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Updated: May 20, 2026
Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
Published on: September 27, 2012
RORγ directly regulates the circadian expression of clock genes and downstream targets in vivo
Yukimasa Takeda1, Raja Jothi, Veronique Birault
1Cell Biology Section, Systems Biology Group, Biostatistics Branch, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Abstract:
In this study, we demonstrate that the lack of retinoic acid-related orphan receptor (ROR) γ or α expression in mice significantly reduced the peak expression level of Cry1, Bmal1, E4bp4, Rev-Erbα and Per2 in an ROR isotype- and tissue-selective manner without affecting the phase of their rhythmic expression. Analysis of RORγ/RORα double knockout mice indicated that in certain tissues RORγ and RORα exhibited a certain degree of redundancy in regulating clock gene expression. Reporter gene analysis showed that RORγ was able to induce reporter gene activity through the RORE-containing regulatory regions of Cry1, Bmal1, Rev-Erbα and E4bp4. Co-expression of Rev-Erbα or addition of a novel ROR antagonist repressed this activation. ChIP-Seq and ChIP-Quantitative real-time polymerase chain reaction (QPCR) analysis demonstrated that in vivo RORγ regulate these genes directly and in a Zeitgeber time (ZT)-dependent manner through these ROREs. This transcriptional activation by RORs was associated with changes in histone acetylation and chromatin accessibility. The rhythmic expression of RORγ1 by clock proteins may lead to the rhythmic expression of RORγ1 target genes. The presence of RORγ binding sites and its down-regulation in RORγ-/- liver suggest that the rhythmic expression of Avpr1a depends on RORγ consistent with the concept that RORγ1 provides a link between the clock machinery and its regulation of metabolic genes.
Insights
Retinoic acid-related orphan receptor (ROR) gamma and alpha regulate core clock gene expression rhythm. RORs directly control target genes, linking the circadian clock to metabolic processes.
Area of Science:
- Chronobiology
- Molecular Biology
- Gene Regulation
Background:
- The circadian clock regulates numerous physiological processes.
- Retinoic acid-related orphan receptors (RORs) are nuclear receptors implicated in various biological functions.
- The precise role of RORs in regulating circadian clock gene expression remains incompletely understood.
Purpose of the Study:
- To investigate the role of RORγ and RORα in the regulation of core circadian clock gene expression.
- To elucidate the mechanisms by which RORs control the rhythmic expression of clock genes.
- To determine if RORs link the circadian machinery to metabolic gene regulation.
Main Methods:
- Analysis of RORγ/RORα double knockout mice.
- Reporter gene assays to assess RORγ-mediated transcriptional activation.
- Chromatin immunoprecipitation sequencing (ChIP-Seq) and ChIP-quantitative real-time polymerase chain reaction (QPCR) to identify direct RORγ targets.
- Assessment of histone acetylation and chromatin accessibility.
Main Results:
- Lack of RORγ or RORα significantly reduced peak expression of core clock genes (Cry1, Bmal1, E4bp4, Rev-Erbα, Per2) in a tissue-specific manner.
- RORγ and RORα showed redundancy in regulating clock gene expression in certain tissues.
- RORγ directly activated reporter gene activity via ROREs in target genes, which was repressed by Rev-Erbα or an ROR antagonist.
- ChIP-Seq and ChIP-QPCR confirmed direct, Zeitgeber time-dependent RORγ regulation of target genes through ROREs.
- ROR-mediated transcriptional activation involved changes in histone acetylation and chromatin accessibility.
- Rhythmic RORγ expression, driven by clock proteins, likely leads to rhythmic expression of its target genes, including Avpr1a, linking the clock to metabolic regulation.
Conclusions:
- RORγ and RORα play a critical role in regulating the amplitude of core circadian clock gene expression.
- RORγ directly binds to ROREs in target genes, mediating rhythmic transcription and influencing chromatin dynamics.
- RORγ acts as a crucial link between the circadian clockwork and the regulation of metabolic genes, such as Avpr1a.
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