PROJECT SUMMARY Daily rhythms in animal behavior, physiology and metabolism are driven by cell-autonomous circadian clocks that are synchronized by environmental cycles, but maintain ~24h rhythms even in their absence. These clocks keep time and control overt rhythms via transcriptional feedback loops. Because clock dysfunction negatively impacts human health and well being, it is imperative to define the mechanisms that drive rhythmic transcription. The goal of this proposal is to understand how a critical event controlling rhythmic transcription, i.e. feedback repression, is achieved using two complementary model systems, the monarch butterfly and Drosophila. In animals, two feedback loop paradigms having orthologous components can be discerned: A Drosophila-like (dl) paradigm in which CLOCK (CLK) activates and PERIOD (PER) represses transcription, and a mammal-like paradigm (ml) in which BMAL1 activates and PER-CRYPTOCHROME (PER-CRY) complexes repress transcription. Monarch butterflies possess an ml clock, but unlike mammals, monarchs carry single copies of both circadian activators and repressors, thus making it an attractive model to dissect circadian mechanisms relevant to mammals. Common features of dl and ml clocks are that PER initiates transcriptional repression `on-DNA' by binding CLK complexes present on E-box regulatory elements, and that the subsequent removal of repressor-activator complexes from E-boxes initiates an `off-DNA' repression phase that is maintained for many hours until activators bind E-boxes to reactivate transcription. How PER initiates on-DNA repression and how off-DNA repression is initiated and maintained remains poorly understood. Our preliminary data demonstrate that PER-dependent repression in monarch requires a conserved region encoded by CLK exon 19 (hereafter e19ar), that Drosophila CLOCKWORK ORANGE (CWO) binds E-boxes in antiphase to CLK-CYC, and that loss of cwo increases trough levels of CLK-CYC binding. Based on published and preliminary data, we will pursue two aims to determine 1) how PER initiates on-DNA repression of CLK- BMAL1 and CLK-CYC transcription with a focus on CLKe19ar, and 2) how PER and CWO collaborate to maintain off-DNA transcriptional repression and promote CLK-CYC/CLK-BMAL1 transcription. Successful completion of these aims will provide mechanistic insight into how circadian repression determines the phase, period and amplitude of transcriptional rhythms. Ultimately, such knowledge may be broadly applied for diagnosis and treatment of many diseases and ailments associated with clock dysfunction.