Abstract
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.
Highlights
Most organisms have evolved an endogenous circadian clock to adjust their metabolic activities to anticipate daily environmental events, such as day/night switches or temperature changes
CHR was identified as a circadian clock component that functions as a transcriptional repressor of B/C complexes [13,14]
We performed the same assay by combing individual parts (CHRNH and CHRHC represent the combinations of CHRN, CHRH, and CHRC) and found CHRHC can repress the transcription activity as strongly as the PER2 protein (Figure 1C)
Summary
Most organisms have evolved an endogenous circadian clock to adjust their metabolic activities to anticipate daily environmental events, such as day/night switches or temperature changes. After translation and post-translational modifications, PERs and CRYs translocate into the nucleus to repress BMAL1/CLOCK complex function [4,5]. A second loop composed of circadian components, such as rev-erb (I confirm) [6] and ror [7] and their coding products is involved in regulating the transcription of arntl gene, which encodes the BMAL1 protein. Other clock genes, such as csnk1d, csnk1e, fblx, and fblx, contribute to setting the clock speed [8,9,10,11] or in entraining the clockwork [12]
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