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NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in Mus musculus.

ABSTRACT: Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock, yet the responsible mechanisms are not well understood. We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity, with phenotypes similar to genetic NRF2 activation. Loss of Nrf2 function in mouse fibroblasts, hepatocytes and liver also altered circadian rhythms, suggesting that NRF2 stoichiometry and/or timing of expression are important to timekeeping in some cells. Consistent with this concept, activation of NRF2 at a circadian time corresponding to the peak generation of endogenous oxidative signals resulted in NRF2-dependent reinforcement of circadian amplitude. In hepatocytes, activated NRF2 bound specific enhancer regions of the core clock repressor gene Cry2, increased Cry2 expression and repressed CLOCK/BMAL1-regulated E-box transcription. Together these data indicate that NRF2 and clock comprise an interlocking loop that integrates cellular redox signals into tissue-specific circadian timekeeping.

SUBMITTER: Wible RS

PROVIDER: S-EPMC5826263 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in <i>Mus musculus</i>.

Wible Ryan S RS Ramanathan Chidambaram C Sutter Carrie Hayes CH Olesen Kristin M KM Kensler Thomas W TW Liu Andrew C AC Sutter Thomas R TR

eLife 20180226

Diurnal oscillation of intracellular redox potential is known to couple metabolism with the circadian clock, yet the responsible mechanisms are not well understood. We show here that chemical activation of NRF2 modifies circadian gene expression and rhythmicity, with phenotypes similar to genetic NRF2 activation. Loss of <i>Nrf2</i> function in mouse fibroblasts, hepatocytes and liver also altered circadian rhythms, suggesting that NRF2 stoichiometry and/or timing of expression are important to ...[more]

PMID: 29481323

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Project description:The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

Project description:The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period--a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations.

Dataset Information

NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in Mus musculus.

Publications

NRF2 regulates core and stabilizing circadian clock loops, coupling redox and timekeeping in <i>Mus musculus</i>.

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