Nicolas Cermakian

Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors.

Circadian rhythms result from feedback loops involving clock genes and their protein products. In mammals, 2 orphan nuclear receptors, REV-ERBα and RORα, play important roles in the transcription of the clock gene Bmal1. The authors now considerably extend these findings with the demonstration that all members of the REV-ERB (α and β) and ROR (α, β, and γ) families repress and activate Bmal1 transcription, respectively. The authors further show that transcription of Bmal1 is the result of competition between REV-ERBs and RORs at their specific response elements (RORE). Moreover, they demonstrate that Reverb genes are similarly expressed in the thymus, skeletal muscle, and kidney, whereas Ror genes present distinct expression patterns. Thus, the results indicate that all members of the REV-ERB and ROR families are crucial components of the molecular circadian clock. Furthermore, their strikingly different patterns of expression in nervous and peripheral tissues provide important insights into functional differences between circadian clocks within the organism.

Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK/BMAL1 activity

Circadian rhythmicity in mammals is under the control of a molecular pacemaker constituted of clock gene products organized in transcriptional autoregulatory loops. Phase resetting of the clock in response to light involves dynamic changes in the expression of several clock genes. The molecular pathways used by light to influence pacemaker-driven oscillation of clock genes remain poorly understood. We explored the functional integration of both light- and clock-responsive transcriptional regulation at the promoter level of the Period (Per) genes. Three Per genes exist in the mouse. Whereas mPer1 and mPer2 are light-inducible in clock neurons of the hypothalamic suprachiasmatic nucleus, mPer3 is not. We have studied the promoter structure of the three mPer genes and compared their regulation. All three mPer promoters contain E-boxes and respond to the CLOCK/brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein 1 (BMAL1) heterodimer. On the other hand, only mPer1 and mPer2 promoters contain bona fide cAMP-responsive elements (CREs) that bind CRE-binding protein (CREB) from suprachiasmatic nucleus protein extracts. The mPer1 promoter is responsive to synergistic activation of the cAMP and mitogen-activated protein kinase pathways, a physiological response that requires integrity of the CRE. In contrast, activation of mPer promoters by CLOCK/BMAL1 occurs regardless of an intact CRE. Altogether, these results constitute strong evidence that CREB acts as a pivotal endpoint of signaling pathways for the regulation of mPer genes. Our results reveal that signaling-dependent activation of mPer genes is distinct from the CLOCK/BMAL1-driven transcription required within the clock feedback loop.

Molecular circadian rhythms in central and peripheral clocks in mammals

The last decade has seen tremendous progress in our understanding of the organization and function of the circadian clock. A number of so‐called clock genes were discovered, and these genes and their protein products were shown to organize into feedback loops to give a near 24 h rhythmicity. However, the mechanism is much more complicated. First, many new clock components have been identified, increasing both our understanding and the overall complexity of the mechanism. Second, there is now evidence that transcription may not play a central role in determining the functioning of the clock: the identification of post‐translational modifications of the clock proteins has revealed new levels of control. Finally, chromatin remodeling seems to be crucial in the regulation of the expression of major clock components. This review describes the recent advances in our knowledge of the molecular clockwork in mammals; in particular, the contribution of new clock components and of post‐transcriptional and post‐translational events to circadian timekeeping are discussed.

THE CROSSTALK BETWEEN PHYSIOLOGY AND CIRCADIAN CLOCK PROTEINS

In mammals, many physiological processes present diurnal variations, and most of these rhythms persist even in absence of environmental timing cues. These endogenous circadian rhythms are generated by intracellular timing mechanisms termed circadian clocks. In mammals, the master clock is located in the suprachiasmatic nuclei (SCN), but other brain regions and most peripheral tissues contain circadian clocks. These clocks are responsive to environmental cues, in particular light/dark and feeding/fasting cycles. In the last few years, tissue-specific knock-out and transgenic mouse models have helped to define the physiological roles of specific clocks. Recent reports indicate that the clock-physiology connection is bi-directional, and physiological cues, in particular the energetic status of the cell, can feed into the clockwork. This effect was discovered unexpectedly in molecular analyses of clock protein modifications. Beyond the positive and negative transcription/translation feedback loops of the molecular oscillator lies another level of complexity. Post-translational modifications of clock proteins are both critical for the timing of the clock feedback mechanism and to provide regulatory fine-tuning. This review summarizes recent advances in our understanding of the roles of peripheral clocks and of post-translational modifications occurring on clock proteins. These two matters are at the intersection of physiology, metabolism, and the circadian system. (Author correspondence: nicolas.cermakian@mcgill.ca)

A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock

The primary hallmark of circadian clocks is their ability to entrain to environmental stimuli. The dominant, and therefore most physiologically important, entraining stimulus comes from environmental light cycles. Here we describe the establishment and characterization of a new cell line, designated Z3, which derives from zebrafish embryos and contains an independent, light-entrainable circadian oscillator. Using this system, we show distinct and differential light-dependent gene activation for several central clock components. In particular, activation of Per2 expression is shown to be strictly regulated and dependent on light. Furthermore, we demonstrate that Per1, Per2, and Per3 all have distinct responses to light–dark (LD) cycles and light-pulse treatments. We also show that Clock, Bmal1, and Bmal2 all oscillate under LD and dark–dark conditions with similar kinetics, but only Clock is significantly induced while initiating a light-induced circadian oscillation in Z3 cells that have never been exposed to a LD cycle. Finally, our results suggest that Per2 is responsible for establishing the phase of a circadian rhythm entraining to an alternate LD cycle. These findings not only underscore the complexity by which central clock genes are regulated, but also establishes the Z3 cells as an invaluable system for investigating the links between light-dependent gene activation and the signaling pathways responsible for vertebrate circadian rhythms.

Circadian regulation of cell cycle and apoptosis proteins in mouse bone marrow and tumor

Proapoptotic drugs such as docetaxel displayed least toxicity and highest antitumor efficacy following dosing during the circadian rest phase in mice, suggesting that cell cycle and apoptotic processes could be regulated by the circadian clock. In study 1, mouse bone marrow and/or tumor were obtained every 4 h for 24 h in C3H/HeN mice with or without MA13/C mammary adenocarcinoma in order to determine the circadian patterns in cell‐cycle phase distribution and BCL‐2 anti‐apoptotic protein expression. In study 2, mouse bone marrow from B6D2F1 mice was sampled every 3 h for 24 h in order to confirm the BCL‐2 rhythm and to study its relation with 24 h changes in the expression of proapoptotic BCL‐2‐associated X protein (BAX) protein and clock genes mPer2, mBmal1, mClock, and mTim mRNAs. The rhythms in G1‐, S‐ or G2/M‐phase cells were shifted in tumor compared with bone marrow. In the tumor, the mean proportion of G2/M‐phase cells increased by 75% from late rest to late activity span (P from cosinor = 0.001). No 24 h rhythm was found for BCL‐2 in tumors. In contrast to this, in the bone marrow, mean BCL‐2 expression varied 2.8‐fold in B6D2F1 mice (P=0.025) and 3‐ or 4.5‐fold in tumor‐bearing and nontumor‐bearing C3H/HeN mice, with a peak during the early rest span (P=0.024 and P<0.001, respectively). BAX varied fivefold during the 24 h span with a major peak occurring near mid‐activity (P=0.007). The mean mRNAs of mPer2, mClock, and mBmal1 varied twofold to threefold over the 24 h, with high values during the activity span (P<0.05). In the tumor, the circadian organization in cell‐cycle phase distribution was shifted and BCL2 rhythm was ablated. Conversely, a molecular circadian clock likely regulated BCL‐2 and BAX expression in the bone marrow, increasing cellular protection against apoptosis during the rest span.

A molecular perspective of human circadian rhythm disorders

A large number of physiological variables display 24-h or circadian rhythms. Genes dedicated to the generation and regulation of physiological circadian rhythms have now been identified in several species, including humans. These clock genes are involved in transcriptional regulatory feedback loops. The mutation of these genes in animals leads to abnormal rhythms or even to arrhythmicity in constant conditions. In this view, and given the similarities between the circadian system of humans and rodents, it is expected that mutations of clock genes in humans may give rise to health problems, in particular sleep and mood disorders. Here we first review the present knowledge of molecular mechanisms underlying circadian rhythmicity, and we then revisit human circadian rhythm syndromes in light of the molecular data.

Crosstalk between the circadian clock circuitry and the immune system

Various features, components, and functions of the immune system present daily variations. Immunocompetent cell counts and cytokine levels present variations according to the time of day and the sleep-wake cycle. Moreover, different immune cell types, such as macrophages, natural killer cells, and lymphocytes, contain a circadian molecular clockwork. The biological clocks intrinsic to immune cells and lymphoid organs, together with inputs from the central pacemaker of the suprachiasmatic nuclei via humoral and neural pathways, regulate the function of cells of the immune system, including their response to signals and their effector functions. Consequences of this include, for example, the daily variation in the response to an immune challenge (e.g., bacterial endotoxin injection) and the circadian control of allergic reactions. The circadian-immune connection is bidirectional, because in addition to this circadian control of immune functions, immune challenges and immune mediators (e.g., cytokines) were shown to have strong effects on circadian rhythms at the molecular, cellular, and behavioral levels. This tight crosstalk between the circadian and immune systems has wide-ranging implications for disease, as shown by the higher incidence of cancer and the exacerbation of autoimmune symptoms upon circadian disruption. (Author correspondence: g.mazzoccoli@operapadrepio.it)

Environmental stimulus perception and control of circadian clocks.

Circadian rhythms are regulated by clocks located in specific structures of the central nervous system, such as the suprachiasmatic nucleus (SCN) in mammals, and by peripheral oscillators present in various other tissues. Recent discoveries have elucidated the control of central and peripheral clocks by environmental signals. The major synchroniser in animals is light. In mammals, a subset of retinal ganglion cells receive light signals that are transmitted to the SCN via the retinohypothalamic tract. Photoreception is probably elicited by a novel opsin, melanopsin, although cryptochromes may also play a role. These signals feed directly to the SCN master clock, which then provides timing cues to peripheral clocks. In contrast to mammals, peripheral tissues in the fly and in the fish are directly photoreceptive. However, alternative routes exist. Some peripheral clocks in mammals can be specifically entrained in an SCN-independent manner by restricting food during the light period.

Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression.

CLOCK and BMAL1 [brain and muscle ARNT (arylhydrocarbon receptor nuclear translocator)-like protein 1] are central components of the molecular clock in mammals and belong to the bHLH (basic helix-loop-helix)/PAS [PER (Period)/ARNT/SIM (single-minded)] family. Features of their dimerization have never been investigated. Here, we demonstrate that PAS domain function requires regions extending over the short PAS core repeats. Strikingly, while deleting PAS core repeats does not overtly affect dimerization, it abolishes the transcriptional activity of the heterodimer. Interestingly, these deletions also abolish co-dependent phosphorylation of CLOCK and BMAL1, suggesting a link between the phosphorylation status of the heterodimer and its transactivation potential. We demonstrate that NPAS2 (neuronal PAS domain protein 2) and BMAL2 also undergo similar posttranslational modifications, thereby establishing the mechanism proposed for CLOCK-BMAL1 as a common feature of transcriptional activators in the circadian clock. The discovery of two novel splice variants of BMAL2 confirms the crucial role of the PAS domain and further strengthens the view that co-dependent phosphorylation is of functional significance. In agreement with this, we demonstrate that CRY1-2 (cryptochromes 1-2) affect transactivation and phosphorylation of transcriptional activators of the clock. Furthermore, CRY proteins stabilize the unphosphorylated forms of BMAL1(BMAL2) thereby shifting the phosphorylated/unphosphorylated ratio towards a predominantly unphosphorylated (transcriptionally inactive) form. In contrast, PER proteins, which are weak repressors, are without effect. From these results, we propose a general mechanism for the inhibition of CLOCK(NPAS2)-BMAL1(BMAL2) circadian transcriptional activation by CRY1-2.