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Updated: Jun 19, 2026
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Published on: September 27, 2012
Circadian clock-controlled intestinal expression of the multidrug-resistance gene mdr1a in mice
Yuichi Murakami1, Yuko Higashi, Naoya Matsunaga
1Pharmaceutics, Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Background & Aims:
P-glycoprotein, the product of the multidrug resistance (mdr) gene, functions as a xenobiotic transporter contributing to the intestinal barrier. Although intestinal expression of the mdr1a gene and its efflux pump function has been shown to exhibit 24-hour variation, the mechanism of the variations remains poorly understood. Here, we demonstrated that the molecular components of the circadian clock act as regulators to control 24-hour variation in the expression of the mdr1a gene.
Methods:
Luciferase reporter assay and gel mobility shift assay were used to study the mechanism of transcriptional regulation of the mdr1a gene by clock gene products. The messenger RNA levels and protein abundances in colon 26 cells and mouse intestine were measured by quantitative real-time polymerase chain reaction and Western blotting, respectively.
Results:
Hepatic leukemia factor (HLF) and E4 promoter binding protein-4 (E4BP4) regulated transcription of the mdr1a gene by competing with each other for the same DNA binding site. Molecular and biochemical analyses of HLF- and E4BP4-down-regulated colon 26 cells and the intestinal tract of Clock mutant mice suggested that these 2 proteins consisted of a reciprocating mechanism in which HLF activated the transcription of the mdr1a gene, whereas E4BP4 periodically suppressed transcription at the time of day when E4BP4 was abundant.
Conclusions:
The intestinal expression of the mdr1a gene is influenced by the circadian organization of molecular clockwork. Our present findings provide a link between the circadian timekeeping system and xenobiotic detoxification.
Insights
The circadian clock regulates daily variations in mdr1a gene expression, impacting intestinal xenobiotic transport. Hepatic leukemia factor and E4BP4 compete to control mdr1a gene transcription.
Area of Science:
- Chronobiology
- Molecular Biology
- Pharmacology
Background:
- P-glycoprotein, encoded by the multidrug resistance (mdr) gene, is a xenobiotic transporter crucial for the intestinal barrier.
- Daily variations in intestinal mdr1a gene expression and P-glycoprotein efflux function are known but mechanistically unclear.
Purpose of the Study:
- To elucidate the mechanism behind the 24-hour variation in mdr1a gene expression.
- To investigate the role of circadian clock components in regulating intestinal mdr1a gene expression.
Main Methods:
- Luciferase reporter and gel mobility shift assays to study transcriptional regulation.
- Quantitative real-time PCR and Western blotting to measure mRNA and protein levels in cells and mouse intestine.
Main Results:
- Hepatic leukemia factor (HLF) and E4 promoter binding protein-4 (E4BP4) compete for the same DNA binding site to regulate mdr1a transcription.
- HLF activates mdr1a transcription, while E4BP4 periodically suppresses it.
- Analyses in colon 26 cells and Clock mutant mice confirmed a reciprocating regulatory mechanism.
Conclusions:
- Circadian clockwork organization dictates intestinal mdr1a gene expression.
- Establishes a link between the body's internal clock and the detoxification of foreign substances.
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