Related Experiment Video
Updated: Nov 23, 2025
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Published on: September 27, 2012
A Sextuple Knockout Cell Line System to Study the Differential Roles of CRY, PER, and NR1D in the
Yi-Ying Chiou1, Tzu-Ying Li1, Yanyan Yang2
1Graduate Institute of Biochemistry, National Chung Hsing University, Taichung City, Taiwan.
Abstract:
The transcription-translation feedback loop (TTFL) is the core mechanism of the circadian rhythm. In mammalian cells, CLOCK-BMAL1 proteins activate the downstream genes by binding on the E-box sequence of the clock-controlled genes. Among these gene products, CRY1, CRY2, PER1, PER2, NR1D1, and NR1D2 can regulate the CLOCK-BMAL1-mediated transcription to form the feedback loop. However, the detailed mechanism of the TTFL is unclear because of the complicated inter-regulation of these proteins. Here, we generated a cell line lacking CRY1, CRY2, PER1, PER2, NR1D1, and NR1D2 (Cry/Per/Nr1d_KO) to study TTFL. We compared the Dbp transcription after serum-shock and dexamethasone-shock between Cry/Per/Nr1d_KO cells and cells expressing endogenous CRY (Per/Nr1d_KO) or NR1D (Cry/Per_KO). Furthermore, we found that CRY1-mediated repression of Dbp could persist more than 24 h in the absence of other proteins in the negative limb of the TTFL. Our Cry/Per/Nr1d_KO cells is a suitable system for the studying of differential roles of CRY, PER, and NR1D in the TTFL.
Insights
This study created a knockout cell line to investigate the circadian rhythm's transcription-translation feedback loop (TTFL). The findings reveal persistent CRY1-mediated repression, aiding the study of TTFL components.
Area of Science:
- Molecular Biology
- Chronobiology
- Genetics
Background:
- The circadian rhythm is regulated by the transcription-translation feedback loop (TTFL).
- Mammalian TTFL involves CLOCK-BMAL1 activating clock-controlled genes, including CRY, PER, and NR1D proteins, which then repress CLOCK-BMAL1 activity.
- The intricate inter-regulation of these proteins complicates a full understanding of the TTFL mechanism.
Purpose of the Study:
- To generate and utilize a novel cell line lacking key TTFL repressors (CRY1, CRY2, PER1, PER2, NR1D1, NR1D2) to dissect the TTFL.
- To compare transcriptional dynamics, specifically Dbp gene expression, in knockout versus partially rescued cell lines following stimuli.
- To elucidate the distinct roles of CRY, PER, and NR1D proteins in regulating the TTFL.
Main Methods:
- Generation of a quadruple knockout cell line (Cry/Per/Nr1d_KO) lacking CRY1, CRY2, PER1, PER2, NR1D1, and NR1D2.
- Comparison of Dbp transcription levels after serum and dexamethasone shock in Cry/Per/Nr1d_KO cells versus Per/Nr1d_KO and Cry/Per_KO cells.
- Analysis of the persistence of CRY1-mediated repression in the absence of other negative feedback loop components.
Main Results:
- The Cry/Per/Nr1d_KO cell line provides a system to study the TTFL without the influence of major repressors.
- CRY1-mediated repression of Dbp transcription was observed to persist for over 24 hours, even without other negative limb proteins.
- Differential roles of CRY, PER, and NR1D proteins in the TTFL were investigated using this system.
Conclusions:
- The generated Cry/Per/Nr1d_KO cell line is a valuable tool for dissecting the mammalian circadian TTFL.
- CRY1 plays a significant role in sustained repression within the TTFL.
- This system facilitates the study of the specific contributions of CRY, PER, and NR1D proteins to circadian rhythm regulation.
