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A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery.

ABSTRACT: Circadian clocks in mammals are built on a negative feedback loop in which the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1) drives the expression of its own inhibitors, the PERIOD and CRYPTOCHROME proteins. Reactivation of CLOCK-BMAL1 occurs at a specific time several hours after PERIOD and CRYPTOCHROME protein turnover, but the mechanism underlying this process is unknown. We found that mouse BMAL1 complexes include TRAP150 (thyroid hormone receptor-associated protein-150; also known as THRAP3). TRAP150 is a selective coactivator for CLOCK-BMAL1, which oscillates under CLOCK-BMAL1 transcriptional control. TRAP150 promotes CLOCK-BMAL1 binding to target genes and links CLOCK-BMAL1 to the transcriptional machinery at target-gene promoters. Depletion of TRAP150 caused low-amplitude, long-period rhythms, identifying it as a positive clock element. The activity of TRAP150 defines a positive feedback loop within the clock and provides a potential mechanism for timing the reactivation of circadian transcription.

SUBMITTER: Lande-Diner L

PROVIDER: S-EPMC3791755 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery.

Lande-Diner Laura L Boyault Cyril C Kim Jin Young JY Weitz Charles J CJ

Proceedings of the National Academy of Sciences of the United States of America 20130916 40

Circadian clocks in mammals are built on a negative feedback loop in which the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1) drives the expression of its own inhibitors, the PERIOD and CRYPTOCHROME proteins. Reactivation of CLOCK-BMAL1 occurs at a specific time several hours after PERIOD and CRYPTOCHROME protein turnover, but the mechanism underlying this process is unknown. We found that mouse BMAL1 complexes include TRAP150 ...[more]

PMID: 24043798

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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:Controlling bilirubin to a low level is necessary in physiology because of its severe neurotoxicity. Therefore, it is of great interest to understand the regulatory mechanisms for bilirubin homeostasis. In this study, we uncover a critical role for circadian clock in regulation of bilirubin detoxification and homeostasis. Methods: The mRNA and protein levels of Bmal1 (a core clock gene), metabolic enzymes and transporters were measured by qPCR and Western blotting, respectively. Luciferase reporter, mobility shift and chromatin immunoprecipitation were used to investigate transcriptional gene regulation. Experimental hyperbilirubinemia was induced by injection of bilirubin or phenylhydrazine. Unconjugated bilirubin (UCB) and conjugated bilirubin were assessed by ELISA. Results: We first demonstrated diurnal variations in plasma UCB levels and in main bilirubin-detoxifying genes Ugt1a1 and Mrp2. Of note, the circadian UCB levels were antiphase to the circadian expressions of Ugt1a1 and Mrp2. Bmal1 ablation abrogated the circadian rhythms of UCB and bilirubin-induced hepatotoxicity in mice. Bmal1 ablation also decreased mRNA and protein expressions of both Ugt1a1 and Mrp2 in mouse livers, and blunted their circadian rhythms. A combination of luciferase reporter, mobility shift, and chromatin immunoprecipitation assays revealed that Bmal1 trans-activated Ugt1a1 and Mrp2 through specific binding to the E-boxes in the promoter region. Further, Bmal1 ablation caused a loss of circadian time-dependency in bilirubin clearance and sensitized mice to chemical induced-hyperbilirubinemia. Moreover, bilirubin stimulated Bmal1 expression through antagonism of Rev-erbα, constituting a feedback mechanism in bilirubin detoxification. Conclusion: These data supported a dual role for circadian clock in regulation of bilirubin detoxification, generating circadian variations in bilirubin level via direct transactivation of detoxifying genes Ugt1a1 and Mrp2, and defending the body against hyperbilirubinemia via Rev-erbα antagonism. Thereby, our study provided a potential mechanism for management of bilirubin related diseases.

Dataset Information

A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery.

Publications

A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery.

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