CHRONO participates in multi-modal repression of circadian transcriptional complexes

Abstract

AbstractThe mammalian protein CHRONO was previously identified to be a rhythmically expressed repressor of the circadian transcriptional activator complex CLOCK:BMAL1. Mice and cells lacking CHRONO display a lengthened circadian period and altered circadian gene expression. Currently, however, we lack specific mechanistic understanding of CHRONO’s activity and function. Here we define an evolutionarily conserved minimal repressive domain (MRD) of CHRONO and demonstrate this domain’s capacity to repress CLOCK:BMAL1 activity through interaction with the BMAL1 C-terminal transactivation domain (TAD). Notably, this binding region overlaps with the binding site for CRY and coactivators CBP/p300, with CHRONO capable of competing with both of these classical regulators of BMAL1 for TAD binding, highlighting this as a hotspot for BMAL1 regulation.Additionally, we investigate the previously unexplored interaction between CHRONO and another major circadian repressor, 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, adding another layer to our understanding of PERIOD protein regulation. Taken together, these data show a more substantive role for CHRONO within molecular circadian timekeeping than previously posited and provide a platform for further investigation into CHRONO’s role within the circadian repressive complex.