Circadian rhythm is an endogenous, self-sustainable oscillation that participates in regulating organisms’ physiological activities. Key to this oscillation is a negative feedback by the main clock components Periods and Cryptochromes that repress the transcriptional activity of BMAL1/CLOCK (defined in the Abbreviations) 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 and a region on the C-terminus of BMAL1 where Cryptochrome and CBP (defined in the Abbreviations) bind. Our resultsindicate that CHRONO and PER differentially function as BMAL1/CLOCK-dependent repressors. Besides, the N-terminus of CHRONO is important for its nuclear localization. We further develop a repression model of how PER, CRY, and CHRONO proteins associate with BMAL1, respectively.

AbstractCircadian 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.

Bipolar disorder (BPD) is a psychiatric disorder characterized by alternate episodes of mania and depression. Disruption of normal circadian clock and abnormal sleep cycles are common symptoms of BPD patients. Lithium salt is currently an effective clinical therapeutic drug for BPD. Animal and cellular studies have found that lithium salt can upregulate the expression of the clock gene Per2 , but the mechanism is unknown. We aim to understand the mechanism underlying the Per2 upregulation by lithium treatment. By taking approaches of both comparative transcriptome analysis and comparative qPCR analysis between human and murine cells, Lumicycle assay, luciferase assay and RT-qPCR assay showed that lithium could significantly upregulate the expression of Per2 in both mouse and human cells, and significantly inhibit the expression of E4bp4 , which encodes a transcriptional inhibitor of Per2 . After knocking out the cis-element upstream on the Per2 promoter that responds to E4BP4, the upregulation effect on Per2 by lithium disappeared. When E4bp4 gene was knocked out, the upregulation effect on Per2 by lithium salt disappeared. This study has found that lithium upregulates Per2 expression by reducing the expression of transcription factor E4BP4, but the mechanism of lithium salt downregulation of E4BP4 remains to be further studied. Our study provides a new therapeutic target and approaches for treating BPD.

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