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Entrainment Dissociates Transcription and Translation of a Circadian Clock Gene in Neurospora
Abstract
Circadian systems coordinate the daily sequence of events in cells, tissues, and organisms. In constant conditions, the biological clock oscillates with its endogenous period, whereas it is synchronized to the 24 hr light:dark cycle in nature. Here, we investigate light entrainment of Neurospora crassa to photoperiods that mimic seasonal changes. Clock gene (frequency, or frq) RNA levels directly reflect the light environment in all photoperiods, whereas the FRQ protein follows neither RNA levels nor light transitions. Induction of frq RNA and protein can be dissociated by as much as 6 hr, depending on photoperiod. The phase of entrainment at the physiological level (e.g., asexual spore development) correlates with FRQ protein. Thus, a dissociation of transcription, translation, and protein stability is fundamental to circadian entrainment of Neurospora. Our findings suggest that simple feedback models are insufficient to explain the molecular circadian mechanisms under entrained conditions and that clock control of light input pathways involves posttranscriptional regulation. The regulators mediating the dissociation between RNA and protein levels are still unknown and will be the key to understanding both circadian timing at the molecular level and how the clock exerts control over many cellular processes.
... In constant light, levels of both frq mRNA and FRQ are elevated and arrhythmic, indicating that the circadian clock is not functioning [5], [3]. Dark-to-light transfers induce rapid increases in both frq and wc-1 mRNA levels, while lightto-dark transfers cause rapid decreases in the level of frq [5], [33]. These light-induced changes in mRNA levels provide the molecular basis for the entrainment of the clock by light-dark (LD) cycles [19]. ...
... Experiments have shown that, like many circadian species, Neurospora exhibits systematic variations in the phase of entrainment with photoperiod length [33], [24]. Such behaviour is consistent with the mathematical theory of coupled oscillators [11], [26]. ...
... Figure 6 of the main paper plots simulations of the frq 1 , frq 7 and frq S1531 period-temperature profiles p(T ) obtained by modifying the parameters contributing to the net FRQ loss rates r and r ′ for model 2. All the profiles are in qualitative agreement with experimental data [30]. Table 11 shows the FRQ pathway parameters used to generate the simulations, together with the corresponding values of r and r ′ computed using (33) and (34). For the simulated short-period frq 1 mutant, r and r ′ are seen to be greater than in the wild-type, indicating a decrease in the stability of the FRQ forms. ...
Supplementary Information
... This term acts in two ways. Firstly, it raises the forward rate of the reaction P W P L W in equations (S.4) and (S.5): this models the rise in the relative concentration of FAD-bound WC-1 in complex with the LREs at the frq promoter observed with increasing light levels [5], and the resulting enhanced transcription of frq [6,7]. Through this mechanism, it also increases the transcription rate of wc-1 through the second term of (S.3), reflecting the loss of wc-1 light-responses in wc-1 mutant backgrounds [8]. ...
... The fourth term C ALRLD checks that frq mRNA exhibits both acute dawn and dusk responses and that wc-1 mRNA exhibits an acute dawn response, as reported in [7]: ...
... B. The effect of removing the wc-1 loop on the dusk sensitivity-photoperiod profile of ϕ F RQ . Note the pronounced increase in sensitivity for larger P values as the relative coupling strength a 7 Figure S3: Dependence of the dusk sensitivities plotted in Figure 5 on T-cycle length. Conidiation phase ϕ F RQ has a high sensitivity across the range shown, indicating a dusk-driven response. ...
Supplementary Information. This file contains Supplementary Figures S1-S6 together with details of the modelling, parameter optimisation and sensitivity analysis methods used in this work.
... the study by Tan et al. (2004) has been one of the few efforts to monitor frq dynamics under more realistic photo-cycle conditions. In this article, we have focused on the effects of light on frq expression. ...
... Using frq-luc and classic entraining conditions of 12L:12D, we observe in replicate cultures (Fig. 3) that the waveform of luciferase levels differs significantly from the cosine curve described under free-running conditions (compare with Fig. 1). After each lights-on event in the 12L:12D treatment, there is a dramatic increase in signal, consistent with the reported acute light induction of frq expression (Crosthwaite et al., 1995;Tan et al., 2004). Typically, the waveform during the light stage shows biphasic kinetics for frq-luc. ...
... Such variability is reproducible in all of the replicate plates of an experiment (Fig. 3), but another experiment with the same protocol may show very different behavior (e.g., compare during 12L:12D between h 48 and 84 of Fig. 6A). Following lights-off on a 12L:12D treatment, there is always a dramatic decrease in signal after about a 30-to 60-min delay, consistent with prior biochemical data (Tan et al., 2004). Compared with the free-running rhythm in DD, the peak to trough amplitude of the entrained cycle is much larger (up to 4 times larger). ...
The role of the frq gene in the Neurospora crassa circadian rhythm has been widely studied, but technical limitations have hindered a thorough analysis of frq circadian expression waveform. Through our experiments, we have shown an improved precision in defining Neurospora's circadian rhythm kinetics using a codon optimized firefly luciferase gene reporter linked to a frq promoter. In vivo examination of this real-time reporter has allowed for a better understanding of the relationship of the light responsive elements of the frq promoter to its circadian feedback components. We provide a detailed phase response curve showing the phase shifts induced by a light pulse applied at different points of the circadian cycle. Using the frq-luc reporter, we have found that a 12-h light:12-h dark cycle (12L:12D) results in a luciferase expression waveform that is more complex and higher in amplitude than that seen in free-running conditions of constant darkness (DD). When using a lighting regime more consistent with solar timing, rather than a square wave pattern, one observes a circadian waveform that is smoother, lower in amplitude, and different in phasing. Using dim light in place of darkness in these experiments also affects the resulting waveform and phasing. Our experiments illustrate Neurospora's circadian kinetics in greater detail than previous methods, providing further insight into the complex underlying biochemical, genetic, and physiological mechanisms underpinning the circadian oscillator.
... In the Boolean framework, the times at which solutions switch between 0 and 1 emerge as natural phase measures. For continuous time courses—such as those generated by DE models—the point in the circadian cycle at which the expression level of a molecular species decreases below a set threshold can be employed as a phase marker [25,56,57]. This suggested using the time at which each species decreases below its discretization threshold as a phase measure for the DE simulations, and the time of the 1 ! ...
... 2370 Report. Digital clocks O. E. Akman et al. causing a transition from a predominately dusk-locked system to a dawn-locked one [57]. In particular, the Boolean 2-loop Arabidopsis circuit exactly reproduces the dual light response in the Y gene, in which the acute peak tracks dawn, and the circadian peak tracks dusk. ...
... The success of the logic models in recovering the correct DE configurations from synthetic data suggested that for a fixed abstract topology, our optimization procedure has the capacity to determine the logic network most consistent with a given dataset. We tested this finding further by optimizing the 3-loop Arabidopsis logic circuit to highly sampled experimental time series recorded using luciferase (LUC) imaging in constant light from a wild-type strain [57]. All possible LCs were considered, corresponding to a network inference carried out assuming no prior biological knowledge. ...
The gene networks that comprise the circadian clock modulate biological function across a range of scales, from gene expression to performance and adaptive behaviour. The clock functions by generating endogenous rhythms that can be entrained to the external 24-h day?night cycle, enabling organisms to optimally time biochemical processes relative to dawn and dusk. In recent years, computational models based on differential equations have become useful tools for dissecting and quantifying the complex regulatory relationships underlying the clock's oscillatory dynamics. However, optimizing the large parameter sets characteristic of these models places intense demands on both computational and experimental resources, limiting the scope of in silico studies. Here, we develop an approach based on Boolean logic that dramatically reduces the parametrization, making the state and parameter spaces finite and tractable. We introduce efficient methods for fitting Boolean models to molecular data, successfully demonstrating their application to synthetic time courses generated by a number of established clock models, as well as experimental expression levels measured using luciferase imaging. Our results indicate that despite their relative simplicity, logic models can (i) simulate circadian oscillations with the correct, experimentally observed phase relationships among genes and (ii) flexibly entrain to light stimuli, reproducing the complex responses to variations in daylength generated by more detailed differential equation formulations. Our work also demonstrates that logic models have sufficient predictive power to identify optimal regulatory structures from experimental data. By presenting the first Boolean models of circadian circuits together with general techniques for their optimization, we hope to establish a new framework for the systematic modelling of complex clocks.
... Experimental advances have revealed much information underlying adaptation mechanisms in a response to the environmental cycles as well as mechanisms for the generation of oscillation (1). Light up-regulates the expression of white collar-1 (wc-1) and white collar-2 (wc-2) genes in Neurospora that causes frequency (frq) gene expression to activate78910111213. Indeed, the amount of frq gene transcript reaches about 10 times that under constant darkness (DD) conditions within 30 min [10]. ...
... Light up-regulates the expression of white collar-1 (wc-1) and white collar-2 (wc-2) genes in Neurospora that causes frequency (frq) gene expression to activate78910111213. Indeed, the amount of frq gene transcript reaches about 10 times that under constant darkness (DD) conditions within 30 min [10]. Similarly, light up-regulates the expression of period1 (per1) and period2 (per2) genes in mammals [14– 18]. ...
... Modeling predicts autonomous oscillations can be entrained by light-dark (LD) cycles in which negative feedback regulation on gene expression and induction of gene by light are assumed [28]. However, above-mentioned induction of genes by light does not continue for many hours under constant light89101112131415. This molecular response to light is called light adaptation [1,12,13]. ...
Periods of biological clocks are close to but often different from the rotation period of the earth. Thus, the clocks of organisms must be adjusted to synchronize with day-night cycles. The primary signal that adjusts the clocks is light. In Neurospora, light transiently up-regulates the expression of specific clock genes. This molecular response to light is called light adaptation. Does light adaptation occur in other organisms? Using published experimental data, we first estimated the time course of the up-regulation rate of gene expression by light. Intriguingly, the estimated up-regulation rate was transient during light period in mice as well as Neurospora. Next, we constructed a computational model to consider how light adaptation had an effect on the entrainment of circadian oscillation to 24-h light-dark cycles. We found that cellular oscillations are more likely to be destabilized without light adaption especially when light intensity is very high. From the present results, we predict that the instability of circadian oscillations under 24-h light-dark cycles can be experimentally observed if light adaptation is altered. We conclude that the functional consequence of light adaptation is to increase the adjustability to 24-h light-dark cycles and then adapt to fluctuating environments in nature.
... The conidiation rhythm can be entrained by both light and temperature cycles, exhibiting either systematic or driven entrainment depending on the forcing protocol used[15]. In 24 hr light-dark (LD) cycles, the phase of entrainment (judged by the time of conidiation onset) coincides with the middle of the night in both long and short days[16]. The phase of the clock thus varies systematically with photoperiod: both dusk and dawn signals are integrated to set phase rather than phase being determined solely by either signal alone[15]. ...
... In constant light, levels of both frq mRNA and FRQ are elevated and arrhythmic, indicating that the central FREQUENCY-WHITE COLLAR (FRQ-WC) clock is not functioning[31,32]. In 24 hr LD cycles, acute light responses give rise to frq mRNA profiles that directly reflect the light environment in different photoperiods; by contrast, the FRQ protein profile appears to determine the onset of conidiation[16]. ...
... Furthermore, despite the fact the cost function only assesses goodness-of-fit in simulated 12:12 LD cycles, the optimal solution is a good match to data in long and short days also. As reported experimentally, in all photoperiods for which the clock is stably entrained, frq and wc-1 transcripts exhibit rapid induction at dawn while frq expression falls rapidly at dusk, with both transcripts converging to an equilibrium level during the light phase in long days[16]. By contrast, FRQ protein displays markedly smoother changes in expression level, increasing slowly from a minimum level around dawn to a peak level around dusk before degrading back down to its minimum at a roughly constant rate. ...
Robustness is a central property of living systems, enabling function to be maintained against environmental perturbations. A key challenge is to identify the structures in biological circuits that confer system-level properties such as robustness. Circadian clocks allow organisms to adapt to the predictable changes of the 24-hour day/night cycle by generating endogenous rhythms that can be entrained to the external cycle. In all organisms, the clock circuits typically comprise multiple interlocked feedback loops controlling the rhythmic expression of key genes. Previously, we showed that such architectures increase the flexibility of the clock's rhythmic behaviour. We now test the relationship between flexibility and robustness, using a mathematical model of the circuit controlling conidiation in the fungus Neurospora crassa.
The circuit modelled in this work consists of a central negative feedback loop, in which the frequency (frq) gene inhibits its transcriptional activator white collar-1 (wc-1), interlocked with a positive feedback loop in which FRQ protein upregulates WC-1 production. Importantly, our model reproduces the observed entrainment of this circuit under light/dark cycles with varying photoperiod and cycle duration. Our simulations show that whilst the level of frq mRNA is driven directly by the light input, the falling phase of FRQ protein, a molecular correlate of conidiation, maintains a constant phase that is uncoupled from the times of dawn and dusk. The model predicts the behaviour of mutants that uncouple WC-1 production from FRQ's positive feedback, and shows that the positive loop enhances the buffering of conidiation phase against seasonal photoperiod changes. This property is quantified using Kitano's measure for the overall robustness of a regulated system output. Further analysis demonstrates that this functional robustness is a consequence of the greater evolutionary flexibility conferred on the circuit by the interlocking loop structure.
Our model shows that the behaviour of the fungal clock in light-dark cycles can be accounted for by a transcription-translation feedback model of the central FRQ-WC oscillator. More generally, we provide an example of a biological circuit in which greater flexibility yields improved robustness, while also introducing novel sensitivity analysis techniques applicable to a broader range of cellular oscillators.
... Under LD conditions, vvd is rhythmic, but when transferred to DD, vvd oscillation is only seen in the first 24 h. For wc-1, rhythmicity has been observed in LD and though its promoter shows rhythms in DD, its mRNA levels are not predicted to be highly rhythmic [26,33,58,62,63]. In addition, we used the expression patterns from fungal tissue cultured in LD conditions. ...
... To verify the rhythmic gene expression in our samples, we measured expression of a small subset of genes through RT-QPCR. As target genes, we chose the four clock gene homologs for which experimental evidence in N. crassa has been reported [63,[75][76][77][78]: frq, vvd, wc-1 and wc-2 ( Table 2). We added the O. kimflemingiae homolog for cry as a fifth candidate gene ( Table 1). ...
Various parasite-host interactions that involve adaptive manipulation of host behavior display time-of-day synchronization of certain events. One example is the manipulated biting behavior observed in Carpenter ants infected with Ophiocordyceps unilateralis sensu lato. We hypothesized that biological clocks play an important role in this and other parasite-host interactions. In order to identify candidate molecular clock components, we used two general strategies: bioinformatics and transcriptional profiling. The bioinformatics approach was used to identify putative homologs of known clock genes. For transcriptional profiling, RNA-Seq was performed on 48 h time courses of Ophiocordyceps kimflemingiae (a recently named species of the O. unilateralis complex), whose genome has recently been sequenced. Fungal blastospores were entrained in liquid media under 24 h light-dark (LD) cycles and were harvested at 4 h intervals either under LD or continuous darkness. Of all O. kimflemingiae genes, 5.3% had rhythmic mRNAs under these conditions (JTK Cycle, ≤ 0.057 statistical cutoff). Our data further indicates that a significant number of transcription factors have a peaked activity during the light phase (day time). The expression levels of a significant number of secreted enzymes, proteases, toxins and small bioactive compounds peaked during the dark phase or subjective night. These findings support a model whereby this fungal parasite uses its biological clock for phase-specific activity. We further suggest that this may be a general mechanism involved in parasite-host interactions.
... In the light period of a LD cycle frq levels are high, whereas they rapidly drop following the LD transition (61,159). Then, in the second half of the dark period frq levels rise again, reflecting the reactivation of the WCC. ...
... However, the exact molecular mechanism of clock functioning under entrained conditions is still not entirely clear, and the simple transcriptional-translational feedback model is not sufficient to explain all molecular events. Tan et al. (159) showed that transcription 4 GYÖ NGYÖ SI AND KÁ LDI and translation of FRQ are dissociated in photocycles (up to a delay of 6 h) and the delay depends on the light portion of the cycle. These interesting data suggest that additional posttranscriptional mechanisms play an important role in clock regulation under entrained conditions. ...
Significance:
Both circadian rhythm and the production of reactive oxygen species (ROS) are fundamental features of aerobic eukaryotic cells. The circadian clock enhances the fitness of organisms by enabling them to anticipate cycling changes in the surroundings. ROS generation in the cell is often altered in response to environmental changes, but oscillations in ROS levels may also reflect endogenous metabolic fluctuations governed by the circadian clock. On the other hand, an effective regulation and timing of antioxidant mechanisms may be crucial in the defense of cellular integrity. Thus, an interaction between the circadian timekeeping machinery and ROS homeostasis or signaling in both directions may be of advantage at all phylogenetic levels.
Recent advances:
The Frequency-White Collar-1 and White Collar-2 oscillator (FWO) of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Several members of the ROS homeostasis were found to be controlled by the circadian clock, and ROS levels display circadian rhythm in Neurospora. On the other hand, multiple data indicate that ROS affect the molecular oscillator.
Critical issues:
Increasing evidence suggests the interplay between ROS homeostasis and oscillators that may be partially or fully independent of the FWO. In addition, ROS may be part of a complex cellular network synchronizing non-transcriptional oscillators with timekeeping machineries based on the classical transcription-translation feedback mechanism.
Future directions:
Further investigations are needed to clarify how the different layers of the bidirectional interactions between ROS homeostasis and circadian regulation are interconnected.
... As the gene networks responsible for the circadian rhythms are different from organism to organism, so are the dependencies that are responsible for the mechanisms of entrainment. The examples given above correspond to those mechanisms discovered to be used by Neurospora [36] and Drosophila [26] respectively. ...
... To model the effect of light signals on the gene network, one is led to experimental evidence that light intensity has a positive effect on the transcription of the frq gene [36]. This has been incorporated into deterministic models of entrainment and used to study the effect of varying the sensitivity of this effect [15]. ...
Abstract Circadian rhythms occur in almost all living things, providing an organism with a biological clock that gives an estimate of external time. The phenomenon,of entrainment is a particularly interesting property of circadian rhythms in which the period of an organism’s circadian rhythm is adapted to match the natural oscillations of the environment. We applied the technique of stochastic simulation to modelling the entrainment of circadian rhythms. The biochemical processes that determine the behaviour of a circadian rhythm in nature tend to be reliant on tens to hundreds of molecules [9] With the concentrations of molecules being so low, the phenomenon of molecular noise can have a significant impact on the behaviour of the system [24]. While deterministic simulations of these processes have been used to demonstrate interesting results [15], corresponding,stochastic techniques more accurately represent the mechanics,of the processes due to their simulation of molecular noise [16]. Stochastic simulation of biologically incorrect models of entrainment and deterministic simulation of biologically accurate models have been carried out ([33] and [15] respectively), but the application of stochastic simulation to biologically accurate models,of entrainment of circadian rhythms is demonstrated,for the first time in this dissertation. Stochastic Petri Nets (SPNs) were used to implement computational models of circadian rhythms, as described in [32]. To implement models that incorporated the effects of entrainment, a new software tool was developed that was based on the extended Petri Net Kernel (PNK2e) [5]. The new version of the PNK2e described in this dissertation enables modelling and simulation of biochemical networks with time-dependent reaction rates. Its useful application is not limited to modelling biochemical networks, but extends to any system that can be effectively modelled,by stochastic processes. This software will be released under the Open Source agreement like its predecessor. A series of experiments were designed to investigate the effect of modelling entrainment of circadian rhythms,as a system of stochastic processes. The design of
... Together, these data suggest that O 2 dÀ pool that is accessible for SOD-1 affects the entrained phase of conidiation. Even under entrained conditions, timing of conidiation is controlled by the circadian clock [49]. To investigate whether menadione-sensitive banding is dependent on a functional FWO, we also tested the effect of menadione in the frq-deficient frq 10 , bd strain. ...
... Liquid cultures were entrained to 12/12-h light/dark cycles and frq RNA levels were determined. Expression of frq is relatively high during the light period, whereas it rapidly drops after the LD transfer [49] (Supplementary Fig. S15). However, in the second half of the dark period frq levels start to rise again, reflecting the relief of repression on the WCC. ...
Reactive oxygen species may serve as signals coupling metabolism to other cell functions. Beside being byproducts of normal metabolism, they are generated at elevated levels under environmental stress situations. We analyzed how reactive oxygen species affect the circadian clock in the model organism Neurospora crassa. In light-dark cycles, an increase in the levels of reactive oxygen species advanced the phase of both the conidiation rhythm and the expression of the clock gene frequency. Our results indicate a dominant role of the superoxide anion in the control of the phase. Elevation of superoxide production resulted in the activation of protein phosphatase 2A, a regulator of the positive element of the circadian clock. Our data indicate that even under non-stress conditions, reactive oxygen species affect circadian timekeeping. Reduction of their basal levels results in delay of the phase in light-dark cycles and a longer period under constant conditions. We show that under entrained conditions the phase depends on the temperature and reactive oxygen species contribute to this effect. Our results suggest that the superoxide anion is an important factor controlling the circadian oscillator, and is able to reset the clock most probably by activating protein phosphatase 2A, thereby modulating the activity of the White Collar Complex.
... While many researchers in the field have focused on rhythmic regulation of mRNA as a central mechanism in biological clock function (including the present study), translational control of clock components and their outputs that is independent of transcriptional regulation have been described in many model systems, including vertebrates. For example, in the filamentous fungus Neurospora crassa, circadian regulation of the clock gene frq and its protein FRQ can be separated by entrainment to long and short photoperiods, suggesting independent regulation of these two components (Tan et al., 2004). In Gonyaulax, circadian regulation of GAPDH levels and activity, a common clock-regulated protein (Morre et al., 2002), appears to be independent of mRNA levels (Fagan et al., 1999), and, while AANAT activity is rhythmically regulated in the sheep pineal gland, no rhythm in mRNA levels can be determined (Coon et al., 1999). ...
... Translational control of clock components and their outputs that are independent of transcriptional regulation have been described in many model systems, including vertebrates. For example, in the filamentous fungus Neurospora crassa, circadian regulation of the clock gene frq and its protein FRQ can be separated by entrainment to long and short photoperiods, suggesting independent regulation of these two components (Tan et al., 2004). In Gonyaulax, circadian regulation of GAPDH levels and activity, a common clock-regulated protein (Morre et al., 2002), appears to be independent of mRNA levels (Fagan et al., 1999), and, while AANAT activity is rhythmically regulated in the sheep pineal gland, no rhythm in mRNA levels can be determined (Coon et al., 1999). ...
The genetic identification of molecular mechanisms responsible for circadian rhythm generation has advanced tremendously over the past 25 years. However the molecular identities of the avian clock remain largely unexplored. The present studies seek to determine candidate clock components in the avian species Gallus domesticus. Construction and examination of the transcriptional profiles of the pineal gland and retina using DNA microarray analysis provided a clear view into the avian clock mechanism. Investigation of the pineal and retina transcriptomes determined the mRNA profiles of several thousand genes over the course of one day in LD (daily) and one day in DD (circadian) conditions. Several avian orthologs of mammalian clock genes were identified and many exhibited oscillating patterns of mRNA abundance including several of the putative avian clock genes. Comparison of the pineal transcriptional profile to that of the retina revealed several intriguing candidate genes that may function as core clock components. Including the putative avian clock genes and several others implicated in phototransduction, metabolism, and immune response. A more detailed examination of several candidate photoisomerase/photopigment genes identified from our transcriptional profiling was conducted. These include peropsin (rrh), RGR-opsin (rgr), melanopsin (opn4) and cryptochrome 2 (cry2) genes. This analysis revealed several interesting patterns of mRNA distribution and regulation for these genes in the chick. First, the mRNA of all 4 genes is located within the Inner Nuclear Layer (INL) and Retinal Ganglion cell Layers (RGL) of the ocular retina, where circadian photoreception is present. Second, opn4 and cry2 mRNA is expressed in the photoreceptor layer of the chick retina where melatonin biosynthesis occurs. Lastly, the mRNA for all 4 candidate photopigment genes is regulated on a circadian basis in the pineal gland. As a whole these data yield significant insight into the mechanisms of the avian circadian system and present several candidate genes that may function to integrate photic information, and/or regulate circadian rhythm generation in birds.
... Blue light appears to acutely suppress ytvA promoter activity in a process that resembles masking, a noncircadian response of the organism to a zeitgeber of the circadian clock. Masking is not incompatible with the presence of a functional clock and can also be modulated by the clock (17)(18)(19)(20). We developed an assay based on ytvA promoter activity to quantify masking by red or blue light (Fig. 2D). ...
Circadian clocks are pervasive throughout nature, yet only recently has this adaptive regulatory program been described in nonphotosynthetic bacteria. Here, we describe an inherent complexity in the Bacillus subtilis circadian clock. We find that B. subtilis entrains to blue and red light and that circadian entrainment is separable from masking through fluence titration and frequency demultiplication protocols. We identify circadian rhythmicity in constant light, consistent with the Aschoff's rule, and entrainment aftereffects, both of which are properties described for eukaryotic circadian clocks. We report that circadian rhythms occur in wild isolates of this prokaryote, thus establishing them as a general property of this species, and that its circadian system responds to the environment in a complex fashion that is consistent with multicellular eukaryotic circadian systems.
... The steady-state level depended on the light intensity. In the realm of circadian rhythm, the photoperiod is generally adjusted from 4 to 20 h and, when combined with dark periods, is a total of 24 h to replicate the change of day and night [28][29][30]. ...
Understanding how Aspergillus oryzae responds to light is critical for developing efficient light regulation strategies in the brewing and waste treatment industries. Although continuous light is known to restrict A. oryzae, little is known about A. oryzae’s sensitivity to light with photoperiod. In this study, we used pulse wave modulation (PWM) to generate nine pulsed blue light (PBL) treatments with varying peak light intensities and frequencies. The effect of PBL on A. oryzae was then compared to that of continuous blue light (CBL). Our findings showed that A. oryzae GDMCC 3.31 mycelium developed faster and produced more conidia under PBL with specific peak intensities and frequencies than under CBL treatment when the light dose and average light intensity were held constant. The colony diameter and conidia count under the two PBL treatments (PL-20_40%_1 Hz and PL-400_20%_10 kHz) were 1.13 and 1.22 times greater than under the CBL treatments, respectively. This different response may be mainly attributed to A. oryzae’s adaptation to the light–dark cycles in nature. Furthermore, an interactive effect was found between peak light intensity and frequency. This work includes pulsed wave modulation as a new factor that influences the A. oryzae photoresponse and recommends it in the development of light regulation methods for fermentation.
... For this reason, frq mRNA degradation kinetics were also examined with an alternative protocol. Light-grown, age-matched liquid Bird cultures of wild-type and Dprd-2 were shifted into the dark and sampled every 10 min to measure frq turnover; transcription of frq ceases immediately on transfer to darkness (Heintzen et al., 2001;Tan et al., 2004). All tissue manipulation in the dark was performed under dim red lights, which do not reset the Neurospora clock . ...
Circadian clocks in fungi and animals are driven by a functionally conserved transcription–translation feedback loop. In Neurospora crassa , negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock’s positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 ( period-2 ) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1 prd-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I .
... Ipomoea nil; Heide et al., 1988) or show an intermediate behaviour (e.g. 'noon-tracking' clocks, as in Neurospora crassa; Tan et al., 2004). These distinct circadian behaviours are illustrated in Fig EV9 for a transcript that peaks at dusk in 12/12 light/dark conditions. ...
Plants respond to seasonal cues, such as the photoperiod, to adapt to current conditions and to prepare for environmental changes in the season to come. To assess photoperiodic responses at the protein level, we quantified the proteome of the model plant Arabidopsis thaliana by mass spectrometry across four photoperiods. This revealed coordinated changes of abundance in proteins of photosynthesis, primary and secondary metabolism, including pigment biosynthesis, consistent with higher metabolic activity in long photoperiods. Higher translation rates in the daytime than the night likely contribute to these changes via rhythmic changes in RNA abundance. Photoperiodic control of protein levels might be greatest only if high translation rates coincide with high transcript levels in some photoperiods. We term this proposed mechanism ´translational coincidence´, mathematically model its components, and demonstrate its effect on the Arabidopsis proteome. Datasets from a green alga and a cyanobacterium suggest that translational coincidence contributes to seasonal control of the proteome in many phototrophic organisms. This may explain why many transcripts but not their cognate proteins exhibit diurnal rhythms.
... These concepts are exemplified by the circadian system in Neurospora crassa. Light drives rapid expression of the RNA of clock gene frequency at any time of day whereas the FREQUENCY protein is produced at a phase that correlates with mid-dark or mid-light phase rather than the time when the lights turn on or off [11]. The outward behavior (spore formation) also correlates with the midpoint of the dark or light phase rather than the transitions between them [12]. ...
Circadian clocks in plants, animals, fungi, and in photosynthetic bacteria have been well-described. Observations of circadian rhythms in non-photosynthetic Eubacteria have been sporadic, and the molecular basis for these potential rhythms remains unclear. Here, we present the published experimental and bioinformatical evidence for circadian rhythms in these non-photosynthetic Eubacteria. From this, we suggest that the timekeeping functions of these organisms will be best observed and studied in their appropriate complex environments. Given the rich temporal changes that exist in these environments, it is proposed that microorganisms both adapt to and contribute to these daily dynamics through the process of temporal mutualism. Understanding the timekeeping and temporal interactions within these systems will enable a deeper understanding of circadian clocks and temporal programs and provide valuable insights for medicine and agriculture.
... Ipomoea nil; Heide et al, 1988) or show an intermediate behaviour (e.g. "noon-tracking" clocks, as in Neurospora crassa; Tan et al, 2004). These distinct circadian behaviours are illustrated in Fig EV7 for a transcript that peaks at dusk in 12/12 light/dark conditions. ...
Plants respond to seasonal cues such as the photoperiod, to adapt to current conditions and to prepare for environmental changes in the season to come. To assess photoperiodic responses at the protein level, we quantified the proteome of the model plantArabidopsis thalianaby mass spectrometry across four photoperiods. This revealed coordinated changes of abundance in proteins of photosynthesis, primary and secondary metabolism, including pigment biosynthesis, consistent with higher metabolic activity in long photoperiods. Higher translation rates in the day than the night likely contribute to these changes, via an interaction with rhythmic changes in RNA abundance. Photoperiodic control of protein levels might be greatest only if high translation rates coincide with high transcript levels in some photoperiods. We term this proposed mechanism "translational coincidence", mathematically model its components, and demonstrate its effect on theArabidopsisproteome. Datasets from a green alga and a cyanobacterium suggest that translational coincidence contributes to seasonal control of the proteome in many phototrophic organisms. This may explain why many transcripts but not their cognate proteins exhibit diurnal rhythms.
... Such disruption results in reduced binding of the light-activated WCC to LREs, precluding further light-activated transcription and thereby, resulting in efficient photoadaptation. Under continuous or prolonged light exposure, light responses are attenuated, but importantly the degree of photoadaptation seems to vary between loci (Tan, Dragovic, Roenneberg, & Merrow, 2004;Wu et al., 2014). The length of the VVD photocycle is also critical for allowing the proper rise in FRQ levels during the light part of the day (Dasgupta et al., 2015), as increasing light intensities are reached. ...
Night follows day and as a consequence, organisms have evolved molecular machineries that allow them to anticipate and respond to the many changes that accompany these transitions. Circadian clocks are precise yet plastic pacemakers that allow the temporal organization of a plethora of biological process. Circadian clocks are widespread across the tree of life and while their exact molecular components differ among phyla, they tend to share common design principles. In this review, we discuss the circadian system of the filamentous fungus Neurospora crassa. Historically, this fungus has served a key role in the genetic and molecular dissection of circadian clocks, aiding in their detailed mechanistic understanding. Recent studies have provided new insights into the daily molecular dynamics that constitute the Neurospora circadian oscillator, some of which have questioned traditional paradigms describing timekeeping mechanisms in eukaryotes. In addition, recent reports support the idea of a dynamic network of transcription factors underlying the rhythmicity of thousands of genes in Neurospora, many of which oscillate only under specific conditions. Besides Neurospora, which harbors the best characterized circadian system among filamentous fungi, the recent characterization of the circadian system of the plant-pathogenic fungus Botrytis cinerea has provided additional insights into the physiological impact of the clock and potential additional functions of clock proteins in fungi. Finally, we speculate on the presence of FRQ or FRQ-like proteins in diverse fungal lineages.
... The peak:trough expression ratio supported by frq::luc-PEST was , 20-fold. This activity profile of frq::luc-PEST corresponds closely to the previously reported temporal expression profile of frq RNA in light/dark cycles [31,32], indicating that the destabilized luciferase is a faithful reporter of the transcription dynamics of the frq promoter. In contrast, the activity of the stable luciferase encoded by frq::luc increased steadily throughout the light phase and then decreased during the dark phase. ...
We show that firefly luciferase is a stable protein when expressed at 25°C in Neurospora, which limits its use as transcription reporter. We created a short-lived luciferase by fusing a PEST signal to its C-terminus (LUC-PEST) and applied the LUC-PEST reporter system to record in vivo transcription dynamics associated with the Neurospora circadian clock and its blue-light photosensory system over the course of several days. We show that the tool is suitable to faithfully monitor rapid, but also subtle changes in transcription in a medium to high throughput format.
... However, if frq or FRQ are involved in systematic circadian entrainment that occurs in T = 24 h photoperiod cycles, then they should be expressed with different kinetics in different cycles. We found this to be true for the protein but not the RNA (Figure 6.2C; Tan et al., 2004b). The frq RNA-kinetics are independent of photoperiod (and night length, scotoperiod). ...
... On reflection, it is clear that the feedback loop could also be reset through light-triggered decay of a clock component that peaks at night; because per peak expression occurs at night, we correctly predicted (Crosthwaite et al. 1995) that the Drosophila clock would be reset in this way, as is the case (see, e.g., HunterEnsor et al. 1996).Although the model implies a wonderful simplicity in the responses to light, this may be misleading because the total light response is not just transcriptional. There is gating, as described below, but beyond this is the fact that FRQ accumulates and is phosphorylated in the light but is not significantly degraded until transfer to dark (Collett et al. 2002;Tan et al. 2004). Also in the dark, frq and FRQ synthesis reflects the feedback loop: If after transfer to darkness, there is not enough FRQ to complete the negative feedback loop, still more FRQ will be made. ...
Neurospora has proven to be a tractable model system for understanding the molecular bases of circadian rhythms in eukaryotes. At the core of the circadian oscillatory system is a negative feedback loop in which two transcription factors, WC-1 and WC-2, act together to drive expression of the frq gene. WC-2 enters the promoter region of frq coincident with increases in frq expression and then exits when the cycle of transcription is over, whereas WC-1 can always be found there. FRQ promotes the phosphorylation of the WCs, thereby decreasing their activity, and phosphorylation of FRQ then leads to its turnover, allowing the cycle to reinitiate. By understanding the action of light and temperature on frq and FRQ expression, the molecular basis of circadian entrainment to environmental light and temperature cues can be understood, and recently a specific role for casein kinase 2 has been found in the mechanism underlying circadian temperature-compensation. These data promise molecular explanations for all of the canonical circadian properties of this model system, providing biochemical answers and regulatory logic that may be extended to more complex eukaryotes including humans.
... Thus, with the information to date, each individual FRQ molecule can be seen as acting as a molecular eggtimer in the circadian clock of Neurospora crassa. Yet, abundance rhythms and degradation kinetics of the FRQ pool have been shown to vary dependent on the photoperiod under entrained conditions [50]. At first glance this appears to contradict the concept of a fixed timer at the center of the clock. ...
Various post-translational modifications have been identified that play a role in the function of circadian clocks. Among these, phosphorylation has been investigated extensively. It was shown that phosphorylation influences half-life, subcellular localisation, transcriptional activity and conformation of clock components over the course of a circadian day. Recent observations also indicate that time-of-day specific sequential phosphorylation of the Neurospora crassa clock protein FREQUENCY is crucial for measuring time and thus for establishing a robust circadian rhythm. The circadian clock of Neurospora is one of the best-investigated molecular clocks to date. In this review, we summarise the data on what is known so far about the role of phosphorylation of proteins involved in the Neurospora circadian clock.
... Photoperiodic responses are not restricted to plants. A study performed with Neurospora demonstrated uncoupling of frequency transcription and FRQ translation in different photoperiods (Tan et al. 2004). The mRNA accumulation reflected the external light conditions, but FRQ protein accumulation could be uncoupled by up to 6 h and was locked to the middle of the dark period. ...
The albumin D site-binding protein (DBP) governs circadian transcription of a number of hepatic detoxification and metabolic enzymes prior to the activity phase and subsequent food intake of mice. However, the behavior of mice is drastically affected by the photoperiod. Therefore, continuous adjustment of the phase of circadian Dbp expression is required in the liver. Here we describe a direct impact of CRYPTOCHROME1 (CRY1) on the phase of Dbp expression. Dbp and the nuclear receptor Rev-Erbalpha are circadian target genes of BMAL1 and CLOCK. Surprisingly, dynamic CRY1 binding to the Dbp promoter region delayed BMAL1 and CLOCK-mediated transcription of Dbp compared with Rev-Erbalpha. Extended presence of CRY1 in the nucleus enabled continuous uncoupling of the phase of Dbp from Rev-Erbalpha expression upon change from short to longer photoperiods. CRY1 thus maintained the peak of DBP accumulation close to the activity phase. In contrast, Rev-Erbalpha expression was phase-locked to the circadian oscillator and shaped by accumulation of its own gene product. Our data indicate that fine-tuning of circadian transcription in the liver is even more sophisticated than expected.
... Die frq-Transkription wird durch Licht stark induziert. Daher spiegelt die frq-mRNA-Menge in Licht-Dunkel-(LD-)Zyklen unmittelbar die Lichtverhältnisse wider, der Rhythmus ist scheinbar allein lichtgesteuert [86]. Weil aber die Phase der Konidienbildung in LD-oder Temperaturzyklen entrained wird, muss die circadiane Uhr unter diesen Bedingungen weiterlaufen. ...
Die circadianen Uhren eukaryontischer Organismen bestehen aus einem Netzwerk miteinander verknüpfter Rückkopplungsschleifen. Ein grundlegendes Prinzip darin ist eine negative Transkriptions-Translations-Rückkopplungsschleife, in der ein Protein die Transkription seiner eigenen mRNA zeitverzögert reprimiert. Daher spielt die Regulation der nukleocytoplasmatischen Verteilung einzelner Proteine im molekularen Mechanismus der Uhren eine wichtige Rolle. FREQUENCY (FRQ) ist eine der zentralen Komponenten der circadianen Uhr von Neurospora crassa. Es hemmt zum einen im Zellkern die Transkription seiner eigenen mRNA (negative Rückkopplung), zum anderen fördert es im Cytosol die Bildung des White Collar Komplexes (WCC) bestehend aus den Transkriptionsfaktoren White Collar (WC) 1 und 2 (positive Rückkopplung), der wiederum die Transkription von frequency-mRNA bewirkt. Die zeitliche Abfolge beider Funktionen ist exakt festgelegt, daher muss die subzelluläre Verteilung von FRQ präzise reguliert werden. Um das korrekte Funktionieren der Uhr zu gewährleisten, ist FRQ zum größten Teil im Cytosol und nur zum kleineren Teil im Zellkern lokalisiert, obwohl es ein funktionales Kernlokalisationssignal (NLS) besitzt. frq9 exprimiert durch Leserasterverschiebung ein um ca. 30% C-terminal verkürztes Protein (FRQ9), das überwiegend im Zellkern vorkommt. Der C-Terminus ist also für die unerwartete cytoplasmatische Lokalisation von FRQ verantwortlich. In der vorliegenden Arbeit wird untersucht, welche Teile des C-Terminus die FRQ-Lokalisation beeinflussen und wie die subzelluläre Verteilung bewirkt wird. Die Verteilung kann nicht einer eng umgrenzten Region (z. B. einem putativen Kernexportsignal, NES) im C-Terminus zugeschrieben werden und spiegelt sehr wahrscheinlich ein dynamisches Gleichgewicht aus Kernimport und -export wider. Ein Teil der FRQ-Population könnte durch Modifikation im Zellkern diesem shuttling-Prozess entzogen werden, so dass er nicht wieder in den Zellkern gelangen kann (z. B. durch Maskierung eines Kernlokalisationssignals, NLS). Phosphorylierungen sind ein wichtiger Regulationsmechanismus für FRQ-Funktionen und könnten auch an der Festlegung der subzellulären Lokalisation beteiligt sein. Um in vivo-Phosphorylierungsstellen und Bindungspartner von FRQ massenspektrometrisch identifizieren zu können, wurden verschiedene Affinitätschromatographiemethoden auf ihre Verwendbarkeit in N. crassa getestet. Eine einzelne Methode führte in keinem Fall zu ausreichender Anreicherung von FRQ, aber eine Doppelstrategie aus His6/Ni2+- und StrepTag II/StrepTactin-Aufreinigung erscheint viel versprechend, da beide das Protein aus Totalextrakten depletieren können und die Puffer miteinander kompatibel sind. Schließlich wird ein Satz neuer Expressionsvektoren für die Transformation von N. crassa vorgestellt. Jeder dieser Vektoren komplementiert die Histidinauxotrophie der his-3-Mutante und besitzt einen N. crassa-Promotor, einen Polylinker sowie einen Transkriptionsterminator mit Polyadenylierungsstelle. Eukaryotic circadian clocks are made up by networks of interconnected feedback loops. One basic principle therein is a transcriptional-translational negative feedback loop in which a protein represses with a time delay transcription of its own mRNA. Therefore, regulation of nucleo-cytoplasmic partitioning of distinct proteins plays an important role in the clocks molecular mechanisms. FREQUENCY (FRQ) is one of the central components of the Neurospora crassa circadian clock. On the one hand, it represses in the nucleus transcription of its own mRNA (negative feedback). On the other hand, it promotes in the cytosol accumulation of White Collar Complex (WCC) consisting of the transcription factors White Collar (WC) 1 and 2 (positive feedback) which in turn causes transcription of frq mRNA. The temporal order of both functions is exactly determined so subcellular distribution of FRQ needs to be regulated precisely. To ensure proper functioning of the clock, most of the protein localises to the cytosol and only a minor portion is in the nucleus although FRQ contains a functional nuclear localisation signal (NLS). frq9 expresses due to a frame shift mutation an approx. 30% C-terminally shortened protein (FRQ9). FRQ9 appears predominantly in the nucleus so the C-terminus accounts for the unexpected cytosolic localisation of FRQ. This study examines which parts of the C-terminus determine localisation and how the subcellular distribution is achieved. FRQ distribution cannot be attributed to a closely confined region in the C-terminus (e.g. a putative nuclear export signal, NES) and presumably reflects a dynamic equilibrium of nuclear import and export. Phosphorylation in the nucleus may withdraw a part of the FRQ population from this shuttling process so it cannot enter the nucleus again (e. g. by masking of the nuclear localisation signal, NLS). Phosphorylation is an important mechanism regulating FRQ functions and may be involved in determining subcellular localisation as well. In order to identify in vivo phosphorylation sites and binding partners by mass spectrometry several affinity chromatography methods were tested for their applicability with Neurospora. One single method has not proven to be sufficient for proper FRQ enrichment but a double strategy of His6/Ni2+ and StrepTag II/StrepTactin purification seems promising since both can deplete the protein from total extracts and their buffers are compatible with each other. Finally, a new set of expression vectors for N. crassa transformation is presented. Each vector complements histidine auxotrophy of the his-3 mutant and contains a N. crassa promoter, polylinker and a transcriptional terminator with polyadenylation site.
... Although many researchers in the field have focused on rhythmic regulation of mRNA as a central mechanism in biological clock function (including the present study), translational control of clock components and their outputs that are independent of transcriptional regulation have been described in many model systems, including vertebrates. For example, in the filamentous fungus Neurospora crassa, circadian regulation of the clock gene frq and its protein FRQ can be separated by entrainment to long and short photoperiods, suggesting independent regulation of these two components (67). In Gonyaulax, circadian regulation of GAPDH levels and activity, a common clock-regulated protein (68), appears to be independent of mRNA levels (69), and whereas AANAT activity is rhythmically regulated in the sheep pineal gland, no rhythm in mRNA levels can be determined (54). ...
Previous transcriptome analyses have identified candidate molecular components of the avian pineal clock, and herein we employ high density cDNA microarrays of pineal gland transcripts to determine oscillating transcripts in the chick retina under daily and constant darkness conditions. Subsequent comparative transcriptome analysis of the pineal and retinal oscillators distinguished several transcriptional similarities between the two as well as significant differences. Rhythmic retinal transcripts were classified according to functional categories including phototransductive elements, transcription/translation factors, carrier proteins, cell signaling molecules, and stress response genes. Candidate retinal clock transcripts were also organized relative to time of day mRNA abundance, revealing groups accumulating peak mRNA levels across the circadian day but primarily reaching peak values at subjective dawn or subjective dusk.
Comparison of the chick retina transcriptome to the pineal transcriptome under constant conditions yields an interesting group of conserved genes. This group includes putative clock elements cry1 and per3 in addition to several previously unidentified and uninvestigated genes exhibiting profiles of mRNA abundance that varied markedly under daily and constant conditions. In contrast, many transcripts were differentially regulated, including those believed to be involved in both melatonin biosynthesis and circadian clock mechanisms. Our results indicate an intimate transcriptional relationship between the avian pineal and retina in addition to providing previously uncharacterized molecular elements that we hypothesize to be involved in circadian rhythm generation.
... Both frq mRNA and FRQ responses depend on time of day and are further modulated acutely by light (reviewed in Liu, 2003). Phosphorylated FRQ accumulates in the light but may not be significantly degraded until transfer to dark (Collett et al., 2002;Tan et al., 2004a). Depending on the duration of light prior to darkness, the synthesis/decay kinetics of FRQ can vary substantially. ...
The eukaryotic filamentous fungus Neurospora crassa has proven to be a durable and dependable model system for the analysis of the cellular and molecular bases of circadian rhythms. Pioneering genetic analyses identified clock genes, and beginning with the cloning of frequency (frq), work over the past 2 decades has revealed the molecular basis of a core circadian clock feedback loop that has illuminated our understanding of circadian oscillators in microbes, plants, and animals. In this transcription/translation-based feedback loop, a heterodimer of the White Collar-1 (WC-1) and WC-2 proteins acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene. FRQ dimerizes and feeds back to block the activity of its activators (making a negative feedback loop), as well as feeding forward to promote the synthesis of its activator, WC-1. Phosphorylation of FRQ by several kinases leads to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle. Light resetting of the clock can be understood through the rapid light induction of frq expression and temperature resetting through the influence of elevated temperatures in driving higher levels of FRQ. Several FRQ- and WC-independent, noncircadian FRQ-less oscillators (FLOs) have been described, each of which appears to regulate aspects of Neurospora growth or development. Overall, the FRQ/white collar complex feedback loop appears to coordinate the circadian system through its activity to regulate downstream-target clock-controlled genes, either directly or via regulation of driven FLOs.
... Neurospora clock null mutants, for example, can still produce self-sustained circadian rhythms under certain conditions (see references in [12]), and can still be systematically entrained by temperature cycles, as described above [8,13]. The data obtained by following both physiology and gene expression under entrained conditions indicates dissociation between transcription (showing direct regulation by the external cycle) and clock-regulated outputs (showing systematic entrainment, as described above for temperature entrainment) [14,15]. There is much to be discovered both in prokaryotes and eukaryotes well beyond transcriptional regulation, despite the overwhelming evidence for the involvement of transcription factors in circadian systems. ...
One of the big questions in biological rhythms research is how a stable and precise circa-24 hour oscillation is generated on the molecular level. While increasing complexity seemed to be the key, a recent report suggests that circa-24 hour rhythms can be generated by just four molecules incubated in a test tube.
... Much progress has been made in our knowledge of the bio-chemistry involved in the generation of these rhythms. The basic current concept (Fig. 1 , top left panel) is that of a negativefeedback model (transcription-translation loop, or TTL), in which it is assumed that the proteins (with some time lag) attenuate their own production (Hardin et al., 1990; Reppert and Weaver, 2002; but see Tan et al., 2004, Tomita et al., 2004). Lema et al. (2000) transformed the conceptual negative feedback model into a mathematical model consisting of 2 equations: ...
In our attempts to understand the circadian system, we unavoidably rely on abstractions. Instead of describing the behavior of the circadian system in all its complexity, we try to derive basic features from which we form a global concept on how the system works. Such a basic concept is a model of reality. The author discusses why it is advantageous or even necessary to transform conceptual models into mathematical formulations. As examples to demonstrate those advantages, the author reviews 4 types of mathematical models: negative feedback models thought to operate within pacemaker cells, models on coupling between pacemaker cells to generate pacemaker output, oscillator models describing the behavior of the composite circadian pacemaker, and models describing how the circadian pacemaker influences behavior.
... Rather, thermosensitive splicing of per regulates the adaptation of locomotor activity to seasonally warm and long versus cold and short days. Accordingly, splicing and translational control of frq may contribute to finetuning of clock functions in Neurospora, modulating, in particular, adaptation of the circadian clock to seasonal variations of photoperiod and temperature (Tan et al. 2004a,b). How may levels and ratios of l-FRQ versus s-FRQ contribute to temperature compensation of period length? ...
Expression levels and ratios of the long (l) and short (s) isoforms of the Neurospora circadian clock protein FREQUENCY (FRQ) are crucial for temperature compensation of circadian rhythms. We show that the ratio of l-FRQ versus s-FRQ is regulated by thermosensitive splicing of intron 6 of frq, a process removing the translation initiation site of l-FRQ. Thermosensitivity is due to inefficient recognition of nonconsensus splice sites at elevated temperature. The temperature-dependent accumulation of FRQ relative to bulk protein is controlled at the level of translation. The 5'-UTR of frq RNA contains six upstream open reading frames (uORFs) that are in nonconsensus context for translation initiation. Thermosensitive trapping of scanning ribosomes at the uORFs leads to reduced translation of the main ORF and allows adjustment of FRQ levels according to ambient temperature.
... As previously reported, all transcripts are light induced, with maximal induction between 15 min and 1 h after lights on, albeit wc-1 induction in wild type was weak in our experiments. In the absence of transcript oscillations , we asked whether FRQ protein levels might dictate the LL rhythm.Figure 4C (top panels) shows a gradual increase of FRQ protein levels in LL in both wildtype and vvd mutant, peaking only after 12–16 h in LL, consistent with previous observations in wild type (Collett et al. 2001; Tan et al. 2004a). These data suggest that the initial phase of rhythmic conidiation in LL most likely arises from changing FRQ protein levels, but other as yet undiscovered processes may contribute to the extended overt rhythmicity we see in LL. ...
A light-entrainable circadian clock controls development and physiology in Neurospora crassa. Existing simple models for resetting based on light pulses (so-called nonparametric entrainment) predict that constant light should quickly send the clock to an arrhythmic state; however, such a clock would be of little use to an organism in changing photoperiods in the wild, and we confirm that true, albeit dampened, rhythmicity can be observed in extended light. This rhythmicity requires the PAS/LOV protein VIVID (VVD) that acts, in the light, to facilitate expression of an oscillator that is related to, but distinguishable from, the classic FREQUENCY/WHITE-COLLAR complex (FRQ/WCC)-based oscillator that runs in darkness. VVD prevents light resetting of the clock at dawn but, by influencing frq RNA turnover, promotes resetting at dusk, thereby allowing the clock to run through the dawn transition and take its phase cues from dusk. Consistent with this, loss of VVD yields a clock whose performance follows the simple predictions of earlier models, and overexpression of VVD restores rhythmicity in the light and sensitivity of phase to the duration of the photoperiod.
... Based on the transcription-translation loop concept, the induced changes will alter the phase of the molecular rhythm, thereby adjusting the biological clock to the external environment. Recent results show that transcription and translation can be dissociated in different photoperiods, indicating that entrainment at the molecular level might be more complex 56 . ...
Circadian clocks control the daily life of most light-sensitive organisms - from cyanobacteria to humans. Molecular processes generate cellular rhythmicity, and cellular clocks in animals coordinate rhythms through interaction (known as coupling). This hierarchy of clocks generates a complex, approximately 24-hour temporal programme that is synchronized with the rotation of the Earth. The circadian system ensures anticipation and adaptation to daily environmental changes, and functions on different levels - from gene expression to behaviour. Circadian research is a remarkable example of interdisciplinarity, unravelling the complex mechanisms that underlie a ubiquitous biological programme. Insights from this research will help to optimize medical diagnostics and therapy, as well as adjust social and biological timing on the individual level.
... frq transcription is strongly induced by light (for review, see Dunlap and Loros 2004). As a consequence, frq RNA abundance directly reflects the light environment in LD cycles, suggesting that frq transcription is lightdriven or masked by light (Tan et al. 2004). Yet, since the phase of conidiation is systematically entrained in photoperiod and T cycles, the circadian clock is running under such conditions. ...
Circadian clocks are self-sustained oscillators modulating rhythmic transcription of large numbers of genes. Clock-controlled gene expression manifests in circadian rhythmicity of many physiological and behavioral functions. In eukaryotes, expression of core clock components is organized in a network of interconnected positive and negative feedback loops. This network is thought to constitute the pacemaker that generates circadian rhythmicity. The network of interconnected loops is embedded in a supra-net via a large number of interacting factors that affect expression and function of core clock components on transcriptional and post-transcriptional levels. In particular, phosphorylation and dephosphorylation of clock components are critical processes ensuring robust self-sustained circadian rhythmicity and entrainment of clocks to external cues. In cyanobacteria, three clock proteins have the capacity to generate a self-sustained circadian rhythm of autophosphorylation and dephosphorylation independent of transcription and translation. This phosphorylation rhythm regulates the function of these clock components, which then facilitate rhythmic gene transcription, including negative feedback on their own genes. In this article, we briefly present the mechanism of clock function in cyanobacteria. We then discuss in detail the contribution of transcriptional feedback and protein phosphorylation to various functional aspects of the circadian clock of Neurospora crassa.
Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a “lights-on timer” logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks.
In response to a light stimulus, the mammalian circadian clock first dramatically increases the expression of Per1 mRNA, and then drops to a baseline even when light persists. This phenomenon is known as light adaptation, which has been experimentally proven to be related to the CRTC1-SIK1 pathway in suprachiasmatic nucleus (SCN). However, the role of this light adaptation in the circadian rhythm remains to be elucidated. To reveal the in-depth function of light adaptation and the underlying dynamics, we proposed a mathematical model for the CRTC1-SIK1 network and coupled it to a mammalian circadian model. The simulation result proved that the light adaptation is achieved by the self-inhibition of the CRTC1/CREB complex. Also, consistently with experimental observations, this adaptation mechanism can limit the phase response to short-term light stimulus, and it also restricts the rate of the phase shift in a jet lag protocol to avoid overly rapid re-entrainment. More importantly, this light adaptation is predicted to prevent the singularity behavior in the cell population, which represents the abolishment of circadian rhythmicity due to desynchronization of oscillating cells. Furthermore, it has been shown to provide refractoriness to successive stimuli with short gap. Therefore, we concluded that the light adaptation generated by the CRTC1-SIK1 pathway in the SCN provides a robust mechanism, allowing the circadian system to maintain homeostasis in the presence of light perturbations. These results not only give new insights into the dynamics of light adaptation from a computational perspective but also lead us to formulate hypotheses about the related physiological significance.
Many organisms have oscillators with a period of about 24 hours, called “circadian clocks”. They employ negative biochemical feedback loops that are self-contained within a single cell (requiring no cell-to-cell interaction). Circadian singularity behavior is a phenomenon of the abolishment of circadian rhythmicities by a critical stimulus. These behaviors have been found experimentally in Neurospora, human and hamster, by temperature step-up or light pulse. Two alternative models have been proposed to explain this phenomenon: desynchronization of cell populations, and loss of oscillations in all cells by resetting the each cell close to a steady state. In this work, we use a mathematical model to investigate the dynamical mechanism of circadian singularity behavior in Neurospora. Our findings suggest that the arrhythmic behavior after the critical stimulus is caused by the collaboration of the desynchronization and the loss of oscillation amplitude. More importantly, we found that the stable manifold of the unstable equilibrium point, instead of the steady state itself, plays a crucial role in circadian singularity behavior.
Three properties are most often attributed to the circadian clock: a ca. 24-h free-running rhythm, temperature compensation of the circadian rhythm, and its entrainment to zeitgeber cycles. Relatively few experiments, however, are performed under entrainment conditions. Rather, most chronobiology protocols concern constant conditions. We have turned this paradigm around and used entrainment to study the circadian clock in organisms where a free-running rhythm is weak or lacking. We describe two examples therein: Caenorhabditis elegans and Saccharomyces cerevisiae. By probing the system with zeitgeber cycles that have various structures and amplitudes, we can demonstrate the establishment of systematic entrained phase angles in these organisms. We conclude that entrainment can be utilized to discover hitherto unknown circadian clocks and we discuss the implications of using entrainment more broadly, even in model systems that show robust free-running rhythms.
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Circadian clocks are molecular timekeepers that provide organisms with a means to predict and prepare for environmental change. The filamentous fungus Neurospora crassa has provided an excellent model system in which the underlying molecular basis of circadian clocks has been elucidated. In Neurospora, and in other eukaryotes, circadian rhythmicity emerges from a network of positive and negative feedback regulation acting on clock genes and proteins. An essential attribute of the clock is that it can detect and respond to the daily cycle of light and dark and temperature change and integrate these environmental time cues to give an accurate depiction of the external day. In Neurospora many of the molecules that sense the daily changes in light and temperature are known. In this review we describe Neurospora’s clock mechanism and how it is tuned to the real world by light and temperature.
Clock genes, which are central to circadian regulation, have been found in all organisms. How clocks are synchronized with, or entrained to, a 24-h day, and adjusted to different seasonal daylengths is, however, still a puzzle.
In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the
cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate
(MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq. Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays
(EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and
dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.
Circadian timing is a fundamental biological process, underlying cellular physiology in animals, plants, fungi, and cyanobacteria. Circadian clocks organize gene expression, metabolism, and behavior such that they occur at specific times of day. The biological clocks that orchestrate these daily changes confer a survival advantage and dominate daily behavior, for example, waking us in the morning and helping us to sleep at night. The molecular mechanism of circadian clocks has been sketched out in genetic model systems from prokaryotes to humans, revealing a combination of transcriptional and posttranscriptional pathways, but the clock mechanism is far from solved. Although Saccharomyces cerevisiae is among the most powerful genetic experimental systems and, as such, could greatly contribute to our understanding of cellular timing, it still remains absent from the repertoire of circadian model organisms. Here, we use continuous cultures of yeast, establishing conditions that reveal characteristic clock properties similar to those described in other species. Our results show that metabolism in yeast shows systematic circadian entrainment, responding to cycle length and zeitgeber (stimulus) strength, and a (heavily damped) free running rhythm. Furthermore, the clock is obvious in a standard, haploid, auxotrophic strain, opening the door for rapid progress into cellular clock mechanisms.
As scientists, we strive for highly controlled conditions. The real world, however, is noisy. Complex networks are a coping mechanism for an erratic environment.
Die circadianen Uhren von Eukaryoten bestehen aus Proteinen, deren Zusammenspiel vernetzte positive und negative Rückkopplungsschleifen erzeugt. Diese steuern metabolische und physiologische Funktionen sowie das Verhalten der Organismen in einem robusten Rhythmus, der ungefähr der 24-stündigen Erdrotation entspricht. In der Uhr von Neurospora crassa, aktiviert der heterodimere Transkriptionsfaktor WHITE COLLAR-Komplex (WCC) als positives Element das Gen seines negativen Regulators, frequency (frq). Das Protein FREQUENCY (FRQ) rekrutiert Casein-Kinase 1a (CK-1a) und vermittelt die Phosphorylierung des WCC, wodurch dieser inaktiviert wird. FRQ selbst wird im Verlauf eines circadianen Tages hyperphosphoryliert und abgebaut. Dadurch wird die Inaktivierung von WCC aufgehoben und ein neuer Zyklus beginnt. FRQ wird im Verlauf einer circadianen Periode an mehr als 75 Stellen phosphoryliert. Die genaue Funktion dieser hohen Anzahl an Phosphorylierungen ist bisher nicht bekannt. In dieser Arbeit konnte gezeigt werden, dass FRQ eine zweiteilige Bindestelle für Casein-Kinase 1a (CK-1a) enthält, die von zwei kurzen Segmenten, FCD1 und FCD2, gebildet wird. FCD1 ist in der basischen N-terminalen Domäne von FRQ lokalisiert, während FCD2 im sauren Mittelteil des Proteins liegt. Beide Segmente werden für eine stabile Interaktion von CK1a mit FRQ benötigt. Des Weiteren weisen die gesammelten Daten darauf hin, dass durch Interaktion des basischen, positiv geladenen N-Terminus von FRQ mit dem negativ geladenen Rest des Proteins FCD1 und FCD2 in räumliche Nähe zueinander gebracht werden und so eine Bindestelle für CK-1a generiert wird. Durch fortschreitende,CK-1a-vermittelte Phosphorylierung des N-Terminus im Laufe eines circadianen Tages wird die elektrostatische Interaktion von N-Terminus und restlichem Protein immer schwächer und damit auch die Interaktion von FRQ und CK-1a. Einhergehend damit verliert FRQ mit fortschreitender Hyperphosphorylierung zunehmend die Fähigkeit, die CK1-abhängige Inaktivierung des WCC zu vermitteln. Die Ergebnisse dieser Arbeit zeigen so einen Mechanismus auf, wie die Interaktion von CK-1a mit FRQ im Laufe eines circadianen Tages reguliert wird. Eukaryotic circadian clocks are comprised of proteins functioning together to produce a network of positive and negative feedback loops. These control a multitude of different metabolical, physiological and behavioral functions in a robust rhythm mimicking the 24-hour rhythm of the earth rotation. In the circadian feedback loop of Neurospora crassa, the heterodimeric transcription factor WHITE COLLAR complex (WCC) as the positive element activates the gene of its negative regulator, frequency (frq). FREQUENCY (FRQ) recruits CASEIN KINASE 1a (CK-1a) and mediates the phosphorylation of WCC, thereby inactivating it. FRQ is hyperphosphorylated and degraded over the course of the circadian day, thereby abrogating the inhibition of WCC so that the cycle starts again. FRQ itself is phosphorylated during the circadian period at more than 75 sites. The functions of most of these phosphorylations are not known in detail. In this work it could be shown that FRQ contains a bipartite binding site for CK-1a, consisting of two short segments, FCD1 and FCD2. FCD1 is located in the basic N-terminal part of FRQ, while FCD2 is located in the acidic middle domain. Both segments are necessary for stable interaction with CK-1a. Further data hint at the possibility that FCD1 and FCD2 are brought into close contact by interaction of the basic, positively charged N-terminal part with the acidic, negatively charged middle domain of FRQ, thereby forming a binding site for CK-1a. CK-1a-mediated phosphorylation of the FRQ N-terminus in the course of a circadian day weakens the ionic interaction of N-terminus and middle part of FRQ, which also leads to the weakening of the FRQ-CK-1a interaction. Hyperphosphorylated FRQ thereby loses its ability to mediate the CK-1a dependent inactivation of WCC. The results of this work propose a mechanism by which the interaction of FRQ and CK-1a is regulated in the course of the circadian day.
Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.
Recent advances in understanding circadian (daily) rhythms in the genera Neurospora, Gonyaulax, and Synechococcus are reviewed and new complexities in their circadian systems are described. The previous model, consisting of a unidirectional flow of information from input to oscillator to output, has now expanded to include multiple input pathways, multiple oscillators, multiple outputs; and feedback from oscillator to input and output to oscillator. New posttranscriptional features of the frq/white-collar oscillator (FWC) of Neurospora are described, including protein phosphorylation and degradation, dimerization, and complex formation. Experimental evidence is presented for frq-less oscillator(s) (FLO) downstream of the FWC. Mathematical models of the Neurospora system are also discussed.
Oscillations are found throughout the physical and biological worlds. Their interactions can result in a systematic process of synchronization called entrainment, which is distinct from a simple stimulus-response pattern. Oscillators respond to stimuli at some times in their cycle and may not respond at others. Oscillators can also be driven if the stimulus is strong (or if the oscillator is weak); i.e., they restart their cycle every time they receive a stimulus. Stimuli can also directly affect rhythms without entraining the underlying oscillator (masking): Drivenness and masking are often difficult to distinguish. Here we use the circadian biological clock to explore properties of entrainment. We confirm previous results showing that the residual circadian system in Neurospora can be entrained in a mutant of the clock gene frequency (frq⁹, a strain deficient in producing a functional FRQ protein). This finding has implications for understanding the evolution of circadian programs. By comparing data sets from independent studies, we develop a template for analyzing, modeling, and dissecting the interactions of entrained and masked components. These insights can be applied to oscillators of all periodicities.
• clock gene
• light
• masking
• model
• temperature
The daily recurrence of activity and rest are so common as to seem trivial. However, they reflect a ubiquitous temporal programme called the circadian clock. In the absence of either anatomical clock structures or clock genes, the timing of sleep and wakefulness is disrupted. The complex nature of circadian behaviour is evident in the fact that phasing of the cycle during the day varies widely for individuals, resulting in extremes colloquially called 'larks' and 'owls'. These behavioural oscillations are mirrored in the levels of physiology and gene expression. Deciphering the underlying mechanisms will provide important insights into how the circadian clock affects health and disease.
The metronomic predictability of the environment has elicited strong selection pressures for the evolution of endogenous circadian clocks. Circadian clocks drive molecular and behavioural rhythms that approximate the 24 h periodicity of our environment. Found almost ubiquitously among phyla, circadian clocks allow preadaptation to rhythms concomitant with the natural cycles of the Earth. Cycles in light intensity and temperature for example act as important cues that couple circadian clocks to the environment via a process called entrainment. This review summarizes our current understanding of the general and molecular principles of entrainment in the model organism Neurospora crassa, a simple eukaryote that has one of the best-studied circadian systems and light-signalling pathways.
Circadian rhythms are endogenously generated by a central pacemaker and are synchronized to the environmental LD cycle. The rhythms can be resynchronized, or reentrained, after a shift of the LD cycle, as in traveling across time zones. The authors have performed high-resolution mapping of the pacemaker to analyze the reentrainment process using rat pineal melatonin onset (MT(on)) and melatonin offset (MT(off)) rhythms as markers. Following LD (12:12) delays of 3, 6, and 12 h, MT(on) was phase locked immediately, whereas MT(off) shifted rapidly during the initial 1 through 3 cycles. In all animals, the MT(off) shifted beyond their expected phase positions in the new LD cycle, which resulted in a transient expansion of melatonin secretion duration for several cycles. It took MT(off) only 1, 2, or 3 cycles to complete most of the required phase shifts after 3, 6, or 12 h of the LD cycle delays, respectively. However, the final stabilization of phase relationships of both MT(on) and MT(off) required at least 6 cycles for rats experiencing a 3-h LD delay and much longer for the rest. These results reaffirmed the notion that both onset and offset phases of melatonin rhythms are important markers for the pacemaker and demonstrated that the reentrainment of the central pacemaker to a delay shift of the LD cycle is a 3-step process: an immediate phase lock of onset and a rapid delay shift of offset rhythms, overshoot of the offset, and, finally, a slow adjustment of both onset and offset phases. This study represents the 1st detailed analysis of the pacemaker behavior during reentrainment using melatonin and supports the notion that the eventual adaptation of the circadian pacemaker to a new time zone is a time-consuming process.
Two genes, period (per) and timeless (tim), are required for production of circadian rhythms in Drosophila. The proteins encoded by these genes (PER and TIM) physically interact, and the timing of their association and nuclear localization
is believed to promote cycles of per and tim transcription through an autoregulatory feedback loop. Here it is shown that TIM protein may also couple this molecular pacemaker
to the environment, because TIM is rapidly degraded after exposure to light. TIM accumulated rhythmically in nuclei of eyes
and in pacemaker cells of the brain. The phase of these rhythms was differentially advanced or delayed by light pulses delivered
at different times of day, corresponding with phase shifts induced in the behavioral rhythms.
The effects of 24 hr light-dark cycles on the circadian conidiation rhythm inNeurospora crassa were compared among will-typefrq
+ and clock mutantsfrq
+,frq
3,frq
7,frq
9 andfrq
11. The minimum length of the light period necessary for complete entrainment to the light-dark cycles was almost 2 hr infrq
+,frq
3 andfrq
7 strains. The minimum duration of the dark period necessary for the appearance of circadian conidiation was almost 4 hr in
all of the strains except thefrq
11 strain. The phase of the conidiation rhythm was dependent on the light to dark transition in thefrq
1 strain in all light-dark cycles examined and in thefrq
+ andfrq
3 strains when the light period was shorter than 16 hr. In contrast, the phase of thefrq
7 strain was dependent on the light to dark transition when the light period was shorter than 10 hr.
The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq(9) and frq(10)) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq(9), chol-1 frq(10), chol-1 wc-1, chol-1 wc-2, cel frq(9), cel frq(10), and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.
To understand how light entrains circadian clocks, we examined the effects of light on a gene known to encode a state variable of a circadian oscillator, the frequency (frq) gene. frq is rapidly induced by short pulses of visible light; clock resetting is correlated with frq induction and is blocked by drugs that block the synthesis of protein or translatable RNA. The speed and magnitude of frq induction suggest that this may be the initial clock-specific event in light resetting. Light induction overcomes frq negative autoregulation so that frq expression can remain high in constant light. These data explain how a simple unidirectional signal (light and the induction of frq) may be turned into a bidirectional clock response (time of day-specific advances and delays). This light entrainment model is easily generalized and may be the common mechanism by which the intracellular feedback cycles that comprise circadian clocks are brought into synchrony with external cycles in the real world.
The frequency (frq) locus of Neurospora crassa was originally identified in searches for loci encoding components of the circadian clock. The frq gene is now shown to encode a central component in a molecular feedback loop in which the product of frq negatively regulated its own transcript, which resulted in a daily oscillation in the amount of frq transcript. Rhythmic messenger RNA expression was essential for overt rhythmicity in the organism and no amount of constitutive expression rescued normal rhythmicity in frq loss-of-function mutants. Step reductions in the amount of FRQ-encoding transcript set the clock to a specific and predicted phase. These results establish frq as encoding a central component in a circadian oscillator.
In the filamentous fungus Neurospora crassa, several events in the process of conidiation are influenced by light. Two genes, con-6 and con-10, which were previously shown to be transcriptionally activated during conidiation and by exposure to light, were found to be unexpressed in mycelium maintained in constant darkness or in constant light. However, when mycelium was shifted from darkness to light, transcripts of both genes appeared and were abundant. Upon further illumination both transcripts disappeared--i.e., their continued production was light repressed. When dark-grown mycelium was exposed to a light pulse and reincubated in the dark, expression of con-6 and con-10 exhibited a 20-hr circadian periodicity. Both genes were photoinducible throughout the stages of the circadian cycle. In the mutant strains bd and bd;frq9, con-6 and con-10 were light inducible but were not normally light repressible. Mutant genes such as acon-2, acon-3, and fl that block developmental expression of con-6 and/or con-10 did not prevent their photoinduction.
The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.
Circadian behavioral rhythms in Drosophila depend on the appropriate regulation of at least two genes, period (per) and timeless (tim). Previous studies demonstrated that levels of PER and TIM RNA cycle with the same phase and that the PER and TIM proteins interact directly. Here we show the cyclic expression of TIM protein in adult heads and report that it lags behind peak levels of TIM RNA by several hours. We alsoshow that nuclear expression of TIM depends upon the expression of PER protein. Finally, we report that the expression of TIM, but not PER, is rapidly reduced by light, suggesting that TIM mediates light-induced resetting of the circadian clock. Since both PER and TIM RNA are unaffected by light treatment, the effects of light on TIM appear to be posttranscriptional.
The circadian oscillator in Neurospora is a negative feedback loop involving as principal players the products of the frequency (frq) locus. frq encodes multiple forms of its protein product FRQ, which act to depress the amounts of frq transcript. In this scheme there are two discrete and separable steps to the circadian cycle, negative feedback itself (repression) in which FRQ acts to decrease the levels of its own transcript, and recovery from repression (derepression) in which frq transcript levels return to peak amounts. By introducing an exogenously regulatable frq transgene into a frq loss-of-function strain (frq9), we created an artificial system in which the two separate steps in the circadian cycle can be initiated and followed separately for purposes of observing their kinetics. Under these conditions the frq-FRQ cycle occupies the time scale of a full circadian cycle. During this time, the process of negative feedback of FRQ on frq transcript levels is rapid and efficient; it requires only 3 to 6 h and can be mediated by on the order of 10 molecules of FRQ per nucleus, a level even less than that seen in the normal oscillation. In contrast, recovery from negative feedback requires 14 to 18 h, most of the circadian cycle, during which time de novo FRQ synthesis has stopped, and existing FRQ is progressively posttranslationally modified. Altogether the time required to complete both of these steps is in good agreement with the 22-h observed period length of the normal circadian cycle.
The frequency (frq) gene encodes central components of the transcription/translation-based negative-feedback loop comprising the core of the Neurospora circadian oscillator; posttranscriptional regulation associated with FRQ is surprisingly complex. Alternative use of translation initiation sites gives rise to two forms of FRQ whose levels peak 4-6 hr following the peak of frq transcript. Each form of FRQ is progressively phosphorylated over the course of the day, thus providing a number of temporally distinct FRQ products. The kinetics of these regulatory processes suggest a view of the clock where relatively rapid events involving translational regulation in the synthesis of FRQ and negative feedback of FRQ on frq transcript levels are followed by slower posttranslational regulation, ultimately driving the turnover of FRQ and reactivation of the frq gene.
Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggests involvement of the casein kinase I homolog doubletime (dbt) in the Drosophila circadian pacemaker. We have characterized two novel dbt mutants. Both cause a lengthening of behavioral period and profoundly alter period (per) and timeless (tim) transcript and protein profiles. The PER profile shows a major difference from the wild-type program only during the morning hours, consistent with a prominent role for DBT during the PER monomer degradation phase. The transcript profiles are delayed, but there is little effect on the protein accumulation profiles, resulting in the elimination of the characteristic lag between the mRNA and protein profiles. These results and others indicate that light and post-transcriptional regulation play major roles in defining the temporal properties of the protein curves and suggest that this lag is unnecessary for the feedback regulation of per and tim protein on per and tim transcription.
FREQUENCY (FRQ) is a critical element of the circadian system of Neurospora. The white collar genes are important both for light reception and circadian function. We show that the responsiveness of the light input pathway is circadianly regulated. This circadian modulation extends to light-inducible components and functions that are not rhythmic themselves in constant conditions. FRQ interacts genetically and physically with WHITE COLLAR-1, and physically with WHITE COLLAR-2. These findings begin to address how components of the circadian system interact with basic cellular functions, in this case with sensory transduction.
FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1 epsilon. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frq Delta PEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frq Delta PEST-1 RNA and FRQ Delta PEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components.
Visible light is thought to reset the Neurospora circadian clock by acting through heterodimers of the WHITE COLLAR-1 and WHITE COLLAR-2 proteins to induce transcription of the frequency gene. To characterize this photic entrainment we examined frq expression in constant light, under which condition the mRNA and protein of this clock gene were strongly induced. In continuous illumination FRQ accumulated in a highly phosphorylated state similar to that seen at subjective dusk, the time at which a step from constant light to darkness sets the clock. Examination of frq expression in several wc-2 mutant alleles surprisingly revealed differential regulation when frq expression was compared between constant light, following a light pulse, and darkness (clock-driven expression). Construction of a wc-2 null strain then demonstrated that WC-2 is absolutely required for both light and clock-driven frq expression, in contrast to previous expectations based on presumptive nulls containing altered Zn-finger function. Additionally, we found that frq light signal transduction differs from that of other light-regulated genes. Thus clock and light-driven frq expression is differentially regulated by, but dependent on, WC-2.
In mammals, circadian control of physiology and behavior is driven by a master pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus. We have used gene expression profiling to identify cycling transcripts in the SCN and in the liver. Our analysis revealed approximately 650 cycling transcripts and showed that the majority of these were specific to either the SCN or the liver. Genetic and genomic analysis suggests that a relatively small number of output genes are directly regulated by core oscillator components. Major processes regulated by the SCN and liver were found to be under circadian regulation. Importantly, rate-limiting steps in these various pathways were key sites of circadian control, highlighting the fundamental role that circadian clocks play in cellular and organismal physiology.
The filamentous fungus Neurospora crassa is a model organism for the genetic dissection of blue light photoreception and circadian rhythms. WHITE COLLAR-1 (WC-1) and WC-2 are considered necessary for all light responses, while FREQUENCY (FRQ) is required for light-regulated asexual development (conidia formation); without any of the three, self-sustained (circadian) rhythmicity in constant conditions fails. Here we show that light-regulated and self-sustained development occur in the individual or mutant white collar strains. These strains resemble wild type in their organization of the daily bout of light-regulated conidiation. Molecular profiles of light- induced genes indicate that the individual white collar-1 and white collar-2 mutants utilize distinct pathways, despite their similar appearance in all aspects. Titration of fluence rate also demonstrates different light sensitivities between the two strains. The data require the existence of an as-yet-unidentified photoreceptor. Furthermore, the extant circadian clock machinery in these mutant strains supports the notion that the circadian system in Neurospora involves components outside the WC-FRQ loop.
Daylength, or photoperiod, is perceived as a seasonal signal for the control of flowering of many plants. The measurement of daylength is thought to be mediated through the interaction of phototransduction pathways with a circadian rhythm, so that flowering is induced (in long-day plants) or repressed (in short-day plants) when light coincides with a sensitive phase of the circadian cycle. To test this hypothesis in the facultative long-day plant, Arabidopsis thaliana, we used varying, non-24-hr light/dark cycles to alter the timing of circadian rhythms of gene expression relative to dawn and dusk. Effects on circadian rhythms were correlated with those on flowering times. We show that conditions that displaced subjective night events, such as expression of the flowering time regulator CONSTANS into the light portion of the cycle, were perceived as longer days. This work demonstrates that the perception of daylength in Arabidopsis relies on adjustments of the phase angle of circadian rhythms relative to the light/dark cycle, rather than on the measurement of the absolute duration of light and darkness.
Human behavior shows large interindividual variation in temporal organization. Extreme "larks" wake up when extreme "owls" fall asleep. These chronotypes are attributed to differences in the circadian clock, and in animals, the genetic basis of similar phenotypic differences is well established. To better understand the genetic basis of temporal organization in humans, the authors developed a questionnaire to document individual sleep times, self-reported light exposure, and self-assessed chronotype, considering work and free days separately. This report summarizes the results of 500 questionnaires completed in a pilot study individual sleep times show large differences between work and free days, except for extreme early types. During the workweek, late chronotypes accumulate considerable sleep debt, for which they compensate on free days by lengthening their sleep by several hours. For all chronotypes, the amount of time spent outdoors in broad daylight significantly affects the timing of sleep: Increased self-reported light exposure advances sleep. The timing of self-selected sleep is multifactorial, including genetic disposition, sleep debt accumulated on workdays, and light exposure. Thus, accurate assessment of genetic chronotypes has to incorporate all of these parameters. The dependence of human chronotype on light, that is, on the amplitude of the light:dark signal, follows the known characteristics of circadian systems in all other experimental organisms. Our results predict that the timing of sleep has changed during industrialization and that a majority of humans are sleep deprived during the workweek. The implications are far ranging concerning learning, memory, vigilance, performance, and quality of life.
Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.
The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. Here we show that the product of the slimb (slmb) gene--a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation--is an essential component of the Drosophila circadian clock. slmb mutants are behaviourally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms.
The possibility that membranes are involved in circadian rhythmicity in the ascomycete fungus Neurospora crassa is discussed. Neurospora is an ideal organism for testing this possibility; knowledge of its biochemistry and genetics is substantial, and mutants affecting circadian rhythms and/or lipid biochemistry are available.
The Neurospora circadian oscillator receives input on temperature and illumination from the environment. Evidence suggests that the Neurospora blue-light photoreceptor may be membrane-localized. Studies using Neurospora inositol and choline auxotrophs, however, appear to rule out hydrolysis of phospholipids by a phospholipase C or a phospholipase D in transduction of light signal information from the photoreceptor to the oscillator. The mechanism of temperature signal transduction remains unknown, but temperature-induced changes in membrane properties could play a role.
Involvement of membrane lipids in the mechanism of the oscillator itself is suggested by correlation of lengthened period with abnormal membrane composition in the Neurospora mutants cel, prd-1 and chol-1. The cel mutant is blocked in fatty acid synthesis; both the period of its rhythm and the fatty acid composition of its membranes are sensitive to environmental conditions, no simple relationship between its membrane composition and its period length has yet been described. The chol-1 mutant is also altered in lipid synthesis; it is a choline auxotroph unable to synthesize phosphatidylcholine unless choline is supplied. The prd-1 mutant was isolated on the basis of long circadian period; it has an abnormal membrane fatty acid composition, although its primary biochemical lesion is unknown.
The levels of linoleic and linolenic acid in the membranes of Neurospora vary with a circadian rhythm, and similar rhythms in fatty acid composition have been reported in a number of species of plants and animals. These rhythms could be part of the circadian oscillator mechanism, or part of an ouput pathway from the circadian oscillator.
Temperature compensation of membrane fluidity by enzymatic alteration of membrane lipid composition may play a role in the temperature compensation of rhythmicity, and this possibility is discussed. Temperature compensation of rhythmicity appears to be lost in three different mutants of Neurospora: cel, chol-1, and null alleles of frq. The cel and chol-1 mutants are defective in lipid synthesis, and are likely to show abnormal adjustment of membrane composition with temperature. The frq locus was originally identified on the basis of altered period length. There is no evidence for defective lipid metabolism in frq mutants, but such a defect has not been ruled out.
The ascomycete Neurospora crassa has the capacity of adapting to a given light quantity, leading to transient blue light responses under continuous light conditions. Here, we present an investigation of this photoadaptation phenomenon. We demonstrated previously that two proteins of the Neurospora blue light signal transduction chain, WC1 and WC2, are subject to light-dependent phosphorylation. WC1 was phosphorylated in parallel with the transient increase in transcript levels of light-regulated genes. Using the light-dependent phosphorylation of WC1 as a marker for an active signalling state of WC1, we show that the transiency of Neurospora blue light responses results from desensitization of the photoreceptor and/or the signalling cascade. Furthermore, a Neurospora mutant was characterized that revealed a specific defect in photoadaptation. In this mutant, the transient expression of light-regulated genes under continuous light, the temporary insensitivity after a light pulse and the capability of differentiating between and adapting to low and high light intensities were abolished. The corresponding protein seems to represent a central component of a negative feedback desensitization mechanism. This negative feedback regulation requires continuous and light-dependent protein de novo biosynthesis.
The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights-on. These rhythms are circadian in nature as they continue approximately under free-running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.
Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.
Circadian clocks can be reset by light stimulation. To investigate the mechanism of this phase shifting, the effects of light pulses on the protein and messenger RNA products of the Drosophila clock gene period (per) were measured. Photic stimuli perturbed the timing of the PER protein and messenger RNA cycles in a manner consistent with the direction and magnitude of the phase shift. In addition, the recently identified clock protein TIM (for timeless) interacted with PER in vivo, and this association was rapidly decreased by light. This disruption of the PER-TIM complex in the cytoplasm was accompanied by a delay in PER phosphorylation and nuclear entry and disruption in the nucleus by an advance in PER phosphorylation and disappearance. These results suggest a mechanism for how a unidirectional environmental signal elicits a bidirectional clock response.
Basal plasma ACTH and corticosterone levels are controlled by the suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, resulting in a daily peak in plasma corticosterone and ACTH. The present study was carried out to investigate the mechanisms employed by the biological clock to control these hormones. Novel environment induced changes in plasma ACTH and corticosterone in intact and SCN-lesioned animals were employed as experimental approach. Placing intact animals in a new environment results in different plasma corticosterone and ACTH responses depending on the clock time of the stimulus. (1) Novel environment (2 h after onset of darkness (ZT14)) results in a fast decrease followed by an increase in corticosterone. This changing pattern in corticosterone secretion was not accompanied by any change in plasma ACTH, suggesting a direct neuronal control of the adrenal cortex. (2) In contrast, novel environment at 2 h after light onset (ZT2) results in a rapid increase in plasma ACTH. Regression analysis of the relation ACTH-corticosterone before and after stress shows a changed pattern at ZT2, although at that time still no significant correlation between ACTH and corticosterone was detected. AT ZT14 this correlation was only present after stress. (3) SCN lesioning results in low basal ACTH at all circadian times combined with elevated corticosterone levels. Here, a new environment results in an immediate increase in corticosterone without inhibition; ACTH also increases rapidly, but attains lower levels than at ZT2 in intact animals. (4) The present results therefore demonstrate SCN modulating corticosterone secretion by affecting ACTH secretion and changing the sensitivity of the adrenal cortex by means of a neuronal input.
Molecular and genetic characterizations of circadian rhythms in Drosophila indicate that function of an intracellular pacemaker requires the activities of proteins encoded by three genes: period (per), timeless (tim), and doubletime (dbt). RNA from two of these genes, per and tim, is expressed with a circadian rhythm. Heterodimerization of PER and TIM proteins allows nuclear localization and suppression of further RNA synthesis by a PER/TIM complex. These protein interactions promote cyclical gene expression because heterodimers are observed only at high concentrations of per and tim RNA, separating intervals of RNA accumulation from times of PER/TIM complex activity. Light resets these molecular cycles by eliminating TIM. The product of dbt also regulates accumulation of per and tim RNA, and it may influence action of the PER/TIM complex. The recent discovery of PER homologues in mice and humans suggests that a related mechanism controls mammalian circadian behavioral rhythms.
Circadian clocks consist of three elements: entrainment pathways (inputs), the mechanism generating the rhythmicity (oscillator), and the output pathways that control the circadian rhythms. It is difficult to assign molecular clock components to any one of these elements. Experiments show that inputs can be circadianly regulated and outputs can feed back on the oscillator. Mathematical simulations indicate that under- or overexpression of a gene product can result in arrhythmicity, whether the protein is part of the oscillator or substantially part of a rhythmically expressed input pathway. To distinguish between these two possibilities, we used traditional circadian entrainment protocols on a genetic model system, Neurospora crassa.
In Neurospora crassa, white collar 1 (WC-1), a transcriptional activator and positive clock element, is rhythmically expressed from a nonrhythmic
steady-state pool of wc-1transcript, consistent with posttranscriptional regulation of rhythmicity. Mutations in frq influence both the level and periodicity of WC-1 expression, and driven FRQ expression not only depresses its own endogenous
levels, but positively regulates WC-1 synthesis with a lag of about 8 hours, a delay similar to that seen in the wild-type
clock. FRQ thus plays dual roles in theNeurospora clock and thereby, with WC-1, forms a second feedback loop that would promote robustness and stability in this circadian
system. The existence also of interlocked loops inDrosophila melanogaster and mouse clocks suggests that such interlocked loops may be a conserved aspect of circadian timing systems.
The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights-on. These rhythms are circadian in nature as they continue approximately under free-running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.
The circadian clock in all organisms is so intimately linked to light reception that it appears as if evolution has simply wired a timer into the mechanism that processes photic information. Several recent studies have provided new insights into the role of light input pathways in the circadian system of Arabidopsis.
Circadian and photoperiodic timing mechanisms were first described in photosynthetic organisms. These organisms depend upon sunlight for their energy, so adaptation to daily and seasonal fluctuations in light must have generated a strong selective pressure. Studies of the endogenous timekeepers of photosynthetic organisms provide evidence for both a fitness advantage and for selective pressures involved in early evolution of circadian clocks. Photoperiodic timing mechanisms in plants appear to use their circadian timers as the ruler by which the day/night length is measured. As in animals, the overall clock system in plants appears to be complex; the system includes multiple oscillators, several input pathways, and a myriad of outputs. Genes have now been isolated from plants that are likely to encode components of the central clockwork or at least that act very close to the central mechanism. Genetic and biochemical analyses of the central clockwork of a photosynthetic organism are most highly advanced in cyanobacteria, where a cluster of clock genes and interacting factors have been characterized.
vvd, a gene regulating light responses in Neurospora, encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock-associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq, consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase-response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.
con-10 and con-6 are two of the conidiation (con) genes of Neurospora crassa that were identified based on their preferential expression during macroconidiophore development. They are also regulated by several other environmental stimuli independent of development, including a transient induction by light. We identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to light. In addition, con-10 and con-6 show a heightened response to photoinduction. We tested the function of the light-responsive circadian clock in the vvd mutant and found no major defect in the circadian rhythm of conidiation or light regulation of a key clock component, frequency (frq). We conclude that vvd is primarily involved in a process of light-dependent gene repression, called light adaptation. Although a number of gene products are known to control light induction in fungi, vvd is the first gene shown to have a role in adaptation to constant light.
Light is the most reliable environmental signal for adjusting biological clocks to the 24-hour day. Mammals receive this signal exclusively through the eyes, but not just via rods and cones. New evidence has been uncovered for a novel photoreceptor that may be responsible for more than just adjusting the clock.
Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecular basis of circadian timing in peripheral tissue is not well understood. We used a custom-made cDNA microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions.
Using two independent tissue sampling and hybridization regimes, we show that approximately 9% of the 2122 genes studied show robust circadian cycling in the liver. These transcripts were categorized by their phase of abundance, defining clusters of day- and night-related genes, and also by the function of their products. Circadian regulation of genes was tissue specific, insofar as novel rhythmic liver genes were not necessarily rhythmic in the brain, even when expressed in the SCN. The rhythmic transcriptome in the periphery is, nevertheless, dependent on the SCN because surgical ablation of the SCN severely dampened or destroyed completely the cyclical expression of both canonical circadian genes and novel genes identified by microarray analysis.
Temporally complex, circadian programming of the transcriptome in a peripheral organ is imposed across a wide range of core cellular functions and is dependent on an interaction between intrinsic, tissue-specific factors and extrinsic regulation by the SCN central pacemaker.
In the fungus Neurospora crassa, the blue light photoreceptor(s) and signaling pathway(s) have not been identified. We examined light signaling by exploiting
the light sensitivity of the Neurospora biological clock, specifically the rapid induction by light of the clock componentfrequency (frq). Light induction offrq is transcriptionally controlled and requires two cis-acting elements (LREs) in the frq promoter. Both LREs are bound by a White Collar–1 (WC-1)/White Collar–2 (WC-2)–containing complex (WCC), and light causes
decreased mobility of the WCC bound to the LREs. The use of in vitro–translated WC-1 and WC-2 confirmed that WC-1, with flavin
adenine dinucleotide as a cofactor, is the blue light photoreceptor that mediates light input to the circadian system through
direct binding (with WC-2) to the frq promoter.
Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase Iepsilon. However, it is not clear how Dbt regulates the levels of Per. Here we show, using a cell culture system, that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin-proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin-proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. Our findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb.
The circadian clock provides a temporal structure that modulates biological functions from the level of gene expression to performance and behaviour. Pioneering work on the fruitfly Drosophila has provided a basis for understanding how the temporal sequence of daily events is controlled in mammals. New insights have come from work on mammals, specifically from studying the daily activity profiles of clock mutant mice; from more detailed recordings of clock gene expression under different experimental conditions and in different tissues; and from the discovery and analysis of a growing number of additional clock genes. These new results are moving the model paradigm away from a simple negative feedback loop to a molecular network. Understanding the coupling and interactions of this network will help us to understand the evolution of the circadian system, advance medical diagnosis and treatment, improve the health of shift workers and frequent travellers, and will generally enable the treatment of clock-related pathologies.
Light and temperature are 2 of the most important environmental influences on all circadian clocks, and Neurospora provides an excellent system for understanding their effects. Progress made in the past decade has led to a basic molecular understanding of how the Neurospora clock works and how environmental factors influence it. The purpose of this review is to summarize what we currently know about the molecular mechanism of light and temperature entrainment in Neurospora.
The circadian system actively synchronizes the temporal sequence of biological functions with the environment. The oscillatory behavior of the system ensures that entrainment is not passive or driven and therefore allows for great plasticity and adaptive potential. With the tools at hand, we now can concentrate on the most important circadian question: How is the complex task of entrainment achieved by anatomical, cellular, and molecular components? Understanding entrainment is equal to understanding the circadian system. The results of this basic research will help us to understand temporal ecology and will allow us to improve conditions for humans in industrialized societies.
The suprachiasmatic nucleus (SCN) of the anterior hypothalamus contains a major circadian pacemaker that imposes or entrains rhythmicity on other structures by generating a circadian pattern in electrical activity. The identification of "clock genes" within the SCN and the ability to dynamically measure their rhythmicity by using transgenic animals open up new opportunities to study the relationship between molecular rhythmicity and other well-documented rhythms within the SCN. We investigated SCN circadian rhythms in Per1-luc bioluminescence, electrical activity in vitro and in vivo, as well as the behavioral activity of rats exposed to a 6-hr advance in the light-dark cycle followed by constant darkness. The data indicate large and persisting phase advances in Per1-luc bioluminescence rhythmicity, transient phase advances in SCN electrical activity in vitro, and an absence of phase advances in SCN behavioral or electrical activity measured in vivo. Surprisingly, the in vitro phase-advanced electrical rhythm returns to the phase measured in vivo when the SCN remains in situ. Our study indicates that hierarchical levels of organization within the circadian timing system influence SCN output and suggests a strong and unforeseen role of extra-SCN areas in regulating pacemaker function.
Circadian timing within the suprachiasmatic nucleus (SCN) is modelled around cell-autonomous, autoregulatory transcriptional/post-translational feedback loops, in which protein products of canonical clock genes Period and Cryptochrome periodically oppose transcription driven by CLOCK:BMAL complexes. Consistent with this model, mCLOCK is a nuclear antigen constitutively expressed in mouse SCN, whereas nuclear mPER and mCRY are expressed rhythmically. Peaking in late subjective day, mPER and mCRY form heteromeric complexes with mCLOCK, completing the negative feedback loop as levels of mPer and mCry mRNA decline. Circadian resetting by light or non-photic resetting (mediated by neuropeptide Y) involves acute up- and down-regulation of mPer mRNA, respectively. Expression of Per mRNA also peaks in subjective day in the SCN of the ground squirrel, indicating common clock and entrainment mechanisms for nocturnal and diurnal species. Oscillation within the SCN is dependent on intercellular signals, in so far as genetic ablation of the VPAC2 receptor for vasoactive intestinal polypeptide (VIP) suspends SCN circadian gene expression. The pervasive effect of the SCN on peripheral physiology is underscored by cDNA microarray analysis of the circadian gene expression in liver, which involves ca. 10% of the genome and almost all aspects of cell function. Moreover, the same molecular regulatory mechanisms driving the SCN appear also to underpin peripheral cycles.
The timing of cell proliferation is a key factor contributing to the regulation of normal growth. Daily rhythms of cell cycle progression have been documented in a wide range of organisms. However, little is known about how environmental, humoral, and cell-autonomous factors contribute to these rhythms. Here, we demonstrate that light plays a key role in cell cycle regulation in the zebrafish. Exposure of larvae to light-dark (LD) cycles causes a range of different cell types to enter S phase predominantly at the end of the day. When larvae are raised in constant darkness (DD), a low level of arrhythmic S phase is observed. In addition, light-entrained cell cycle rhythms persist for several days after transfer to DD, both observations pointing to the involvement of the circadian clock. We show that the number of LD cycles experienced is essential for establishing this rhythm during larval development. Furthermore, we reveal that the same phenomenon exists in a zebrafish cell line. This represents the first example of a vertebrate cell culture system where circadian rhythms of the cell cycle are observed. Thus, we implicate the cell-autonomous circadian clock in the regulation of the vertebrate cell cycle by light.