CHRONO participates in multi-modal repression of circadian transcriptional complexes

The mammalian protein CHRONO is a rhythmically expressed repressor of the circadian transcriptional activator complex CLOCK:BMAL1, and was proposed to be a novel component of the circadian clock. However, lack of specific mechanistic understanding of the activity and function of CHRONO meant that its role within the circadian machinery was opaque. Here we fill this knowledge gap, confirming an evolutionarily conserved minimal repressive domain (MRD) of CHRONO that interacts with specific regions in the BMAL1 C-terminal transactivation domain (TAD) to repress CLOCK:BMAL1 activity. Notably, this binding region overlaps with the binding site for the repressor CRY and coactivators CBP/p300, with CHRONO capable of competing with both of these classical regulators of BMAL1 for TAD binding, highlighting CHRONO as a direct regulator of BMAL1 function. Additionally, we investigate the interaction between CHRONO and the major circadian repressor, PERIOD2 (PER2). We show that CHRONO reduces PER2 stability through interaction between the CHRONO C-terminus and the Casein Kinase 1 (CK1)-binding domain of PER2. This results in competition between CHRONO and CK1 for binding at this site on PER2, with CHRONO binding inhibiting CK1 phosphorylation of PER2 at the stabilising S662 residue. Taken together, these data show a more substantive and complex role for CHRONO in molecular circadian timekeeping than previously posited, suggesting that CHRONO acts to fine-tune cellular timekeeping by modulating multiple protein-protein interactions that are critical for maintenance of circadian rhythmicity.