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1.  A New Mutation Affecting FRQ-Less Rhythms in the Circadian System of Neurospora crassa 
PLoS Genetics  2011;7(6):e1002151.
We are using the fungus Neurospora crassa as a model organism to study the circadian system of eukaryotes. Although the FRQ/WCC feedback loop is said to be central to the circadian system in Neurospora, rhythms can still be seen under many conditions in FRQ-less (frq knockout) strains. To try to identify components of the FRQ-less oscillator (FLO), we carried out a mutagenesis screen in a FRQ-less strain and selected colonies with altered conidiation (spore-formation) rhythms. A mutation we named UV90 affects rhythmicity in both FRQ-less and FRQ-sufficient strains. The UV90 mutation affects FRQ-less rhythms in two conditions: the free-running long-period rhythm in choline-depleted chol-1 strains becomes arrhythmic, and the heat-entrained rhythm in the frq10 knockout is severely altered. In a FRQ-sufficient background, the UV90 mutation causes damping of the free-running conidiation rhythm, reduction of the amplitude of the FRQ protein rhythm, and increased phase-resetting responses to both light and heat pulses, consistent with a decreased amplitude of the circadian oscillator. The UV90 mutation also has small but significant effects on the period of the conidiation rhythm and on growth rate. The wild-type UV90 gene product appears to be required for a functional FLO and for sustained, high-amplitude rhythms in FRQ-sufficient conditions. The UV90 gene product may therefore be a good candidate for a component of the FRQ-less oscillator. These results support a model of the Neurospora circadian system in which the FRQ/WCC feedback loop mutually interacts with a single FLO in an integrated circadian system.
Author Summary
All eukaryotes (including humans), and some bacteria, have evolved internal biological clocks that control activity and physiology in a daily (circadian) cycle. The molecular oscillators that drive these circadian rhythms are said to depend on rhythmic expression and feedback regulation of a small set of “clock genes.” However, there is increasing evidence that there is more to the story than these well-studied feedback loops. In the fungus Neurospora crassa, rhythms can still be seen in mutants that are missing one of the clock genes, frq. There is currently a controversy as to whether there are many different frq-less oscillators and whether they interact with the frq clock. To identify the molecular mechanism that drives these frq-less rhythms, we started with a frq-less strain and mutagenized it to look for genes that affect the frq-less rhythms. We found a new mutation that not only disrupted two frq-less rhythms but also affected the rhythm when the frq gene is present. Our results suggest there is only one frq-less oscillator, and it interacts with the frq clock. Our new mutation may identify a gene that is critical to both oscillators. We suggest that a similar clock architecture may be common to all organisms.
PMCID: PMC3121751  PMID: 21731506
2.  Quantitative analysis of regulatory flexibility under changing environmental conditions 
Day length changes with the seasons in temperate latitudes, affecting the many biological rhythms that entrain to the day/night cycle: we measure these effects on the expression of Arabidopsis clock genes, using RNA and reporter gene readouts, with a new method of phase analysis.Dusk sensitivity is proposed as a simple, natural and general mathematical measure to analyse and manipulate the changing phase of a clock output relative to the change in the day/night cycle.Dusk sensitivity shows how increasing the numbers of feedback loops in the Arabidopsis clock models allows more flexible regulation, consistent with a previously-proposed, general operating principle of biological networks.The Arabidopsis clock genes show flexibility of regulation that is characteristic of a three-loop clock model, validating aspects of the model and the operating principle, but some clock output genes show greater flexibility arising from direct light regulation.
The analysis of dynamic, non-linear regulation with the aid of mechanistic models is central to Systems Biology. This study compares the predictions of mechanistic, mathematical models of the circadian clock with molecular time-series data on rhythmic gene expression in the higher plant Arabidopsis thaliana. Analysis of the models helps us to understand (explain and predict) how the clock gene circuit balances regulation by external and endogenous factors to achieve particular behaviours. Such multi-factorial regulation is ubiquitous in, and characteristic of, living systems.
The Earth's rotation causes predictable changes in the environment, notably in the availability of sunlight for photosynthesis. Many biological processes are driven by the environmental input via sensory pathways, for example, from photoreceptors. Circadian clocks provide an alternative strategy. These endogenous, 24-h rhythms can drive biological processes that anticipate the regular environmental changes, rather than merely responding. Many rhythmic processes have both light and clock control. Indeed, the clock components themselves must balance internal timing with external inputs, because circadian clocks are reset daily through light regulation of one or more clock components. This process of entrainment is complicated by the change in day length. When the times of dawn and dusk move apart in summer, and closer together in winter, does the clock track dawn, track dusk or interpolate between them?
In plants, the clock controls leaf and petal movements, the opening and closing of stomatal pores, the discharge of floral fragrances, and many metabolic activities, especially those associated with photosynthesis. Centuries of physiological studies have shown that these rhythms can behave differently. Flowering in Ipomoea nil (Pharbitis nil, Japanese morning glory) is controlled by a rhythm that tracks the time of dusk, to give a classic example. We showed that two other rhythms associated with vegetative growth track dawn in this species (Figure 5A), so the clock system allows flexible regulation.
The relatively small number of components involved in the circadian clockwork makes it an ideal candidate for mathematical modelling. Molecular genetic studies in a variety of model eukaryotes have shown that the circadian rhythm is generated by a network of 6–20 genes. These genes form feedback loops generating a rhythm in mRNA production. A single negative feedback loop in which a gene encodes a protein that, after several hours, turns off transcription is capable of generating a circadian rhythm, in principle. A single light input can entrain the clock to ‘local time', synchronised with a light–dark cycle. However, real circadian clocks have proven to be more complicated than this, with multiple light inputs and interlocked feedback loops.
We have previously argued from mathematical analysis that multi-loop networks increase the flexibility of regulation (Rand et al, 2004) and have shown that appropriately deployed flexibility can confer functional robustness (Akman et al, 2010). Here we test whether that flexibility can be demonstrated in vivo, in the model plant, A. thaliana. The Arabidopsis clock mechanism comprises a feedback loop in which two partially redundant, myb transcription factors, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of their activator, TIMING OF CAB EXPRESSION 1 (TOC1). We previously modelled this single-loop circuit and showed that it was not capable of recreating important data (Locke et al, 2005a). An extended, two-loop model was developed to match observed behaviours, incorporating a hypothetical gene Y, for which the best identified candidate was the GIGANTEA gene (GI) (Locke et al, 2005b). Two further models incorporated the TOC1 homologues PSEUDO-RESPONSE REGULATOR (PRR) 9 and PRR7 (Locke et al, 2006; Zeilinger et al, 2006). In these circuits, a morning oscillator (LHY/CCA1–PRR9/7) is coupled to an evening oscillator (Y/GI–TOC1) via the original LHY/CCA1–TOC1 loop.
These clock models, like those for all other organisms, were developed using data from simple conditions of constant light, darkness or 12-h light–12-h dark cycles. We therefore tested how the clock genes in Arabidopsis responded to light–dark cycles with different photoperiods, from 3 h light to 18 h light per 24-h cycle (Edinburgh, 56° North latitude, has 17.5 h light in midsummer). The time-series assays of mRNA and in vivo reporter gene images showed a range of peak times for different genes, depending on the photoperiod (Figure 5C). A new data analysis method, mFourfit, was introduced to measure the peak times, in the Biological Rhythms Analysis Software Suite (BRASS v3.0). None of the genes showed the dusk-tracking behaviour characteristic of the Ipomoea flowering rhythm. The one-, two- and three-loop models were analysed to understand the observed patterns. A new mathematical measure, dusk sensitivity, was introduced to measure the change in timing of a model component versus a change in the time of dusk. The one- and two-loop models tracked dawn and dusk, respectively, under all conditions. Only the three-loop model (Figure 5B) had the flexibility required to match the photoperiod-dependent changes that we found in vivo, and in particular the unexpected, V-shaped pattern in the peak time of TOC1 expression. This pattern of regulation depends on the structure and light inputs to the model's evening oscillator, so the in vivo data supported this aspect of the model. LHY and CCA1 gene expression under short photoperiods showed greater dusk sensitivity, in the interval 2–6 h before dawn, than the three-loop model predicted, so these data will help to constrain future models.
The approach described here could act as a template for experimental biologists seeking to understand biological regulation using dynamic, experimental perturbations and time-series data. Simulation of mathematical models (despite known imperfections) can provide contrasting hypotheses that guide understanding. The system's detailed behaviour is complex, so a natural and general measure such as dusk sensitivity is helpful to focus on one property of the system. We used the measure to compare models, and to predict how this property could be manipulated. To enable additional analysis of this system, we provide the time-series data and experimental metadata online.
The circadian clock controls 24-h rhythms in many biological processes, allowing appropriate timing of biological rhythms relative to dawn and dusk. Known clock circuits include multiple, interlocked feedback loops. Theory suggested that multiple loops contribute the flexibility for molecular rhythms to track multiple phases of the external cycle. Clear dawn- and dusk-tracking rhythms illustrate the flexibility of timing in Ipomoea nil. Molecular clock components in Arabidopsis thaliana showed complex, photoperiod-dependent regulation, which was analysed by comparison with three contrasting models. A simple, quantitative measure, Dusk Sensitivity, was introduced to compare the behaviour of clock models with varying loop complexity. Evening-expressed clock genes showed photoperiod-dependent dusk sensitivity, as predicted by the three-loop model, whereas the one- and two-loop models tracked dawn and dusk, respectively. Output genes for starch degradation achieved dusk-tracking expression through light regulation, rather than a dusk-tracking rhythm. Model analysis predicted which biochemical processes could be manipulated to extend dusk tracking. Our results reveal how an operating principle of biological regulators applies specifically to the plant circadian clock.
PMCID: PMC3010117  PMID: 21045818
Arabidopsis thaliana; biological clocks; dynamical systems; gene regulatory networks; mathematical models; photoperiodism
3.  Neurospora WC-1 Recruits SWI/SNF to Remodel frequency and Initiate a Circadian Cycle 
PLoS Genetics  2014;10(9):e1004599.
In the negative feedback loop comprising the Neurospora circadian oscillator, the White Collar Complex (WCC) formed from White Collar-1 (WC-1) and White Collar-2 (WC-2) drives transcription of the circadian pacemaker gene frequency (frq). Although FRQ-dependent repression of WCC has been extensively studied, the mechanism by which the WCC initiates a circadian cycle remains elusive. Structure/function analysis of WC-1 eliminated domains previously thought to transactivate frq expression but instead identified amino acids 100–200 as essential for frq circadian expression. A proteomics-based search for coactivators with WCC uncovered the SWI/SNF (SWItch/Sucrose NonFermentable) complex: SWI/SNF interacts with WCC in vivo and in vitro, binds to the Clock box in the frq promoter, and is required both for circadian remodeling of nucleosomes at frq and for rhythmic frq expression; interestingly, SWI/SNF is not required for light-induced frq expression. These data suggest a model in which WC-1 recruits SWI/SNF to remodel and loop chromatin at frq, thereby activating frq expression to initiate the circadian cycle.
Author Summary
Circadian clocks govern behavior in a wide variety of organisms. These clocks are assembled in such a way that proteins encoded by a few dedicated “clock genes” form a complex that acts to reduce their own expression. That is, the genes and proteins participate in a negative feedback loop, and so long as the feedback has delays built in, this system will oscillate. The feedback loops that underlie circadian rhythms in fungi and animals are quite similar in many ways, and while much is known about the proteins themselves, both those that activate the dedicated clock genes and the clock proteins that repress their own expression, relatively little is known about how the initial expression of the clock genes is activated. In Neurospora, a fungal model for these clocks, the proteins that activate expression of the clock gene “frequency” bind to DNA far away from where the coding part of the gene begins, and a mystery has been how this action-at-a-distance works. The answer revealed here is that the activating proteins recruit other proteins to unwrap the DNA and bring the distal site close to the place where the coding part of the gene begins.
PMCID: PMC4177678  PMID: 25254987
4.  Extension of a genetic network model by iterative experimentation and mathematical analysis 
Molecular Systems Biology  2005;1:2005.0013.
We extend the current model of the plant circadian clock, in order to accommodate new and published data. Throughout our model development we use a global parameter search to ensure that any limitations we find are due to the network architecture and not to our selection of the parameter values, which have not been determined experimentally. Our final model includes two, interlocked loops of gene regulation and is reminiscent of the circuit structures previously identified by experiments on insect and fungal clocks. It is the first Arabidopsis clock model to show such good correspondence to experimental data.Our interlocked feedback loop model predicts the regulation of two unknown components. Experiments motivated by these predictions identify the GIGANTEA gene as a strong candidate for one component, with an unexpected pattern of light regulation.*
This study involves an iterative approach of mathematical modelling and experiment to develop an accurate mathematical model of the circadian clock in the higher plant Arabidopsis thaliana. Our approach is central to systems biology and should lead to a greater, quantitative understanding of the circadian clock, as well as being more widely relevant to research into genetic networks.
The day–night cycle caused by the Earth's rotation affects most organisms, and has resulted in the evolution of the circadian clock. The circadian clock controls 24-h rhythms in processes from metabolism to behaviour; in higher eukaryotes, the circadian clock controls the rhythmic expression of 5–10% of genes. In plants, the clock controls leaf and petal movements, the opening and closing of stomatal pores, the discharge of floral fragrances and many metabolic activities, especially those associated with photosynthesis.
The relatively small number of components involved in the central circadian network makes it an ideal candidate for mathematical modelling of complex biological regulation. Genetic studies in a variety of model organisms have shown that the circadian rhythm is generated by a central network of between 6 and 12 genes. These genes form feedback loops generating a rhythm in mRNA production. One negative feedback loop in which a gene encodes a protein that, after several hours, turns off transcription is, in principle, capable of creating a circadian rhythm. However, real circadian clocks have proven to be more complicated than this, with interlocked feedback loops. Networks of this complexity are more easily understood through mathematical modelling.
The clock mechanism in the model plant, A. thaliana, was first proposed to comprise a feedback loop in which two partially redundant genes, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of their activator, TIMING OF CAB EXPRESSION 1 (TOC1). We previously modelled this preliminary network and showed that it was not capable of recreating several important pieces of experimental data (Locke et al, 2005). Here, we extend the LHY/CCA1–TOC1 network in new mathematical models. To check the effects of each addition to the network, the outputs of the extended models are compared to published data and to new experiments.
As is the case for most biological networks, the parameter values in our model, such as the translation rate of TOC1 protein, are unknown. We employ here an optimisation method, which works well with noisy and varied data and allows a global search of parameter space. This should ensure that the limitations we find in our networks are due to the network structure, and not to our parameter choices.
Our final interlocked feedback loop model requires two hypothetical components, genes X and Y (Figure 4), but is the first Arabidopsis clock model to exhibit such a good correspondence with experimental data. The model simulates a residual short-period oscillation in the cca1;lhy mutant, as characterised by our experiments. No single-loop model is able to do this. Our model also matches experimental data under constant light (LL) conditions and correctly senses photoperiod. The model predicts an interlocked feedback loop structure similar to that seen in the circadian clock mechanisms of other organisms.
The interlocked feedback loop model predicts a distinctive pattern of Y mRNA accumulation in the wild type (WT) and in the cca1;lhy double mutant, with Y mRNA levels increasing transiently at dawn. We designed an experiment to identify Y based on this prediction. GIGANTEA (GI) mRNA levels fit very well to our predicted profile for Y (Figure 6), identifying GI as a strong candidate for Y.
The approach described here could act as a template for experimental biologists seeking to extend models of small genetic networks. Our results illustrate the usefulness of mathematical modelling in guiding experiments, even if the models are based on limited data. Our method provides a way of identifying suitable candidate networks and quantifying how these networks better describe a wide variety of experimental measurements. The characteristics of new putative genes are thereby obtained, facilitating the experimental search for new components. To facilitate future experimental design, we provide user-friendly software that is specifically designed for numerical simulation of circadian experiments using models for several species (Brown, 2004b).
*Footnote: Synopsis highlights were added on 5 July 2005.
Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis thaliana have revealed a complex clock network. The first part of the network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), fails to account for significant experimental data. We develop an extended model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicted the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCA1. We found this unexpected regulation in RNA levels of the evening-expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component.
PMCID: PMC1681447  PMID: 16729048
biological rhythms; gene network; mathematical modelling; parameter estimation
5.  Methylation of Histone H3 on Lysine 4 by the Lysine Methyltransferase SET1 Protein Is Needed for Normal Clock Gene Expression* 
The Journal of Biological Chemistry  2013;288(12):8380-8390.
Background: Eukaryotic circadian clocks require chromatin modifications and remodeling.
Results: SET1 is required for proper expression of the Neurospora clock gene frequency (frq). SET1 modifies chromatin at frq with the peak in H3K4me3 occurring after the peak in activation.
Conclusion: H3K4 methylation appears to mitigate White Collar complex (WCC)-mediated expression.
Significance: Chromatin is a key component underlying circadian oscillations in gene expression.
The circadian oscillator controls time-of-day gene expression by a network of interconnected feedback loops and is reset by light. The requisite for chromatin regulation in eukaryotic transcription necessitates temporal regulation of histone-modifying and chromatin-remodeling enzymes for proper clock function. CHD1 is known to bind H3K4me3 in mammalian cells, and Neurospora CHD1 is required for proper regulation of the frequency (frq) gene. Based on this, we examined a strain lacking SET1 to determine the role of H3K4 methylation in clock- and light-mediated frq regulation. Expression of frq was altered in strains lacking set1 under both circadian- and light-regulated gene expression. There is a delay in the phasing of H3K4me3 relative to the peak in frq expression. White Collar 2 (WC-2) association with the frq promoter persists longer in Δset1, suggesting a more permissible chromatin state. Surprisingly, SET1 is required for DNA methylation in the frq promoter, indicating a dependence on H3K4me for DNA methylation. The data support a model where SET1 is needed for proper regulation by modulating chromatin at frq.
PMCID: PMC3605655  PMID: 23319591
Chromatin Immunoprecipitation (ChIP); Chromatin Modification; Circadian Rhythms; Gene Regulation; Neurospora
6.  Minimum Criteria for DNA Damage-Induced Phase Advances in Circadian Rhythms 
PLoS Computational Biology  2009;5(5):e1000384.
Robust oscillatory behaviors are common features of circadian and cell cycle rhythms. These cyclic processes, however, behave distinctively in terms of their periods and phases in response to external influences such as light, temperature, nutrients, etc. Nevertheless, several links have been found between these two oscillators. Cell division cycles gated by the circadian clock have been observed since the late 1950s. On the other hand, ionizing radiation (IR) treatments cause cells to undergo a DNA damage response, which leads to phase shifts (mostly advances) in circadian rhythms. Circadian gating of the cell cycle can be attributed to the cell cycle inhibitor kinase Wee1 (which is regulated by the heterodimeric circadian clock transcription factor, BMAL1/CLK), and possibly in conjunction with other cell cycle components that are known to be regulated by the circadian clock (i.e., c-Myc and cyclin D1). It has also been shown that DNA damage-induced activation of the cell cycle regulator, Chk2, leads to phosphorylation and destruction of a circadian clock component (i.e., PER1 in Mus or FRQ in Neurospora crassa). However, the molecular mechanism underlying how DNA damage causes predominantly phase advances in the circadian clock remains unknown. In order to address this question, we employ mathematical modeling to simulate different phase response curves (PRCs) from either dexamethasone (Dex) or IR treatment experiments. Dex is known to synchronize circadian rhythms in cell culture and may generate both phase advances and delays. We observe unique phase responses with minimum delays of the circadian clock upon DNA damage when two criteria are met: (1) existence of an autocatalytic positive feedback mechanism in addition to the time-delayed negative feedback loop in the clock system and (2) Chk2-dependent phosphorylation and degradation of PERs that are not bound to BMAL1/CLK.
Author Summary
Molecular components and mechanisms that connect cell cycle and circadian rhythms are important for the well-being of an organism. Cell cycle machinery regulates the progress of cell growth and division while the circadian rhythm network generates an ∼24 h time-keeping mechanism that regulates the daily processes of an organism (i.e. metabolism, bowel movements, body temperature, etc.). It is observed that cell divisions usually occur during a certain time window of a day, which indicated that there are circadian-gated cell divisions. Moreover, it's been shown that mice are more prone to develop cancer when certain clock genes are mutated resulting in an arrhythmic clock. Recently, a cell cycle checkpoint regulator, Chk2, was identified as a component that influences a core clock component and creates mostly phase advances (i.e., jet lags due to traveling east) in circadian rhythms upon DNA damage. This phase response with minimum delays is an unexpected result, and the molecular mechanism behind this phenomenon remains unknown. Our computational analyses of a mathematical model reveal two molecular criteria that account for the experimentally observed phase responses of the circadian clock upon DNA damage. These results demonstrate how circadian clock regulation by cell cycle checkpoint controllers provides another layer of complexity for efficient DNA damage responses.
PMCID: PMC2677641  PMID: 19424508
7.  Robustness from flexibility in the fungal circadian clock 
BMC Systems Biology  2010;4:88.
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.
PMCID: PMC2913929  PMID: 20576110
8.  Neurospora Clock-Controlled Gene 9 (ccg-9) Encodes Trehalose Synthase: Circadian Regulation of Stress Responses and Development 
Eukaryotic Cell  2002;1(1):33-43.
The circadian clock of Neurospora crassa regulates the rhythmic expression of a number of genes encoding diverse functions which, as an ensemble, are adaptive to life in a rhythmic environment of alternating levels of light and dark, warmth and coolness, and dryness and humidity. Previous differential screens have identified a number of such genes based solely on their cycling expression, including clock-controlled gene 9 (ccg-9). Sequence analysis now shows the predicted CCG-9 polypeptide to be homologous to a novel form of trehalose synthase; as such it would catalyze the synthesis of the disaccharide trehalose, which plays an important role in protecting many cells from environmental stresses. Consistent with this, heat, glucose starvation, and osmotic stress induce ccg-9 transcript accumulation. Surprisingly, however, a parallel role in development is suggested by the finding that inactivation of ccg-9 results in altered conidiophore morphology and abolishes the normal circadian rhythm of asexual macroconidial development. Examination of a clock component, FRQ, in the ccg-9-null strain revealed normal cycling, phosphorylation, and light induction, indicating that loss of the conidiation rhythm is not due to changes in either the circadian oscillator or light input into the clock but pointing instead to a defect in circadian output. These data imply an interplay between a role of trehalose in stress protection and an apparent requirement for trehalose in clock regulation of conidiation under constant environmental conditions. This requirement can be bypassed by a daily light signal which drives a light-entrained rhythm in conidiation in the ccg-9-null strain; this bypass suggests that the trehalose requirement is related to clock control of development and not to the developmental process itself. Circadian control of trehalose synthase suggests a link between clock control of stress responses and that of development.
PMCID: PMC118043  PMID: 12455969
9.  Circadian Output, Input, and Intracellular Oscillators: Insights into the Circadian Systems of Single Cells 
Circadian output comprises the business end of circadian systems in terms of adaptive significance. Work on Neurospora pioneered the molecular analysis of circadian output mechanisms, and insights from this model system continue to illuminate the pathways through which clocks control metabolism and overt rhythms. In Neurospora, virtually every strain examined in the context of rhythms bears the band allele that helps to clarify the overt rhythm in asexual development. Recent cloning of band showed it to be an allele of ras-1 and to affect a wide variety of signaling pathways yielding enhanced light responses and asexual development. These can be largely phenocopied by treatments that increase levels of intracellular reactive oxygen species. Although output is often unidirectional, analysis of the prd-4 gene provided an alternative paradigm in which output feeds back to affect input. prd-4 is an allele of checkpoint kinase-2 that bypasses the requirement for DNA damage to activate this kinase; FRQ is normally a substrate of activated Chk2, so in Chk2PRD-4, FRQ is precociously phosphorylated and the clock cycles more quickly. Finally, recent adaptation of luciferase to fully function in Neurospora now allows the core FRQ/WCC feedback loop to be followed in real time under conditions where it no longer controls the overt rhythm in development. This ability can be used to describe the hierarchical relationships among FRQ-Less Oscillators (FLOs) and to see which are connected to the circadian system. The nitrate reductase oscillator appears to be connected, but the oscillator controlling the long-period rhythm elicited upon choline starvation appears completely disconnected from the circadian system; it can be seen to run with a very long noncompensated 60–120-hour period length under conditions where the circadian FRQ/WCC oscillator continues to cycle with a fully compensated circadian 22-hour period.
PMCID: PMC3671946  PMID: 18419278
10.  CHD1 Remodels Chromatin and Influences Transient DNA Methylation at the Clock Gene frequency 
PLoS Genetics  2011;7(7):e1002166.
Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it is evident that chromatin modifications and chromatin remodeling are integral to this process in eukaryotes. We previously determined that multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In this report, we demonstrate that the Neurospora homologue of chd1 is required for normal remodeling of chromatin at frq and is required for normal frq expression and sustained rhythmicity. Surprisingly, our studies of CHD1 also revealed that DNA sequences within the frq promoter are methylated, and deletion of chd1 results in expansion of this methylated domain. DNA methylation of the frq locus is altered in strains bearing mutations in a variety of circadian clock genes, including frq, frh, wc-1, and the gene encoding the frq antisense transcript (qrf). Furthermore, frq methylation depends on the DNA methyltransferase, DIM-2. Phenotypic characterization of Δdim-2 strains revealed an approximate WT period length and a phase advance of approximately 2 hours, indicating that methylation plays only an ancillary role in clock-regulated gene expression. This suggests that DNA methylation, like the antisense transcript, is necessary to establish proper clock phasing but does not control overt rhythmicity. These data demonstrate that the epigenetic state of clock genes is dependent on normal regulation of clock components.
Author Summary
Circadian rhythms facilitate daily changes in gene expression via a transcriptional negative feedback loop. In eukaryotes, chromatin remodeling is an integral part of transcriptional regulation and is proving to be one of the major determinants for the proper timing and amplitude of clock-gene expression. We describe here the action of chromodomain helicase DNA–binding (CHD1), one of two ATP–dependent chromatin-remodeling enzymes required for normal circadian regulated gene expression of the central clock gene frequency (frq). Molecular analysis of strains lacking chd1 indicates that CHD1 is required for remodeling chromatin structure at the frq locus as a part of the daily clock cycle. Moreover, we discovered DNA methylation in the promoter of frq that diminishes over time in the absence of light/dark cycles and determined that normal DNA methylation appears to require a functional clock. The DNA methyltransferase DIM-2 is responsible for this DNA methylation, and the DNA methylation is required for proper phasing of clock gene expression. Collectively, these data demonstrate a close connection among chromatin remodeling, DNA methylation, and clock gene expression.
PMCID: PMC3140994  PMID: 21811413
11.  Data assimilation constrains new connections and components in a complex, eukaryotic circadian clock model 
Integrating molecular time-series data resulted in a more robust model of the plant clock, which predicts that a wave of inhibitory PRR proteins controls the morning genes LHY and CCA1.PRR5 is experimentally validated as a late-acting component of this wave.The family of sequentially expressed PRR proteins allows flexible entrainment of the clock, whereas a single protein could not, suggesting that the duplication of clock genes might confer this generic, functional advantage.The observed post-translational regulation of the evening protein TOC1 by interaction with ZTL and GI remains consistent with an indirect activation of TOC1 mRNA expression by GI, which was previously postulated from modelling.
Circadian rhythms are present in most eukaryotic organisms including plants. The core genes of the circadian clock are very important for plant physiology as they drive the rhythmic expression of around 30% of Arabidopsis genes (Edwards et al, 2006; Michael et al, 2008). The clock is normally entrained by daily environmental changes in light and temperature. Oscillations also persist under constant environmental conditions in a laboratory. The clock gene circuit in Arabidopsis is based on multiple interlocked feedback loops, which are typical of circadian genetic networks in other organisms (Dunlap and Loros, 2004; Bell-Pedersen et al, 2005). Mechanistic, mathematical models are increasingly useful in analysing and understanding how the observed molecular components give rise to the rhythmic behaviour of this dynamic, non-linear system.
Our previous model of Arabidopsis circadian clock (Locke et al, 2006) presented the core, three-loop structure of the clock, which comprised morning and evening oscillators and coupling between them (Figure 1). The morning loop included the dawn-expressed LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) genes, which negatively regulate their expression through activation of the inhibitor proteins, PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. These were described by a single, combined model component, PRR9/7. The evening loop included the dusk-expressed gene TIMING OF CAB EXPRESSION 1 (TOC1), which negatively regulates itself through inhibition of a hypothetical activator, gene Y. The evening-expressed gene GIGANTEA (GI) contributes to Y function. The morning and evening loops were connected through inhibition of the evening genes by LHY/CCA1 and activation of LHY/CCA1 expression by a hypothetical evening gene X. Here, we extend the previous model of circadian gene expression (Locke et al, 2006) based on recently published data (Figure 1). The new model retains the good match of our previous model to the large volume of molecular time-series data, and improves the behaviour of the model clock system under a range of light conditions and in a wider range of mutants.
The morning loop was extended by adding a hypothetical clock component, the night inhibitor (NI), which acts together with PRR9 and PRR7 to keep the expression of LHY and CCA1 at low levels over a broad interval spanning dusk. This regulation is important to set the phase of LHY/CCA1 expression at dawn. Data from the literature suggested that the PRR5 gene was a candidate for NI, leading us to predict that the sequentially expressed PRR9, PRR7 and PRR5 proteins together formed a wave of inhibitors of LHY and CCA1. This hypothesis was tested under discriminating light conditions, in which the light interval is replaced with the dawn and dusk pulses of light to form a ‘skeleton photoperiod'. Combining this protocol with mutation of the PRR7 and/or PRR5 genes, our new experimental results validated the model predictions and confirmed that PRR5 contributes to the function that we modelled as NI. During revision of this paper, that result received further experimental support (Nakamichi et al, 2010).
Model simulations revealed the functional importance of the inhibitor wave in entraining the clock to the light/dark cycle. Separating PRR9 from the other inhibitors in the model showed how the strong light activation observed for this gene contributes to more rapid entrainment. The observed, post-translation regulation of all three inhibitor proteins by light (Farre and Kay, 2007; Ito et al, 2007; Kiba et al, 2007) was also included in the model. Light-regulated degradation provides a molecular mechanism to explain the later phase of LHY and CCA1 expression under long photoperiods compared with short photoperiods, in line with experimental observations.
The connection between evening and morning loops was revised by including the inhibition of the morning gene PRR9 by the evening component TOC1, based on the data on TOC1-overexpressing plants (Makino et al, 2002; Ito et al, 2005). This inhibition causes a delay of PRR9 expression relative to LHY/CCA1, which allows LHY/CCA1 to reach a higher expression level at dawn. Our simulations showed that a partial mutant that lacks this inhibition of PRR9 by TOC1 is sufficient to cause the higher level of PRR9 and the short circadian period observed in toc1 mutant plants.
The evening loop was extended by introducing the observed, post-translational regulation of the TOC1 protein by the F-box protein ZEITLUPE (ZTL) and stabilization of ZTL by its interaction with GI in the presence of light (Kim et al, 2007). GI's function in the clock model has thus been revised according to the data: GI promotes an inhibition of TOC1 protein function through positive regulation of ZTL. This results, together with negative regulation of Y by TOC1, in indirect activation of TOC1 mRNA expression by GI, which agrees with our earlier experimental data (Locke et al, 2006). Simulations showed that the post-translational regulation of TOC1 by ZTL and GI results in the observed long period of the ztl mutant and fast dampening of rhythms in the lhy/cca1/gi triple mutant.
This is the first mathematical model that incorporates the observed post-translational regulation into the genetic network of the Arabidopsis clock. In addition to specific, mechanistic insights, the model shows a generic advantage from the duplication of clock genes and their expression at different phases. Such clock gene duplications are observed in eukaryotes with larger genomes, such as the mouse. Analogous, functional duplication can be achieved by differential regulation of a single clock gene in distinct cells, as in Drosophila.
Circadian clocks generate 24-h rhythms that are entrained by the day/night cycle. Clock circuits include several light inputs and interlocked feedback loops, with complex dynamics. Multiple biological components can contribute to each part of the circuit in higher organisms. Mechanistic models with morning, evening and central feedback loops have provided a heuristic framework for the clock in plants, but were based on transcriptional control. Here, we model observed, post-transcriptional and post-translational regulation and constrain many parameter values based on experimental data. The model's feedback circuit is revised and now includes PSEUDO-RESPONSE REGULATOR 7 (PRR7) and ZEITLUPE. The revised model matches data in varying environments and mutants, and gains robustness to parameter variation. Our results suggest that the activation of important morning-expressed genes follows their release from a night inhibitor (NI). Experiments inspired by the new model support the predicted NI function and show that the PRR5 gene contributes to the NI. The multiple PRR genes of Arabidopsis uncouple events in the late night from light-driven responses in the day, increasing the flexibility of rhythmic regulation.
PMCID: PMC2964123  PMID: 20865009
Arabidopsis thaliana; biological clocks; circadian rhythms; mathematical model; systems biology
12.  Network Discovery Pipeline Elucidates Conserved Time-of-Day–Specific cis-Regulatory Modules 
PLoS Genetics  2008;4(2):e14.
Correct daily phasing of transcription confers an adaptive advantage to almost all organisms, including higher plants. In this study, we describe a hypothesis-driven network discovery pipeline that identifies biologically relevant patterns in genome-scale data. To demonstrate its utility, we analyzed a comprehensive matrix of time courses interrogating the nuclear transcriptome of Arabidopsis thaliana plants grown under different thermocycles, photocycles, and circadian conditions. We show that 89% of Arabidopsis transcripts cycle in at least one condition and that most genes have peak expression at a particular time of day, which shifts depending on the environment. Thermocycles alone can drive at least half of all transcripts critical for synchronizing internal processes such as cell cycle and protein synthesis. We identified at least three distinct transcription modules controlling phase-specific expression, including a new midnight specific module, PBX/TBX/SBX. We validated the network discovery pipeline, as well as the midnight specific module, by demonstrating that the PBX element was sufficient to drive diurnal and circadian condition-dependent expression. Moreover, we show that the three transcription modules are conserved across Arabidopsis, poplar, and rice. These results confirm the complex interplay between thermocycles, photocycles, and the circadian clock on the daily transcription program, and provide a comprehensive view of the conserved genomic targets for a transcriptional network key to successful adaptation.
Author Summary
As the earth rotates, environmental conditions oscillate between illuminated warm days and dark cool nights. Plants have adapted to these changes by timing physiological processes to specific times of the day or night. Light and temperature signaling and the circadian clock regulate this adaptive response. To determine the contributions of each of these factors on gene regulation, we analyzed microarray time course experiments interrogating light, temperature, and circadian conditions. We discovered that almost all Arabidopsis genes cycle in at least one condition. From a signaling perspective, this suggests that light, temperature, and circadian clock play an important role in modulating many physiological pathways. To clarify the contribution of transcriptional regulation on this process, we mined the promoters of cycling genes to identify DNA elements associated with expression at specific times of day. This confirmed the importance of several DNA motifs such as the G-box and the evening element in the regulation of gene expression by light and the circadian clock, but also facilitated the discovery of new elements linked to a novel midnight regulatory module. Identification of orthologous promoter elements in rice and poplar revealed a conserved transcriptional regulatory network that allows global adaptation to the ever-changing daily environment.
PMCID: PMC2222925  PMID: 18248097
13.  Coupling governs entrainment range of circadian clocks 
Circadian clock oscillator properties that are crucial for synchronization with the environment (entrainment) are studied in experiment and theory.The ratio between stimulus (zeitgeber) strength and oscillator amplitude, and the rigidity of the oscillatory system (relaxation rate upon perturbation) determine entrainment properties. Coupling among oscillators affects both qualities resulting in increased amplitude and rigidity.Uncoupled lung clocks entrain to extreme zeitgeber cycles, whereas the coupled oscillator system in the suprachiasmatic nucleus (SCN) does not; however, when coupling in the SCN is inhibited, larger ranges of entrainment can be achieved.
Daily rhythms in physiology, metabolism and behavior are controlled by an endogenous circadian timing system, which has evolved to synchronize an organism to periodically recurring environmental conditions, such as light–dark or temperature cycles. In mammals, the circadian system relies on cell-autonomous oscillators residing in almost every cell of the body. Cells of the SCN in the anterior hypothalamus are able to generate precise, long-lasting self-sustained circadian oscillations, which drive most rhythmic behavioral and physiological outputs, and which are believed to originate from the fact that the SCN tissue consists of tightly coupled cells (Aton and Herzog, 2005). In contrast, peripheral oscillators, such as lung tissue, exhibit seemingly damped and usually less precise oscillations, which are thought to be brought about by the lack of intercellular coupling.
Precise synchronization of these rhythms within the organism, but also with the environment (so-called entrainment), is an essential part of circadian organization. Entrainment is one of the cornerstones of circadian biology (Roenneberg et al, 2003). In evolution, the phase of a rhythmic variable is selective rather than its endogenous period. Thus, the synchronization of endogenous rhythms to zeitgeber cycles of the environment (resulting in a specific phase of entrainment) is fundamental for the adaptive value of circadian clocks. In this study, we systematically investigated the properties of circadian oscillators that are essential for entrainment behavior and describe coupling as a primary determinant.
As an experimental starting point of this study, we found that the circadian oscillators of lung tissue have a larger range of entrainment than SCN tissue—they readily entrained to extreme experimental temperature cycle of 20 or 28 h, whereas SCN tissue did not (Figure 4). For this purpose, we cultured SCN and lung slices derived from mice that express luciferase as fusion protein together with the clock protein PERIOD2 (Yoo et al, 2004). The detection of luciferase-driven bioluminescence allowed us to follow molecular clock gene activity in real-time over the course of several days.
In theoretical analyses, we show that both the ratio of amplitude and zeitgeber strength and, importantly, inter-oscillator coupling are major determinants for entrainment. The reason for coupling being critical is twofold: (i) Coupling makes an oscillatory system more rigid, i.e., it relaxes faster in response to a perturbation, and (ii) coupling increases the amplitude of the oscillatory system. Both of these consequences of coupling lead to a smaller entrainment range, because zeitgeber stimuli affect the oscillatory system less if the relaxation is fast and the amplitude is high (Figure 1). From these theoretical considerations, we conclude that the lung clock probably constitutes a weak oscillatory system, likely because a lack in coupling leads to a slow amplitude relaxation. (Circadian amplitude is not particularly low in lung (Figure 4).) In contrast, the SCN constitutes a rigid oscillator, whereby coupling and its described consequences probably are the primary causes for this rigidity. We then tested these theoretical predictions by experimentally perturbing coupling in the SCN (with MDL and TTX; O'Neill et al, 2008; Yamaguchi et al, 2003) and find that, indeed, reducing the coupling weakens the circadian oscillatory system in the SCN, which results in an enlargement of the entrainment range (Figure 6).
Why is the SCN designed to be a stronger circadian oscillator than peripheral organs? We speculate that the position of the SCN—as the tissue that conveys environmental timing information (i.e., light) to the rest of the body—makes it necessary to create a circadian clock that is robust against noisy environmental stimuli. The SCN oscillator needs to be robust enough to be protected from environmental noise, but flexible enough to fulfill its function as an entrainable clock even in extreme photoperiods (i.e., seasons). By the same token, peripheral clocks are more protected from the environmental zeitgebers due to intrinsic homeostatic mechanisms. Thus, they do not necessarily need to develop a strong oscillatory system (e.g., by strengthening the coupling), rather they need to stay flexible enough to respond to direct or indirect signals from the SCN, such as hormonal, neural, temperature or metabolic signals. Such a design ensures that only robust and persistent environmental signals trigger an SCN resetting response, while SCN signals can relatively easily be conveyed to the rest of the body. Thus, the robustness in the SCN clock likely serves as a filter for environmental noise.
In summary, using a combination of simulation studies, analytical calculations and experiments, we uncovered critical features for entrainment, such as zeitgeber-to-amplitude ratio and amplitude relaxation rate. Coupling is a primary factor that governs these features explaining important differences in the design of SCN and peripheral oscillators that ensure a robust, but also flexible circadian system.
Circadian clocks are endogenous oscillators driving daily rhythms in physiology and behavior. Synchronization of these timers to environmental light–dark cycles (‘entrainment') is crucial for an organism's fitness. Little is known about which oscillator qualities determine entrainment, i.e., entrainment range, phase and amplitude. In a systematic theoretical and experimental study, we uncovered these qualities for circadian oscillators in the suprachiasmatic nucleus (SCN—the master clock in mammals) and the lung (a peripheral clock): (i) the ratio between stimulus (zeitgeber) strength and oscillator amplitude and (ii) the rigidity of the oscillatory system (relaxation rate upon perturbation) determine entrainment properties. Coupling among oscillators affects both qualities resulting in increased amplitude and rigidity. These principles explain our experimental findings that lung clocks entrain to extreme zeitgeber cycles, whereas SCN clocks do not. We confirmed our theoretical predictions by showing that pharmacological inhibition of coupling in the SCN leads to larger ranges of entrainment. These differences between master and the peripheral clocks suggest that coupling-induced rigidity in the SCN filters environmental noise to create a robust circadian system.
PMCID: PMC3010105  PMID: 21119632
circadian clock; coupling; entrainment; mathematical modeling; oscillator
14.  Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex ▿† 
Eukaryotic Cell  2010;9(10):1549-1556.
Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.
PMCID: PMC2950426  PMID: 20675579
15.  The Circadian Clock Coordinates Ribosome Biogenesis 
PLoS Biology  2013;11(1):e1001455.
The authors identify a new role of the circadian clock in coordinating mRNA translation during ribosome biogenesis, a key process for cell metabolism.
Biological rhythms play a fundamental role in the physiology and behavior of most living organisms. Rhythmic circadian expression of clock-controlled genes is orchestrated by a molecular clock that relies on interconnected negative feedback loops of transcription regulators. Here we show that the circadian clock exerts its function also through the regulation of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator directly regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis.
Author Summary
Most living organisms on earth present biological rhythms that play a fundamental role in the coordination of their physiology and behavior. The discovery of the molecular circadian clock gives important insight into the mechanisms involved in the generation of these rhythms. Indeed, this molecular clock orchestrates the rhythmic transcription of clock-controlled genes involved in different aspects of metabolism, for example lipid, carbohydrate, and xenobiotic metabolisms in the liver. However, we show here that the circadian clock could also exert its function through the coordination of mRNA translation. Namely, the circadian clock influences the temporal translation of a subset of mRNAs by controlling the expression and activation of translation initiation factors, as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. These rhythmically translated mRNAs are mainly involved in ribosome biogenesis, an energy consuming process, which has to be gated to a period when the cell resources are less limited. Moreover, the role of the circadian oscillator in this process is highlighted by its direct regulation of the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus our findings suggest that the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis.
PMCID: PMC3536797  PMID: 23300384
16.  The Neurospora circadian clock: simple or complex? 
The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.
PMCID: PMC1088545  PMID: 11710976
17.  Machine Learning Helps Identify CHRONO as a Circadian Clock Component 
PLoS Biology  2014;12(4):e1001840.
Two independent studies, one of them using a computational approach, identified CHRONO, a gene shown to modulate the activity of circadian transcription factors and alter circadian behavior in mice.
Over the last decades, researchers have characterized a set of “clock genes” that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.
Author Summary
Daily rhythms are ever-present in the living world, driving the sleep–wake cycle and many other physiological changes. In the last two decades, several labs have identified “clock genes” that interact to generate underlying molecular oscillations. However, many aspects of circadian molecular physiology remain unexplained. Here, we used a simple “machine learning” approach to identify new clock genes by searching the genome for candidate genes that share clock-like features such as cycling, broad-based tissue RNA expression, in vitro circadian activity, genetic interactions, and homology across species. Genes were ranked by their similarity to known clock components and the candidates were screened and validated for evidence of clock function in vitro. One candidate, which we renamed CHRONO (Gm129), interacted with the master regulator of the clock, BMAL1, disrupting its transcriptional activity. We found that Chrono knockout mice had prolonged locomotor activity rhythms, getting up progressively later each day. Our experiments demonstrated that CHRONO interferes with the ability of BMAL1 to recruit CBP, a bona fide histone acetylase and key transcriptional coactivator of the circadian clock.
PMCID: PMC3988006  PMID: 24737000
18.  Coupling of a Core Post-Translational Pacemaker to a Slave Transcription/Translation Feedback Loop in a Circadian System 
PLoS Biology  2010;8(6):e1000394.
Analysis of the cyanobacterial circadian biological clock reveals a complex interdependence between a transcription/translation feedback loop and a biochemical oscillator.
Cyanobacteria are the only model circadian clock system in which a circadian oscillator can be reconstituted in vitro. The underlying circadian mechanism appears to comprise two subcomponents: a post-translational oscillator (PTO) and a transcriptional/translational feedback loop (TTFL). The PTO and TTFL have been hypothesized to operate as dual oscillator systems in cyanobacteria. However, we find that they have a definite hierarchical interdependency—the PTO is the core pacemaker while the TTFL is a slave oscillator that quickly damps when the PTO stops. By analysis of overexpression experiments and mutant clock proteins, we find that the circadian system is dependent upon the PTO and that suppression of the PTO leads to damped TTFL-based oscillations whose temperature compensation is not stable under different metabolic conditions. Mathematical modeling indicates that the experimental data are compatible with a core PTO driving the TTFL; the combined PTO/TTFL system is resilient to noise. Moreover, the modeling indicates a mechanism by which the TTFL can feed into the PTO such that new synthesis of clock proteins can phase-shift or entrain the core PTO pacemaker. This prediction was experimentally tested and confirmed by entraining the in vivo circadian system with cycles of new clock protein synthesis that modulate the phosphorylation status of the clock proteins in the PTO. In cyanobacteria, the PTO is the self-sustained core pacemaker that can operate independently of the TTFL, but the TTFL damps when the phosphorylation status of the PTO is clamped. However, the TTFL can provide entraining input into the PTO. This study is the first to our knowledge to experimentally and theoretically investigate the dynamics of a circadian clock in which a PTO is coupled to a TTFL. These results have important implications for eukaryotic clock systems in that they can explain how a TTFL could appear to be a core circadian clockwork when in fact the true pacemaker is an embedded biochemical oscillator.
Author Summary
Many organisms from bacteria to humans have evolved circadian mechanisms for regulating biological processes on a daily time scale. In cyanobacteria, a minimal system for such cyclical regulation can be reconstituted in vitro from three proteins, called KaiA, KaiB, and KaiC. This three-protein oscillator is believed to regulate the cyclical activities in vivo through a post-translational mechanism that involves rhythmic phosphorylation of KaiC. Although this post-translational oscillator (PTO) is sufficient for generating rhythms in vitro, the cyanobacterial circadian system in vivo also includes a transcriptional/translational feedback loop (TTFL). The precise roles of the PTO and the TTFL and their interdependence in forming the complete clock system in vivo are unclear. By manipulating wild-type and mutant clock protein expression in vivo, we here show that the cyanobacterial circadian system is dependent upon the biochemical oscillator provided by the PTO and that suppression of the PTO leads to a residual damped (slave) oscillation that results from the TTFL. Mathematical modeling shows that the experimental data are compatible with a mechanism in which the PTO acts as a pacemaker to drive the activity of the TTFL. Moreover, our analyses suggest a mechanism by which the TTFL can feed back into the PTO such that new synthesis of the Kai proteins entrains the core PTO pacemaker. Therefore, the PTO and TTFL appear to have a definite hierarchical interdependency: the PTO is a self-sustained core pacemaker that can oscillate independently of the TTFL, but the TTFL is a slave oscillator that damps when the phosphorylation status of KaiC in the PTO is clamped. The core circadian pacemaker in eukaryotes is thought to be a TTFL, but our results with cyanobacteria have important implications for eukaryotic clock systems in that they can explain how a TTFL could appear to be the core clock when in fact the true pacemaker is an embedded biochemical oscillator.
PMCID: PMC2885980  PMID: 20563306
19.  Cell Type-Specific Functions of Period Genes Revealed by Novel Adipocyte and Hepatocyte Circadian Clock Models 
PLoS Genetics  2014;10(4):e1004244.
In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.
Author Summary
Various aspects of our daily rhythms in physiology and behavior such as the sleep-wake cycle are regulated by endogenous circadian clocks that are present in nearly every cell. It is generally accepted that these oscillators share a similar biochemical negative feedback mechanism, consisting of transcriptional activators and repressors. In this study, we developed cell-autonomous, metabolically relevant clock models in mouse hepatocytes and adipocytes. Each clock model has an integrated luciferase reporter that allows for kinetic luminescence recording with an inexpensive microplate reader and thus is feasible for most laboratories. These models are amenable to high throughput screening of small molecules or genomic entities for impacts on cell-autonomous clocks relevant to metabolism. We validated these new models by RNA interference via lentivirus-mediated knockdown of known clock genes. As expected, we found that many core clock components have similar functions across cell types. To our surprise, however, we also uncovered previously under-appreciated cell type-specific functions of core clock genes, particularly Per1, Per2, and Per3. Because the circadian system is integrated with, and influenced by, the local physiology that is under its control, our studies provide important implications for future studies into cell type-specific mechanisms of various circadian systems.
PMCID: PMC3974647  PMID: 24699442
20.  Circadian Remodeling of Neuronal Circuits Involved in Rhythmic Behavior 
PLoS Biology  2008;6(3):e69.
Clock output pathways are central to convey timing information from the circadian clock to a diversity of physiological systems, ranging from cell-autonomous processes to behavior. While the molecular mechanisms that generate and sustain rhythmicity at the cellular level are well understood, it is unclear how this information is further structured to control specific behavioral outputs. Rhythmic release of pigment dispersing factor (PDF) has been proposed to propagate the time of day information from core pacemaker cells to downstream targets underlying rhythmic locomotor activity. Indeed, such circadian changes in PDF intensity represent the only known mechanism through which the PDF circuit could communicate with its output. Here we describe a novel circadian phenomenon involving extensive remodeling in the axonal terminals of the PDF circuit, which display higher complexity during the day and significantly lower complexity at nighttime, both under daily cycles and constant conditions. In support to its circadian nature, cycling is lost in bona fide clockless mutants. We propose this clock-controlled structural plasticity as a candidate mechanism contributing to the transmission of the information downstream of pacemaker cells.
Author Summary
Circadian systems evolved as a mechanism that allows organisms to adapt to the environmental changes in light and dark which occur as a consequence of the rotation of Earth. Because of its unique repertoire of genetic tools, Drosophila is a well established model for the study of the circadian clock. Although the biochemical components underlying the molecular oscillations have been characterized in detail, the mechanisms used by the clock neurons to convey information to the downstream pathways remain elusive. In the fruit fly, the small ventral lateral neurons (LNv) are capable of synchronizing other clock cells relying on a neuropeptide named pigment dispersing factor. In this work we introduce a novel mechanism as a possible candidate for contributing to the transmission of information downstream of the small LNvs, involving clock-controlled remodeling of their axonal morphology. By labeling the entire neuronal membrane and analyzing the complexity of the axonal arbor at different times we showed that there is a circadian variation in the complexity of the axonal arbor. This phenomenon was not observed in flies carrying null mutations in two canonical clock genes, underscoring the dependence of the circadian clock for the structural plasticity of its pacemaker neurons.
The circadian clock controls a wide array of biological phenomena ranging from basal transcription to overt behavior. Now, new evidence shows that the clock affects a striking remodeling of the circuit controlling rest-activity cycles inDrosophila.
PMCID: PMC2270325  PMID: 18366255
21.  Systematic Identification of Rhythmic Genes Reveals camk1gb as a New Element in the Circadian Clockwork 
PLoS Genetics  2012;8(12):e1003116.
A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.
Author Summary
The circadian clock is a molecular pacemaker that drives rhythmic expression of genes with a ∼24-hour period. As a result, many physiological processes have daily rhythms. Many of the conserved elements that constitute the circadian clock are known, but the links between the clock and dependent processes have remained elusive. With its amenability to genetic manipulations and a variety of genetic tools, the zebrafish has become an attractive vertebrate model for the quest to identify and characterize novel clock components. Here, we take advantage of another attraction of the zebrafish, the fact that its pineal gland is the site of a central clock which directly receives light input and autonomously generates circadian rhythms that affect the physiology of the whole organism. We show that the systematic design and analysis of genome-wide experiments based on the zebrafish pineal gland can lead to the discovery of new clock elements. We have characterized one novel element, camk1gb, and show that this gene, predominantly expressed within the pineal gland and driven by the circadian clock, links circadian clock timing with locomotor activity in zebrafish larvae.
PMCID: PMC3527293  PMID: 23284293
22.  MicroRNAs shape circadian hepatic gene expression on a transcriptome-wide scale 
eLife  2014;3:e02510.
A considerable proportion of mammalian gene expression undergoes circadian oscillations. Post-transcriptional mechanisms likely make important contributions to mRNA abundance rhythms. We have investigated how microRNAs (miRNAs) contribute to core clock and clock-controlled gene expression using mice in which miRNA biogenesis can be inactivated in the liver. While the hepatic core clock was surprisingly resilient to miRNA loss, whole transcriptome sequencing uncovered widespread effects on clock output gene expression. Cyclic transcription paired with miRNA-mediated regulation was thus identified as a frequent phenomenon that affected up to 30% of the rhythmic transcriptome and served to post-transcriptionally adjust the phases and amplitudes of rhythmic mRNA accumulation. However, only few mRNA rhythms were actually generated by miRNAs. Overall, our study suggests that miRNAs function to adapt clock-driven gene expression to tissue-specific requirements. Finally, we pinpoint several miRNAs predicted to act as modulators of rhythmic transcripts, and identify rhythmic pathways particularly prone to miRNA regulation.
eLife digest
The rising and setting of the sun have long driven the schedules of humans and other mammals. This 24-hr cycle influences many behavioural and physiological changes, including alertness, body temperature, and sleep. A region in the brain acts as a master clock that regulates these daily cycles, which are called circadian rhythms.
Signals from the brain's master clock turn on and off ‘core clock genes’ in cells, which trigger cycles that cause some proteins to be produced in a circadian rhythm. The rhythm is specialized to a particular tissue or organ, and may help them to carry out their designated daily tasks. However, circadian rhythms might also be produced in other ways that do not involve these genes.
Messenger RNA (mRNA) molecules have a central role in the production of proteins, and in the mouse liver, up to 15% of mRNA molecules are produced in circadian cycles. The liver performs essential tasks that control metabolism–including that of carbohydrates, fats, and cholesterol. Precisely timing when certain mRNAs and proteins reach peaks and troughs in their activities to coincide with mealtimes is important for nutrients to be properly processed.
Other RNA molecules called microRNAs influence how mRNA molecules are translated into proteins. Now Du, Arpat et al. have looked at the influence of microRNAs on circadian rhythms in the mouse liver in greater detail. These experiments, which involved ‘knocking out’ a gene that is essential for the production of microRNAs, show that rather than setting the mRNA rhythms, the microRNAs appear to adjust them to meet the specific needs of the liver. Targeting specific microRNA molecules may reveal new strategies to tweak these rhythms, which could help to improve conditions when metabolic functions go wrong.
PMCID: PMC4032493  PMID: 24867642
circadian clocks; miRNA; liver; Dicer knockout; mouse
23.  Robustness of Circadian Clocks to Daylight Fluctuations: Hints from the Picoeucaryote Ostreococcus tauri 
PLoS Computational Biology  2010;6(11):e1000990.
The development of systemic approaches in biology has put emphasis on identifying genetic modules whose behavior can be modeled accurately so as to gain insight into their structure and function. However, most gene circuits in a cell are under control of external signals and thus, quantitative agreement between experimental data and a mathematical model is difficult. Circadian biology has been one notable exception: quantitative models of the internal clock that orchestrates biological processes over the 24-hour diurnal cycle have been constructed for a few organisms, from cyanobacteria to plants and mammals. In most cases, a complex architecture with interlocked feedback loops has been evidenced. Here we present the first modeling results for the circadian clock of the green unicellular alga Ostreococcus tauri. Two plant-like clock genes have been shown to play a central role in the Ostreococcus clock. We find that their expression time profiles can be accurately reproduced by a minimal model of a two-gene transcriptional feedback loop. Remarkably, best adjustment of data recorded under light/dark alternation is obtained when assuming that the oscillator is not coupled to the diurnal cycle. This suggests that coupling to light is confined to specific time intervals and has no dynamical effect when the oscillator is entrained by the diurnal cycle. This intringuing property may reflect a strategy to minimize the impact of fluctuations in daylight intensity on the core circadian oscillator, a type of perturbation that has been rarely considered when assessing the robustness of circadian clocks.
Author Summary
Circadian clocks keep time of day in many living organisms, allowing them to anticipate environmental changes induced by day/night alternation. They consist of networks of genes and proteins interacting so as to generate biochemical oscillations with a period close to 24 hours. Circadian clocks synchronize to the day/night cycle through the year principally by sensing ambient light. Depending on the weather, the perceived light intensity can display large fluctuations within the day and from day to day, potentially inducing unwanted resetting of the clock. Furthermore, marine organisms such as microalgae are subjected to dramatic changes in light intensities in the water column due to streams and wind. We showed, using mathematical modelling, that the green unicellular marine alga Ostreococcus tauri has evolved a simple but effective strategy to shield the circadian clock from daylight fluctuations by localizing coupling to the light during specific time intervals. In our model, as in experiments, coupling is invisible when the clock is in phase with the day/night cycle but resets the clock when it is out of phase. Such a clock architecture is immune to strong daylight fluctuations.
PMCID: PMC2978692  PMID: 21085637
24.  Modeling an Evolutionary Conserved Circadian Cis-Element 
PLoS Computational Biology  2008;4(2):e38.
Circadian oscillator networks rely on a transcriptional activator called CLOCK/CYCLE (CLK/CYC) in insects and CLOCK/BMAL1 or NPAS2/BMAL1 in mammals. Identifying the targets of this heterodimeric basic-helix-loop-helix (bHLH) transcription factor poses challenges and it has been difficult to decipher its specific sequence affinity beyond a canonical E-box motif, except perhaps for some flanking bases contributing weakly to the binding energy. Thus, no good computational model presently exists for predicting CLK/CYC, CLOCK/BMAL1, or NPAS2/BMAL1 targets. Here, we use a comparative genomics approach and first study the conservation properties of the best-known circadian enhancer: a 69-bp element upstream of the Drosophila melanogaster period gene. This fragment shows a signal involving the presence of two closely spaced E-box–like motifs, a configuration that we can also detect in the other four prominent CLK/CYC target genes in flies: timeless, vrille, Pdp1, and cwo. This allows for the training of a probabilistic sequence model that we test using functional genomics datasets. We find that the predicted sequences are overrepresented in promoters of genes induced in a recent study by a glucocorticoid receptor-CLK fusion protein. We then scanned the mouse genome with the fly model and found that many known CLOCK/BMAL1 targets harbor sequences matching our consensus. Moreover, the phase of predicted cyclers in liver agreed with known CLOCK/BMAL1 regulation. Taken together, we built a predictive model for CLK/CYC or CLOCK/BMAL1-bound cis-enhancers through the integration of comparative and functional genomics data. Finally, a deeper phylogenetic analysis reveals that the link between the CLOCK/BMAL1 complex and the circadian cis-element dates back to before insects and vertebrates diverged.
Author Summary
Life on earth is subject to daily light/dark and temperature cycles that reflect the earth rotation about its own axis. Under such conditions, organisms ranging from bacteria to human have evolved molecularly geared circadian clocks that resonate with the environmental cycles. These clocks serve as internal timing devices to coordinate physiological and behavioral processes as diverse as detoxification, activity and rest cycles, or blood pressure. In insects and vertebrates, the clock circuitry uses interlocked negative feedback loops which are implemented by transcription factors, among which the heterodimeric activators CLOCK and CYCLE play a key role. The specific DNA elements recognized by this factor are known to involve E-box motifs, but the low information content of this sequence makes it a poor predictor of the targets of CLOCK/CYCLE on a genome-wide scale. Here, we use comparative genomics to build a more specific model for a CLOCK-controlled cis-element that extends the canonical E-boxes to a more complex dimeric element. We use functional data from Drosophila and mouse circadian experiments to test the validity and assess the performance of the model. Finally, we provide a phylogenetic analysis of the cis-elements across insect and vertebrates that emphasizes the ancient link between CLOCK/CYCLE and the modeled enhancer. These results indicate that comparative genomics provides powerful means to decipher the complexity of the circadian cis-regulatory code.
PMCID: PMC2242825  PMID: 18282089
25.  Tuning the Mammalian Circadian Clock: Robust Synergy of Two Loops 
PLoS Computational Biology  2011;7(12):e1002309.
The circadian clock is accountable for the regulation of internal rhythms in most living organisms. It allows the anticipation of environmental changes during the day and a better adaptation of physiological processes. In mammals the main clock is located in the suprachiasmatic nucleus (SCN) and synchronizes secondary clocks throughout the body. Its molecular constituents form an intracellular network which dictates circadian time and regulates clock-controlled genes. These clock-controlled genes are involved in crucial biological processes including metabolism and cell cycle regulation. Its malfunction can lead to disruption of biological rhythms and cause severe damage to the organism. The detailed mechanisms that govern the circadian system are not yet completely understood. Mathematical models can be of great help to exploit the mechanism of the circadian circuitry. We built a mathematical model for the core clock system using available data on phases and amplitudes of clock components obtained from an extensive literature search. This model was used to answer complex questions for example: how does the degradation rate of Per affect the period of the system and what is the role of the ROR/Bmal/REV-ERB (RBR) loop? Our findings indicate that an increase in the RNA degradation rate of the clock gene Period (Per) can contribute to increase or decrease of the period - a consequence of a non-monotonic effect of Per transcript stability on the circadian period identified by our model. Furthermore, we provide theoretical evidence for a potential role of the RBR loop as an independent oscillator. We carried out overexpression experiments on members of the RBR loop which lead to loss of oscillations consistent with our predictions. These findings challenge the role of the RBR loop as a merely auxiliary loop and might change our view of the clock molecular circuitry and of the function of the nuclear receptors (REV-ERB and ROR) as a putative driving force of molecular oscillations.
Author Summary
Most organisms have evolved an internal clock which allows them to anticipate and react to the light/dark daily rhythm and is able to generate oscillation with a circa 24 hour rhythm. A molecular network involving feedback loops is responsible for the rhythm generation. A large number of clock-controlled genes pass on time messages and control several biological processes. In spite of its medical importance (role in cancer, sleep disorders, diabetes and others) the mechanism of action of the circadian clock and the role of its constituent's feedback loops remains partially unknown. Using a mathematical model, we were able to bring insight in open circadian biology questions. Firstly, increasing the mRNA degradation rate of Per can contribute to increase or decrease of the period which might explain contradictory experimental findings. Secondly, our data points to a more relevant role of the ROR/Bmal/REV-ERB loop. In particular, that this loop can be an oscillator on its own. We provide experimental evidence that overexpression of members of the ROR/Bmal/REV-ERB lead to loss of Bmal reporter mRNA oscillations. The fact that REV-ERB and ROR are nuclear receptors and therefore important regulators in many cellular processes might have important implications for molecular biology and medicine.
PMCID: PMC3240597  PMID: 22194677

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