Daily rhythms in animal behavior, physiology and metabolism are driven by cell-autonomous clocks in brain neurons and peripheral tissues that keep time and control overt rhythms via transcriptional feedback loops (TFLs). Animals possess two TFL paradigms with orthologous components: a Drosophila-like (dl) paradigm in which transcription is activated by CLOCK-CYCLE (CLK-CYC) and repressed by PERIOD (PER) and a mammal-like (ml) paradigm which is activated by CLOCK-BMAL1 and repressed by PER-CRYPTOCHROME (PER-CRY). The most important event controlling TFLs is arguably feedback repression because the duration and extent of repression determine the phase, period and amplitude of transcriptional rhythms. Common features of dl and ml clocks are that PER complexes containing DOUBLETIME/CASEIN KINASE 1 (DBT/CK1) initiate transcriptional repression ‘on-DNA’ by binding CLK complexes, followed by progressive DBT/CK1-dependent phosphorylation of intrinsically disordered regions (IDRs) in PER and CLK that are thought to regulate their biological function, removal of PER-CLK complexes from DNA to initiate ‘off-DNA’ repression, and ultimately PER degradation. However, how PER orchestrates transcriptional repression remains largely unknown. In a collaborative effort with Dr. Christine Merlin’s lab, we showed that the CLK exon 19 region (CLK e19r) acts as a conserved molecular hub to coordinate transcription activation and repression using two complementary model systems, the monarch butterfly Danaus plexippus and the fruit fly Drosophila melanogaster. The monarch has an ml clock, but unlike mammals, carries single copies of clock activators and repressors, making it an attractive model to dissect clock mechanisms relevant to mammals. We discovered that the TRITHORAX (TRX) histone methyltransferase, which binds CLK e19r to activate transcription by methylating histone 3 on lysine 4, is also essential for repressing transcription. Unexpectedly, we found that monarch TRX mediates repression by directly or indirectly methylating the chaperonin Heat Shock Protein (HSP) 68 on arginine 45 (R45), which then promotes PER-CLK binding and repression. We also found that CLOCK-Interacting Protein Circadian (CIPC) binds CLK e19r to repress transcription across animals. We propose that CIPC binding to CLK e19r inhibits transcription by displacing TRX, thereby altering TRX substrate specificity to permit HSP68 R45 methylation and PER-CLK binding and/or removing CLK-CYC from DNA. We are using molecular genetic, cell biological and biochemical approaches to test whether CIPC carries out one or more of these proposed functions. Based on our discovery that HSP68 is required for PER-CLK binding and repression, we propose that HSP68/70 family proteins and their HSP40 partners contribute to PER-dependent repression and alter PER and CLK conformation in a phosphorylation-dependent manner. We are using genetic, biochemical and structural biology approaches to determine how HSP68/70 and HSP40 drive sequential structural changes in PER and CLK proteins to keep circadian time.