Identification of the Repressive Domain of the Negative Circadian Clock Component CHRONO
Circadian rhythm is an endogenous, self-sustainable oscillation that participates in regulating organisms' physiological activities. Key to this oscillation is a negative feedback by main clock components Periods and Cryptochromes that repress transcriptional activity of BMAL1/CLOCK complexes. In addition, a novel repressor, CHRONO, has been identified recently, but details of CHRONO's function during repressing the circadian cycle remain unclear. Here we report that a domain of CHRONO mainly composed of α-helixes is critical to repression through the exploitation of protein-protein interactions according to luciferase reporter assays, co-immunoprecipitation, immunofluorescence, genome editing and structural information analysis via circular dichroism spectroscopy. This repression is fulfilled by interactions between CHRONO with a region on the BMAL1 C-terminus where Cryptochrome and CBP bind. Our results indicate that CHRONO and PER differentially function as BMALA/CLOCK-dependent repressors. We further suggest a repression model of how PER, CRY and CHRONO proteins associate with BMAL1, respectively.
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