Friday, May 23, 2025
5/23/2025
Mechanisms of Circadian Repression - 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 circadian time and control overt rhythms via transcriptional feedback loops (TFLs). Because clock dysfunction negatively impacts human health, characterizing mechanisms that drive TFLs is of critical importance. The goal of this proposal is to understand how feedback repression, a key event controlling rhythmic transcription, is achieved using two complementary model systems; the monarch butterfly and the fruit fly Drosophila melanogaster. Animals possess two TFL paradigms with orthologous components: A Drosophila-like (dl) paradigm in which CLOCK (CLK) activates and PERIOD (PER) represses transcription, and a mammal-like paradigm (ml) in which CLK-BMAL1 activates and PER-CRYPTOCHROME (PER-CRY) complexes repress transcription. Monarch butterflies have an ml clock, but unlike mammals, monarchs carry single copies of clock activator and repressor genes, thus making it an attractive model to dissect clock mechanisms relevant to mammals. Common features of dl and ml clocks are that PER complexes containing CASEIN KINASE 1 (CK1) initiate transcriptional repression `on-DNA' by binding CLK complexes on E-box elements, followed by CK1-dependent PER and CLK phosphorylation, removal of PER-CLK complexes from E-boxes to initiate `off-DNA' repression, and ultimately PER degradation. How PER orchestrates transcriptional repression is poorly understood. We recently identified a region in CLK that acts as a conserved molecular hub to coordinate transcription activation and repression. The TRITHORAX (TRX) histone methyltransferase, which activates transcription by binding this hub, is also essential for repressing transcription by permitting CLK-PER binding. TRX mediates repression by directly or indirectly methylating the chaperonin HSP68, which is required for CLK-PER binding and repression. We also discovered that CLOCK-Interacting Protein Circadian (CIPC) also binds the CLK hub to repress transcription across animals. CIPC and TRX binding to the CLK hub suggests that CIPC inhibits transcription by displacing TRX, altering TRX substrate specificity to permit HSP68 R45 methylation, promoting PER-CLK binding, removing CLK-CYC from DNA and/or promoting co-repressor binding. These hypothetical CIPC functions will be tested in Aim 1. Our discovery that TRX methylation of HSP68 is required for PER-CLK binding and repression suggests that HSP68 acts in concert with progressive phosphorylation of unstructured PER and CLK regions to efficiently drive sequential structural changes that control DNA binding and protein interactions needed to maintain a ~24h circadian cycle. This hypothesis will be tested in Aim 2. 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 diseases and ailments associated with clock dysfunction.
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