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Updated: Jun 1, 2026
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Published on: September 27, 2012
Biochemical analysis of the canonical model for the mammalian circadian clock
Rui Ye1, Christopher P Selby, Nuri Ozturk
1Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Abstract:
The current consensus model for the circadian clock in mammals is based on a transcription-translation feedback loop. In this model, CRY and PER proteins repress their own transcription by suppressing the transactivator function of the CLOCK:BMAL1 heterodimer directly (physical model) and by facilitating post-translational modifications (chemical model). Most of the data for this model come from genetic and cell biological experiments. Here, we have purified all of the core clock proteins and performed in vitro and in vivo biochemical experiments to test the physical model. We find that CLOCK:BMAL1 binds to an E-box sequence in DNA and that CRY binds stably to the CLOCK:BMAL1:E-box ternary complex independently of PER. Both CRY and PER bind to CLOCK and BMAL1 off DNA but, in contrast to CRY, PER does not bind to the CLOCK:BMAL1:E-box complex. Unexpectedly, PER actually interferes with the binding of CRY to the CLOCK:BMAL1:E-box ternary complex. CRY likely destabilizes the CLOCK:BMAL1 heterodimer on DNA by a post-translational mechanism after binding to the complex. These findings support some aspects of the canonical model, but also suggest that some key features of the model need to be revised.
Insights
Mammalian circadian clock models need revision. New biochemical data show CRY protein stabilizes the CLOCK:BMAL1 complex on DNA, while PER protein interferes with this interaction, challenging current understanding of the feedback loop.
Area of Science:
- Molecular Biology
- Biochemistry
- Chronobiology
Background:
- The mammalian circadian clock operates via a transcription-translation feedback loop.
- Core clock proteins CRY and PER are known to repress transcription mediated by the CLOCK:BMAL1 heterodimer.
- Existing models are primarily based on genetic and cell biological data.
Purpose of the Study:
- To biochemically test the physical model of circadian clock regulation.
- To elucidate the precise interactions between core clock proteins (CLOCK, BMAL1, CRY, PER) and DNA.
- To investigate the roles of CRY and PER in repressing CLOCK:BMAL1 transcriptional activity.
Main Methods:
- Purification of core circadian clock proteins.
- In vitro biochemical assays to study protein-DNA and protein-protein interactions.
- In vivo biochemical experiments to validate findings.
Main Results:
- CLOCK:BMAL1 heterodimer binds to E-box DNA sequences.
- CRY protein binds stably to the CLOCK:BMAL1:E-box ternary complex independently of PER.
- PER protein does not bind the CLOCK:BMAL1:E-box complex and interferes with CRY binding; CRY may destabilize CLOCK:BMAL1 on DNA via post-translational modification.
Conclusions:
- Findings support aspects of the canonical circadian clock model.
- Key features of the physical model, particularly the roles of CRY and PER in repressing CLOCK:BMAL1 activity, require revision.
- Biochemical data provide a refined understanding of core clock protein interactions and circadian rhythm regulation.
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