About 20% of Neurospora genes are under control of the circadian clock system at the level of transcript accumulation, and the bulk of the clock-controlled mRNAs have peak accumulation in the late night to early morning. These data suggested the existence of global mechanisms for rhythmic control of gene expression. Consistent with this idea, we found that the Neurospora OS pathway, a phosphorelay signal transduction pathway that responds to changes in osmotic stress, functions as an output pathway from the FRQ/WCC. ChIP/Solexa sequencing with known oscillator proteins revealed that phosophorelay/MAPK pathway components are direct targets of the White Colar Complex (WCC), providing a direct connection between the clock and the output pathway. Activation of the OS pathway by the FRQ/WCC oscillator culminates in rhythmic OS-2 MAPK activity, which through time-of-day-specific activation of downstream effector molecules, controls rhythms in several target clock-controlled genes. Hijacking conserved signaling pathways by the circadian clock provides a new paradigm for global rhythmic control of target genes of the pathway.
Amplitude is a measurable parameter of an oscillator, yet it is often not considered as a variable, Amplitude can be measured in several ways: 1) as an output of an oscillator; 2) directly as the amplitude of a "key" clock protein; or 3) indirectly via a Phase-response curve. Data will be presented for a particular mutant (frq7) of Neurospora which shows how the amplitude was altered in all three of the measures listed above. A model will be presented based on limit-cycle expansion which accounts for these observations.
Temperature affects the amplitude of circadian oscillators in almost all systems studied. Data will be presented to illustrate how an increase in temperature leads to an increase in amplitude of these oscillators from many organisms. This increase in amplitude is proposed to be the mechanism of temperature-compensation, ie. to compensate for the increase in rates at a higher temperature, there is an expansion of the limit-cycle. This model is designated as the temperature-amplitude model, or the "T-A" model. A combination of this model with the one mentioned above predicts how much the midpoint of an oscillator will change when the temperature is raised, a feature not found in other models.
To determine if the temperature-amplitude relationship was a general one, a model callled the "degrade and fire" model was explored. This model simulates the known in vivo oscillations of a synthetic circuit, the arabinose circuit, constructed in E. coli. Data will be presented showing how the change in the activation energy of just one reaction can increase the amplitude of this oscillator, and can convert this non- temperature-compensated oscillator into a temperature-compensated oscillator.
Work done in collaboration with Lev Tsimring.
Light responses and photoadaptation of Neurospora depend on the photosensory light-oxygen-voltage (LOV) domains of the circadian transcription factor White Collar Complex (WCC) and its negative regulator Vivid (VVD). We found that light triggers LOV-mediated dimerization of the WCC. The activated WCC induces expression of VVD, which then disrupts and inactivates the WCC homo-dimers by the competitive formation of WCC-VVD hetero-dimers, leading to photoadaptation. Interaction with VVD protects light-activated WCC from rapid degradation and thus allows a sizable fraction of WCC to equilibrate with the WCC dark form. In the photo-adapted state, VVD synthesis triggered by light-activated WCC is balanced by VVD-dependent inhibition of WCC. The VVD-mediated desensitization and stabilization of the light activated WCC explains on a molecular level how Neurospora can robustly entrain to artificial and natural photoperiods: During the day, expression levels of VVD correlate with light intensity, allowing photoadaptation over several orders of magnitude. At night, previously synthesized VVD serves as a molecular memory of the brightness of the preceding day and suppresses responses to light cues of lower intensity, such as moonlight VVD is essential to discriminate between day and night, even in naturally ambiguous photoperiods with moonlight.
Work done in collaboration with Erik Malzahn, Stilianos Ciprianidis, Krisztina Kaldi, and Tobias Schafmeier.
Transcription/ translation feedback loops are central to all eukaryotic circadian clocks (Dunlap et al, Cold Spring Harbor Symp. 72: 57 - 68, 2007). In the circadian oscillator, the negative feedback loop drives periodic expression of proteins that feed back to reduce their own expression. While canonical clock proteins work exclusively in timing, all systems utilize additional, often essential, proteins that perform other functions in the cell. Among these in Neurospora is an essential putative RNA helicase, FRH. A novel, unbiased genetic screen for circadian negative feedback mutants uncovered a point mutation that completely complements the essential functions of FRH yet is totally arrhythmic, thus genetically separating essential functions from clock-associated roles. We used mass spectrometry to look for interactors of FRH, FRQ, and to follow posttranslational modifications of these proteins over the day. Although few modifications are found on FRH, FRQ is extensively modified with nearly 100 phosphorylations. By examining the phenotypes of strains bearing mutants that have lost these sites individually and in groups, we can begin to see how temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability. For over 60% of the confirmed phosphorylation sites, loss of the individual or neighboring sites have no apparent effect on the free running period length. This suggests that sites may work in groups as dynamically regulated charged domains rather than functioning individually. Some domains promote FRQ stability and lengthen period and other promote turnover and shorten period. Modifications are dynamic such that at near all times of day "FRQ" describes a heterogeneous mix of proteins with the same amino acid sequence but variable and distinguishable structure and surface chemistry. We have also used luciferase as a reporter to follow the FRQ-White Collar based core oscillator under growth/nutritional conditions or in genetic backgrounds (e.g. cel, chol-1, ult) where rhythms in growth are manifest. Under these conditions the FRQ/WCC oscillator cycles with a normal compensated circadian period length even when the overt rhythm of growth moves out of the circadian range.
The circadian clock regulates numerous developmental and physiological processes and it is necessary for optimal plant growth and survival. Up to 30% of the genes are circadian regulated in Arabidopsis. For most of these genes we still do not understand the mechanism by which these phase specific expression patterns are achieved. It has been shown that clock components directly regulate the expression of output genes. Pseudo-response regulators PRR7 and PRR9 are clock components in Arabidopsis involved in a transcriptional feedback loop with the morning expressed MYB transcription factors CIRCADIAN CLOCK ASSOCIATED (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). Expression analysis of the prr5prr7prr9 mutant (Nakamichi et al., 2009) indicated that these PRRs are involved in the regulation of many morning expressed genes. We could show that indeed PRR7 and PRR9 directly repress the expression of morning expressed circadian output genes in Arabidopsis.
- Nakamichi, N., et al., Transcript profiling of an Arabidopsis PSEUDO RESPONSE REGULATOR arrhythmic triple mutant reveals a role for the circadian clock in cold stress response. Plant Cell Physiol, 2009. 50(3): p. 447-62.
Circadian rhythms represent one of the more conspicuous examples of biological rhythms. Manifested at the physiological, behavioral, and cellular levels, these 24-hour rhythms originate at the molecular level, through a complex gene regulatory network. In mammals, the circadian pacemaker is located in the suprachiasmatic nuclei of the hypothalamus (SCN). We have developed deterministic models using non-linear ordinary differential equations that account for the occurrence of autonomous circadian oscillations in single cells, for their entrainment by light-dark cycles, and for their phase shifting by light pulses. The model can be used to unravel the links between molecular alterations (e.g. mutations in clock genes) and clock-related physiological pathologies (such as sleep phase disorders). We have investigated the coupling between the SCN cells and proposed a synchronization mechanism based on periodic neurotransmitter release. Numerical analysis of the model predicts that (1) efficient synchronization is achieved when the average neurotransmitter concentration dampens individual oscillators and (2) phases of individual cells are governed by their intrinsic periods. These results illustrate the possible interplay between the single-cell oscillator and the inter-cellular coupling mechanisms.
The circadian clock is an endogenous 24h timer, found throughout nature. It allows an organism to temporally orchestrate metabolic, physiological, biochemical and developmental processes. It is used to predict and respond accurately to transitions at dusk and dawn. The clock also measures day length (photoperiod) and allows the organism to respond appropriately to seasonal rhythms. One of the key defining features of circadian clocks, temperature compensation, allows the maintenance of robust and accurate clock function over a broad range of physiological temperatures. ROBuST seeks to understand the design principles at play to generate temperature buffered networks. The ROBuST project focuses on interconnected light signalling, circadian and cold acclimation pathways containing in total 56 components. We will describe the use of delayed fluorescence (DF), a recently identified novel output of the circadian clock (Gould et al., 2009), to screen our 56 mutants of interest for circadian clock phenotypes in different light and temperature conditions. Our DF results suggest that buffering against temperature changes requires the alteration of the circadian clock architecture in response to temperature. Additional work has also identified a new function for cry1, cry2 and phyA as regulators of temperature buffering of the clock in a light dependent manner. This new data set will be critical for constraining models and understanding how network architecture changes with temperature.
Several different circadian rhythms, as well as an annual rhythm, have been studied in the marine dinoflagellate Gonyaulax polyedra (now Lingulodinium polyedrum), many features of which may be grist for modeling mills, whatever they may be. The rhythm of bioluminescence provides an easy "hand" for the automation of its measurement in vivo, and the luciferase and luciferin responsible serve as unambiguous biochemical correlates. This presentation will present highlights of several of its features, starting the subject of the title: post transcriptional control of circadian protein expression. The term "temperature compensation" was introduced in this system to replace "temperature independence" used earlier, based on the observation that the rhythm has a Q10 less than 1. Rhythmicity is lost at low temperatures and bright light (also observed in other systems) but reverts simply upon the return to permissive conditions, with the phase always determined by the time at which permissive conditions were restored. As first reported in humans, different rhythms may have different periods under some conditions - still not well defined in any system. And different rhythms have characteristically different phase angle relationships, but these can be altered by conditions. Might circadian phase be affected by a humoral factor(s)? This is of continuing interest in the mammalian SCN, where dozens of peptides have recently been reported; a unicell might be a favorable system to investigate the possibility. And do models for circadian systems have applicability to infra- ultradian rhythms? A circannual rhythm in Gonyaulax may be a good challenge for elucidating mechanisms. These and other aspects of circadian rhythms in Gonyaulax will be presented for discussion.
In most eukaryotic organisms, networks of cell cycle and circadian rhythms coexist and work coordinately to create optimal conditions for cells to grow and adapt to the surrounding environment. Cell cycle regulatory mechanisms include multiple checkpoints for controlled growth and cell divisions. The period of this oscillation, however, varies with external conditions such as nutrient and temperature. The cell cycle machinery is optimized for growth and division, but not for time keeping. Circadian rhythms keep track of time and provide temporal regulations in most eukaryotic organisms with a period of about 24 h. In contrast to the period of the cell cycle, the period of circadian rhythms is relatively insensitive to external conditions such as nutrient and temperature. Cell cycle and circadian rhythms are coupled despite of their apparent disparate functions. The circadian gated cell division cycles are observed in various organisms from cyanobacteria to mammals. However, the implications of this coupling on the physiology of an organism are unknown. We use a mathematical model to study interactions between the cell cycle and the circadian clock and their implications in cell cycle regulations.
The circadian clock is a complex biological process by which a period of about 24 hour is generated by numerous genetic components. Considering the pivotal importance of the time-tracking mechanisms for an organism's fitness, we hypothesized that there could be clock genes that are essential for cellular metabolism or that are minor modulators that have not been identified by conventional genetic screenings. In an attempt to identify and characterize all genetic components of the clock, we have adopted the quantitative genetics approach. Quantitative Traits Loci (QTL) analysis allowed us to identify 43 additive QTLs for period and phase phenotypes in the three mapping populations. Thirty of the identified 43 QTLs (70%) were not linked to any previously characterized clock genes. Moreover, 27 QTLs (63%) were found to contribute to variation of phase (but not period). This result contradicts the widely accepted view of phase, that phase is simply another expression of period in a cycling environment. Based on these results, we propose that there are numerous uncharacterized genes that are involved in entrainment. One of the identified phase QTLs, N6phase6, is responsible for a significant phase shift and will be discussed.
Temperature is a major environmental signal impacting circadian rhythms in multiple ways. Neurospora has played a pioneering role in the description of the molecular basis of both circadian rhythmicity and how the clock receives and responds to environmental signals including changes in temperature, although the molecular mechanisms underlying responses to temperature are not completely understood. Ambient temperature influences rhythmicity in multiple ways including phase resetting, the ability of the clock to operate and compensation or period length stability. Some temperature effects are mediated through the amount, and perhaps kind of FRQ protein made through temperature regulated splicing of a central clock component, frq, resulting in both the total amount of FRQ protein in the cell and the ratio of the two FRQ isoforms. Mutations in two Neurospora clock mutants, prd-3 and chr result in both period and temperature compensation defects and identify subunits of the casein kinase 2 enzyme. Inducible wild-type CK2-beta subunit that allows graded expression results in temperature compensation phenotypes ranging from anti-compensation to a near wild-type compensation that corresponds to the level of expression of the wild-type protein. The clock protein FRQ is identified as a direct substrate of CK2 and FRQ stability is shown to increase with increased temperature in CK2-beta hypomorphs but not with another kinase, CK1, involved in clock function. This work demonstrates that phosphorylation of different FRQ residues can lead to changes in either period length or temperature compensation.
Circadian clocks have conventionally been thought to be built around negative transcriptional feedback, but a number of recent results in both prokaryotic and eukaryotic systems have challenged this assumption. Most dramatically, it has been shown that aspects of the circadian rhythm of the cyanobacterium S. elognatus can be reconstituted in vitro with only the three purified proteins KaiA, KaiB, and KaiC. This talk will review our understanding of the in vitro Kai oscillator and then discuss the implications of this understanding for the functioning of the post-translational oscillator in a living cell where it is coupled to transcriptional feedbacks and for the cyanobacterial clock's remarkable robustness. I will conclude by suggesting that many of the basic principles revealed by careful study of the Kai system should apply to other examples of molecular synchronization oscillators, including possible post-translational components of eukaryotic clocks.
Several experimental studies have altered the natural phase relationship between photic and non-photic zeitgebers, in order to assess their hierarchy in the entrainment of circadian rhythms. In order to interpret the complex results that emerge from these conflicting zeitgeber protocols, we present computer simulations of two coupled oscillator systems forced by two independent zeitgebers. First proposed in 1959 by Pittendrigh and Bruce to model results of their studies on the light and temperature entrainment of eclosion in Drosophila, such a circadian system is also coherent with recent data from many organisms. Our simulations show how the phase of a circadian rhythm varies with a systematic change of the phase relationship between two zeitgebers. "Phase-jumps" and hysteresis in the overt rhythm are shown to arise if the inter-oscillator coupling is high in relation to the zeitgeber strength. Changes in the structural symmetry of the system indicated that these results are expected for a wide range of system configurations, while reproduction of the same phenomena with a simpler model, considering phase effects only, added to the generality of conclusions. We argue that our modeling approach can serve as a conceptual framework for understanding, planning and interpreting conflicting zeitgeber experiments.
Work done in collaboration with Daniel S.C.Damineli and Andreas Bohn (Universidade Nova de Lisboa) and W. Otto Friesen (University of Virginia).
I will survey what some recent mathematical results suggest about the design principles behind circadian clocks. In particular, I will discuss flexibility, robustness, buffering mechanisms against environmental heterogeneity, temperature compensation in physiological entrained conditions and tracking of multiple phases. If time permits I will also discuss new methods for fitting reporter time series data to models.
Homeostatic control mechanisms are essential to keep cells and organisms fit in a changing and challenging environment. An important task is to identify the factors which contribute to the functionality and robustness of homeostatic mechanisms in the presence of environmental perturbations. Kinetic conditions which lead to robust homeostasis and perfect adaptation together with their oscillatory behaviors are described. Analysis of the pathways for nitrate assimilation in plants and Neurospora suggest a homeostatic and oscillatory mechanism for the level of nitrate, apparently to keep the oxidative stress induced by nitrate and nitrate reductase under control during day/night cycles.
Circadian clocks, the native endogenous biological timers, are fascinating modelling systems because they have both unique behaviours specific to the species and dominant universal properties conserved across the kingdoms. Besides their ability to generate an entrainable 24hr-oscillation, circadian clocks show the optimum sensitivity and robustness to external cues. Existing circadian clock models for various organisms including; Synechococcus, Neurospora, Dosophila, mammal and Arabidopsis have been constructed in a similar manner, derived from multiple negative feedback loop circuit. A series of one-loop to three-loop models of Arabidopsis circadian clock published recently strengthen the evidence of multi-loop structure in the system. This conservation of fundamental properties together with the observed similarity in model structure motivated us to investigate the relation between the structure and properties of the model.
Most of the work was done at Institute of Molecular Plant Sciences, Department of Biological Sciences, University of Edinburgh.
Post-translational processes such as phosphorylation, SUMOylation and ubiquitylation control protein activity, localization and turnover. Many core oscillator components of the circadian clock are nuclear localized but how the phase and rate of their entry contribute to clock function is unknown. TOC1, a pseudoresponse regulator (PRR) protein, is a central element in one of the feedback loops of the Arabidopsis clock. Both TOC1 and a closely related protein, PRR5, are nuclear localized, expressed in the same phase, and shorten period when deficient. We now show that TOC1-PRR5 oligomerization enhances TOC1 nuclear accumulation through increased nuclear import. Additionally, PRR5 recruits TOC1 to large subnuclear foci and promotes phosphorylation of the TOC1 N-terminus. Our results demonstrate that nuclear TOC1 is essential for normal clock function and reveal a mechanism to enhance phase-specific TOC1 nuclear accumulation. We have also found that a second post-translational mechanism enhances ZTL protein stability through the activity of an ubiquitous chaperonin that plays a role in the clock different from animal systems. The relationship between this new component, ZTL, and another stabilizer of ZTL (GIGANTEA) is being explored through a mathematical model that focuses on the importance of post-translational processes in regulating clock function.
Modeling work done in collaboration with Janet Best, Tony Gallenstein and Grant Oakley.
The transcription factor LHY acts as part of the central components of the Arabidopsis circadian clock, and is also thought to regulate the expression of a wide range of output genes. In order to identify its full range of target genes, genome-wide binding sites were identified by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq). ). Over 5000 sequences were identified. GO term analysis showed that LHY regulates many biological processes, including aspects of developmental regulation and responses to biotic and abiotic stimuli.
Most of the known clock-associated genes contained binding sites for LHY. Binding was also detected within the promoter of LHY, providing novel evidence for direct, negative autoregulation. This may allow prompt repression of transcription upon expression of the LHY protein, and ensure that LHY transcription only takes place during a very narrow time window.
The identified target genes exhibit a wide range of phases of expression, ranging from early morning to late night. Analysis of motif enrichment within LHY-binding regions identified several sequences, including Evening-element-like sequences and G-box sequences. Further analysis is under way to determine whether different classes of binding sites for LHY or for cofactors are associated with expression at different times of the day.
Cyclic processes in biology span a wide dynamic range, from the sub-second periods of neural spike trains to annual rhythms in animal and plant reproduction. Even an individual cell exposed to a constant environment may exhibit many parallel periodic activities with different frequencies. It is therefore important to elucidate how multiple clocks coordinate their oscillations. Circadian oscillation and cell cycle are the two most essential rhythmic events present in almost all organisms. In several unicellular organisms and higher vertebrates, it has been shown that the circadian system affects whether cell division is permitted. Here, we integrate theoretical and experimental approaches to investigate how the circadian and cell division subsystems are coupled together in single cells of the cyanobacterium Synechococcus elongatus. We simultaneously tracked cell division events and circadian phases of individual cells. We found that the timing of cell divisions is synchronized to circadian signals rather than uniformly distributed throughout the day as expected from un-coupled clocks. This suggests that the circadian clock acts as a 'gate' for cell divisions. We fit the data to a model to determine the gating function that describes when cell cycle progression slows as a function of circadian and cell cycle phases. We infer that cell cycle progression in cyanobacteria slows during a specific circadian interval but is uniform across cell cycle phases. Our model is applicable to the quantification of the coupling between biological oscillators in other organisms.
Work done in Alexander van Oudenaarden laboratory at MIT and in collaboration with Susan Golden at UCSD.