Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock

Rui, Yong; Selby, Christopher P.; Öztürk, Nuri; Annayev, Yunus; Sancar, Aziz

doi:10.1074/jbc.m111.254680

Cited by 113 publications

(148 citation statements)

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“…However, both small molecule (18) and genetic experiments (19) have demonstrated the independent period effects of CKI and FBXL3 posttranslational regulations, as the inhibition of one pathway does not diminish the sensitivity of the other. This observation may be explained by a recent clarification of the canonical clock feedback circuit, where dissociated CRY was revealed as the dominant repressor of CLOCK-BMAL1-mediated E box transcription (5). This distinction helps differentiate between the roles of the otherwise similar PER and CRY proteins, in which the main role of PER in transcriptional repression is likely regulating the timing of CRY's nuclear accumulation.…”

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confidence: 99%

Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock

John

Hirota

Kay

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Posttranslational regulation of clock proteins is an essential part of mammalian circadian rhythms, conferring sensitivity to metabolic state and offering promising targets for pharmacological control. Two such regulators, casein kinase 1 (CKI) and F-box and leucinerich repeat protein 3 (FBXL3), modulate the stability of closely linked core clock proteins period (PER) and cryptochrome (CRY), respectively. Inhibition of either CKI or FBXL3 leads to longer periods, and their effects are independent despite targeting proteins with similar roles in clock function. A mechanistic understanding of this independence, however, has remained elusive. Our analysis of cellular circadian clock gene reporters further differentiated between the actions of CKI and FBXL3 by revealing opposite amplitude responses from each manipulation. To understand the functional relationship between the CKI-PER and FBXL3-CRY pathways, we generated robust mechanistic predictions by applying a bootstrap uncertainty analysis to multiple mathematical circadian models. Our results indicate that CKI primarily regulates the accumulating phase of the PER-CRY repressive complex by controlling the nuclear import rate, whereas FBXL3 separately regulates the duration of transcriptional repression in the nucleus. Dynamic simulations confirmed that this spatiotemporal separation is able to reproduce the independence of the two regulators in period regulation, as well as their opposite amplitude effect. As a result, this study provides further insight into the molecular clock machinery responsible for maintaining robust circadian rhythms.identifiability analysis | gene regulation | sensitivity analysis C ircadian rhythms are autonomous, near-24-h oscillations that coordinate daily changes in physiology and metabolism. Because circadian and metabolic regulators are tightly integrated, circadian disruptions often manifest in metabolic disease (1). Recent efforts have therefore sought to gain a mechanistic understanding of these pathways, such that the metabolic burdens imposed by a 24-h society might be mitigated. Posttranslational regulators, which play key roles in connecting circadian and metabolic processes, serve as likely targets for future therapeutics, demonstrated by the wealth of available circadian-active small molecules (2, 3).Oscillations in circadian gene transcription are generated through a time-delayed transcription-translation negative-feedback loop. In mammals, transcription factors CLOCK and BMAL1 promote transcription of E box-containing genes Period (Per) and Cryptochrome (Cry) (Fig. 1A). PER and CRY protein products form heterodimers to accumulate in the nucleus, in which PER is stoichiometrically limiting (4), and subsequently close the negative feedback loop by inhibiting CLOCK-BMAL1-promoted gene expression. Although steady-state endpoint assays have shown the possibility of nuclear entry of CRY without PER (5-7), experiments from Per1 −/− Per2 −/− mice demonstrated that PER proteins are required for the timely nuclear accumulation of ...

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confidence: 99%

Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock

John

Hirota

Kay

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…1d) appears to be responsible for transcriptional repression in Drosophila and mammals [77][78][79][80][81][82][83]. Specifically, in these organisms, repressors sequester activators in a 1:1 stoichiometric complex, which inhibits the transcriptional activity of activators.…”

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confidence: 99%

Protein sequestration versus Hill‐type repression in circadian clock models

Kim¹

2016

IET syst. biol.

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Circadian (~24hr) clocks are self-sustained endogenous oscillators with which organisms keep track of daily and seasonal time. Circadian clocks frequently rely on interlocked transcriptionaltranslational feedback loops to generate rhythms that are robust against intrinsic and extrinsic perturbations. To investigate the dynamics and mechanisms of the intracellular feedback loops in circadian clocks, a number of mathematical models have been developed. The majority of the models use Hill functions to describe transcriptional repression in a way that is similar to the Goodwin model. Recently, a new class of models with protein sequestration-based repression has been introduced. Here, we discuss how this new class of models differs dramatically from those based on Hill-type repression in several fundamental aspects: conditions for rhythm generation, robust network designs and the periods of coupled oscillators. Consistently, these fundamental properties of circadian clocks also differ among Neurospora, Drosophila, and mammals depending on their key transcriptional repression mechanisms (Hill-type repression or protein sequestration). Based on both theoretical and experimental studies, this review highlights the importance of careful modeling of transcriptional repression mechanisms in molecular circadian clocks.

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“…However, its physical participation in the repressive complex has been controversial (Zheng et al 1999;Miki et al 2012). The biochemical study (Ye et al 2011) revealed that PER (PER1 or PER2) does not bind to the CLOCK-BMAL1-E-box complex and, importantly, provided the first evidence for a ternary CLOCK-BMAL1-CRY complex in vitro and in vivo that is incompatible with PER binding . Most surprisingly, in vitro, it was found that PER causes the dissociation of CRY from the CLOCK-BMAL1-E-box complex (Ye et al 2011), suggesting that it may actually interfere with E-box repression by CRY.…”

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confidence: 99%

“…model has provided a framework for molecular clock research, the mechanism of action of the core clock proteinsin particular the roles of PERs and CRYs, which make up the negative arm of the loop-has remained ill-defined. A recent comprehensive biochemical study with the four core clock proteins and in vivo chromatin immunoprecipitation (ChIP) and transcription analyses revealed an unexpected fact that is inconsistent with the canonical model: CRY binds to the CLOCK-BMAL1-E-box complex in vitro and in vivo independent of PER and inhibits transcription (Ye et al 2011). A subsequent genome-wide ChIP study (Koike et al 2012) and a small molecule inhibitor/computational modeling study (St John et al 2014) supported this finding; namely, that CRY is the dominant repressor in the TTFL.…”

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confidence: 99%

“…The biochemical study (Ye et al 2011) revealed that PER (PER1 or PER2) does not bind to the CLOCK-BMAL1-E-box complex and, importantly, provided the first evidence for a ternary CLOCK-BMAL1-CRY complex in vitro and in vivo that is incompatible with PER binding . Most surprisingly, in vitro, it was found that PER causes the dissociation of CRY from the CLOCK-BMAL1-E-box complex (Ye et al 2011), suggesting that it may actually interfere with E-box repression by CRY. However, genome-wide ChIP experiments have indicated that PER actively participates in repression by binding to E-box promoters in a multiprotein complex that In this highly simplified model, only the core TTFL is shown: The CLOCK-BMAL1 heterodimer binds to the E-boxes in Per and Cry genes and activates their transcription.…”

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confidence: 99%

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Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock

et al. 2014

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The mammalian circadian clock is based on a transcription-translation feedback loop (TTFL) in which CLOCK and BMAL1 proteins act as transcriptional activators of Cryptochrome and Period genes, which encode proteins that repress CLOCK-BMAL1 with a periodicity of~24 h. In this model, the mechanistic roles of CRY and PER are unclear. Here, we used a controlled targeting system to introduce CRY1 or PER2 into the nuclei of mouse cells with defined circadian genotypes to characterize the functions of CRY and PER. Our data show that CRY is the primary repressor in the TTFL: It binds to CLOCK-BMAL1 at the promoter and inhibits CLOCK-BMAL1-dependent transcription without dissociating the complex (''blocking''-type repression). PER alone has no effect on CLOCK-BMAL1-activated transcription. However, in the presence of CRY, nuclear entry of PER inhibits transcription by displacing CLOCK-BMAL1 from the promoter (''displacement''-type repression). In light of these findings, we propose a new model for the mammalian circadian clock in which the negative arm of the TTFL proceeds by two different mechanisms during the circadian cycle.

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Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock

Cited by 113 publications

References 49 publications

Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock

Spatiotemporal separation of PER and CRY posttranslational regulation in the mammalian circadian clock

Protein sequestration versus Hill‐type repression in circadian clock models

Dual modes of CLOCK:BMAL1 inhibition mediated by Cryptochrome and Period proteins in the mammalian circadian clock

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