-effective fungal and mammalian model systems to address our hypotheses. This supplement will support the purchase of a widefield microscope, which will help to address all of the proposed hypotheses above, furthering our long-term goal of elucidating the fundamental principles of circadian timing, revealing the mechanisms of circadian control over cellular physiology.