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Isoform switching facilitates period control in the Neurospora crassa circadian clock

2008; Springer Nature; Volume: 4; Issue: 1 Linguagem: Inglês

10.1038/msb.2008.5

1744-4292

Ozgur E. Akman, James Locke, Sanyi Tang, Isabelle A. Carré, Andrew J. Millar, D.A.J. Rand,

Light effects on plants

Article12 February 2008Open Access Isoform switching facilitates period control in the Neurospora crassa circadian clock Ozgur E Akman Ozgur E Akman Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Systems Biology Centre, University of Warwick, Coventry, UK Mathematics Institute, University of Warwick, Coventry, UK Search for more papers by this author James C W Locke James C W Locke Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Department of Physics, University of Warwick, Coventry, UK Search for more papers by this author Sanyi Tang Sanyi Tang Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Systems Biology Centre, University of Warwick, Coventry, UK Mathematics Institute, University of Warwick, Coventry, UK Search for more papers by this author Isabelle Carré Isabelle Carré Department of Biological Sciences, University of Warwick, Coventry, UK Search for more papers by this author Andrew J Millar Andrew J Millar Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author David A Rand Corresponding Author David A Rand Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Systems Biology Centre, University of Warwick, Coventry, UK Mathematics Institute, University of Warwick, Coventry, UK Search for more papers by this author Ozgur E Akman Ozgur E Akman Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Systems Biology Centre, University of Warwick, Coventry, UK Mathematics Institute, University of Warwick, Coventry, UK Search for more papers by this author James C W Locke James C W Locke Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Department of Physics, University of Warwick, Coventry, UK Search for more papers by this author Sanyi Tang Sanyi Tang Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Systems Biology Centre, University of Warwick, Coventry, UK Mathematics Institute, University of Warwick, Coventry, UK Search for more papers by this author Isabelle Carré Isabelle Carré Department of Biological Sciences, University of Warwick, Coventry, UK Search for more papers by this author Andrew J Millar Andrew J Millar Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author David A Rand Corresponding Author David A Rand Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK Systems Biology Centre, University of Warwick, Coventry, UK Mathematics Institute, University of Warwick, Coventry, UK Search for more papers by this author Author Information Ozgur E Akman1,2,3, James C W Locke1,4, Sanyi Tang1,2,3, Isabelle Carré5, Andrew J Millar1,6 and David A Rand 1,2,3 1Interdisciplinary Programme for Cellular Regulation, University of Warwick, Coventry, UK 2Systems Biology Centre, University of Warwick, Coventry, UK 3Mathematics Institute, University of Warwick, Coventry, UK 4Department of Physics, University of Warwick, Coventry, UK 5Department of Biological Sciences, University of Warwick, Coventry, UK 6School of Biological Sciences, University of Edinburgh, Edinburgh, UK *Corresponding author. Systems Biology Centre, University of Warwick, Coventry CV4 7AL, UK. Tel.: +44 2476 523599; Fax: +44 2476 524182; E-mail: [email protected] Molecular Systems Biology (2008)4:164https://doi.org/10.1038/msb.2008.5 Correction(s) for this article Isoform switching facilitates period control in the Neurospora crassa circadian clock15 April 2008 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q10 of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild-type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic. Synopsis Circadian rhythms are universal, controlling 24-h rhythms of metabolism, physiology and behaviour in organisms ranging from humans to cyanobacteria. The circadian clock controls the expression of many genes, in a proportion between 10% (in the fruit fly, Drosophila melanogaster) and 100% (in cyanobacteria). Circadian rhythms share similar basic properties—they can be entrained by environmental light and temperature signals, for example. Temperature is interesting as an environmental time cue, and the understanding of its effects has been a central theme of circadian clock research since the 1950s. On the one hand, the clock is sensitive to temperature to the extent that it can act as an entraining signal, while on the other it is insensitive in that the rhythmic period is largely invariant over a physiological range of temperatures. This latter phenomenon, known as temperature compensation, is generally considered to be one of the defining properties of the clock. It has been suggested to be a key requirement for stability of the clock's phase relationship to daily environmental cycles under varying temperatures, and recent work has suggested that the small variation observed is a specific, adaptive control of period. Moreover, given that many biological rates roughly double when temperature is raised by 10°C, it is remarkable that such clocks remain rhythmic over a broad temperature range with period Q10 values in the range 0.8–1.2. The Neurospora crassa circadian network is particularly interesting from the point of view of temperature regulation because of the discovery that temperature alters the balance of translation between short and long isoforms of the protein FREQUENCY (FRQ), yielding a network with two parallel negative feedback loops that have opposite temperature dependency. A number of different mutant strains have been developed where effectively only one of the forms is present and their temperature responses have been quantified. The evolution of a molecular mechanism to provide a relatively complex modulation of the translation rates of two different forms of the same protein is a fascinating example of the extent to which circadian clocks can diverge from the minimal delayed negative feedback loop network sufficient to produce autonomous, entrainable oscillations exemplified by the Goodwin oscillator. We introduce a new mathematical model for the N. crassa circadian network, which incorporates recent experimental findings, including the temperature-dependent post-transcriptional modification of the FRQ protein (see Figure 1). This model is used to discuss period control and the functional temperature range of the N. crassa clock. The model reproduces all the key experimental data on temperature dependence and rhythmicity, in both wild-type and mutant strains (some simulations are shown in Figure 3). We present a mechanism (referred to as isoform switching) for period control that utilises the presence of the two parallel, temperature-dependent FRQ loops, suggesting a relatively simple means by which period control, and the consequent buffering of entrainment phase against temperature variations, could have evolved. We argue that this regulatory structure and associated tuning may also increase the temperature range where the clock is robustly rhythmic. Our results support theoretical studies proposing that one of the possible benefits of high loop complexity in clock networks is the increased evolutionary flexibility that such architectures confer. Furthermore, although our period control mechanism is presented in the context of the Neurospora system, we believe that the way in which other systems possessing a temperature-dependent switch in one or more key isoforms achieve compensation and phase robustness may be mathematically similar. Finally, by determining the key parameters contributing to period control in the Neurospora clock, we are able to make predictions regarding the biochemical processes affected in compensation mutants associated with changes in FRQ stability, as well as the biological parameters most likely to produce significant changes to the period profile when perturbed experimentally. In particular, our simulations suggest that the FRQ isoforms may have different efficacies as transcription factors, or be differentially phosphorylated, and that changes in the phosphorylation rates of the isoforms will have opposite effects on the period Q10. Introduction Circadian rhythms are affected by a variety of environmental stimuli. Of these, light and temperature are the major factors mediating entrainment to daily environmental cycles. For the circadian clock to provide an adaptive advantage, it is important for it to maintain the appropriate phase relationship relative to dawn and dusk such that rhythmic biological processes occur at the optimal time of the day. The responses of the clock must ensure that this phase relationship changes appropriately when the clock is subject to regular perturbations, such as the annual change in day length or temperature, while being resilient to the more or less random perturbations resulting from external environmental fluctuations or due to the stochastic environment of the cell. Temperature is interesting as an environmental time cue. On the one hand, the clock is sensitive to temperature to the extent that it can act as an entraining factor, but on the other hand, it is insensitive in that the period p of the free-running clock is largely independent of temperature (Gardner and Feldman, 1981; Mattern et al, 1982), with the Q10 of period falling in the range 0.8–1.2. This latter phenomenon, known as temperature compensation, is generally considered to be one of the defining properties of the circadian clock and has been suggested to be a key requirement for stability of the clock's phase relationship to daily environmental cycles, under varying temperatures (Rand et al, 2004). Furthermore, it has been suggested that deviations from perfect compensation—such as the slight undercompensation observed in Neurospora crassa wild-type (WT) strains—contributes to the setting of an appropriate phase under entrained conditions. This implies that the period–temperature profile may be tuned so as to optimise the seasonal adaptation of the clock (Brunner and Diernfellner, 2006; Diernfellner et al, 2007), suggesting that the key requirement for a well-adapted system is a controlled variation in period rather than perfect compensation. Recent research (Liu et al, 1997; Tan et al, 2004; Diernfellner et al, 2005) has also highlighted the question of why circadian clocks oscillate reliably over such a broad range of temperatures, because for some Neurospora mutant strains there are strict limits to the range of temperatures under which the clock is properly rhythmic. Indeed, given that some parameter values are approximately doubling over the temperature range studied, theoretical considerations suggest that loss of rhythmicity is extremely likely. The Neurospora clock is one of the most comprehensively studied circadian systems (Merrow et al, 2001; Liu et al, 2003). The central components are the rhythmic gene frequency (frq) and the constitutively expressed genes white collar-1 (wc-1) and white collar-2 (wc-2). The protein products of the white collar genes, WC-1 and WC-2, comprise the positive elements of a core negative feedback loop. These form a heterodimeric white collar complex (WCC), which binds to two light-response elements in the frq promoter, activating transcription of frq (Cheng et al, 2001a; Froehlich et al, 2002). When FRQ accumulates beyond a certain level, it interacts with the WCC to inhibit its activation of frq transcription, closing the loop (Cheng et al, 2001a, 2001b; Denault et al, 2001; Merrow et al, 2001; Froehlich et al, 2002). This interaction is mediated by the protein product of an RNA helicase, frh (Cheng et al, 2005). FRQ also positively regulates synthesis of WC-1, forming a positive feedback loop that interlocks with the primary loop (Lee et al, 2000; Cheng et al, 2001a). Here, we discuss the effects of temperature in the context of a new mathematical model for the N. crassa circadian clock. Using this model, we reproduce many of the main features of WT and mutant Neurospora systems, such as their protein profiles, their functional temperature ranges and the dependence of their period upon temperature. We show that although temperature compensation and tuning of the period–temperature profile can be achieved with just one form of FRQ with a temperature-dependent translation profile, the presence of two regulatory loops with opposite temperature dependency controlling the production of two FRQ isoforms greatly simplifies the evolution of such control over the full temperature range, with only a small number of biologically relevant parameters having to be tuned. We also propose that this tuning mechanism—referred to here as isoform switching to reflect its dependence on the switch between the relative abundances of the FRQ forms—naturally increases the functional temperature range of the clock by buffering the system against the loss of robust rhythmicity. The Neurospora circadian clock The Neurospora network is particularly interesting from the point of view of temperature regulation because temperature alters the balance of translation between two isoforms of FRQ expressed as a result of alternative initiation of translation from different start codons on the frq open reading frame (ORF). The translation of a longer form (denoted l-FRQ) is initiated at codon 1 while that of a shorter form (denoted s-FRQ) is initiated at codon 100 (Garceau et al, 1997). Furthermore, the rate of FRQ protein synthesis is strongly influenced by temperature. As temperature is raised, short ORFs within the 5′ untranslated region of the frq locus are ignored, resulting in increased translation of the frq ORF (Diernfellner et al, 2005). Consequently, temperature steps cause rapid changes in the level of FRQ, leading to phase shifts (Liu et al, 1998). In addition, more l-FRQ is produced relative to s-FRQ at higher temperatures as a consequence of thermosensitive splicing of intron 6, which removes the initiation codon for l-FRQ (Colot et al, 2005; Diernfellner et al, 2005). To explore the relationship between this temperature-dependent switch in FRQ isoforms and the dependence of period on temperature, a number of different mutant strains have been developed where effectively only one of the isoforms is present (Liu et al, 1997; Diernfellner et al, 2005). Diernfellner and co-workers constructed mutant strains in which splicing of intron 6 was either constitutive or completely abolished (Diernfellner et al, 2005). When splicing is made constitutive, s-FRQ is synthesised at all temperatures with little l-FRQ produced (this strain will be referred to here as strain A). When splicing is abolished, l-FRQ is efficiently synthesised with only trace amounts of s-FRQ (strain B). Liu et al (1997) constructed similar strains that expressed either s-FRQ alone (strain C) or l-FRQ alone (strain D). Strain D was created by removing the initiation codon for s-FRQ. At low temperatures, a substantial fraction of frq is spliced at intron 6 (Diernfellner et al, 2005), removing the start codon for l-FRQ and therefore preventing its translation. As a result, for strain D there is a substantial reduction in the total amount of FRQ at low temperatures and it becomes arrhythmic (Liu et al, 1997). Strain C was created by introducing a frameshift mutation between the l-FRQ and the s-FRQ AUG. Thus, translation initiation still takes place from the l-FRQ AUG but does not give rise to a functional protein. At high temperatures, almost all frq RNA retains intron 6 and translation is initiated preferentially at the AUG of l-FRQ (Diernfellner et al, 2005). Consequently for strain C, at high temperatures, the amount of FRQ being translated is substantially reduced, and as a result the strain becomes arrhythmic (Liu et al, 1997). Results A mathematical model of the Neurospora clock incorporating the two FRQ isoforms We constructed a model of the Neurospora network incorporating the two genes frq and wc-1 in which the FRQ protein pathway is bifurcated into separate parallel pathways for s-FRQ and l-FRQ. The model is shown schematically in Figure 1. It does not include the genes wc-2 and frh or the light-responsive clock gene vvd, as these factors are not known to function in temperature responses. Care was taken to achieve a good fit to experimental time courses as shown in Figures 5 and 6 of the Supplementary information. Figure 1.A schematic representation of the regulatory network underlying the mathematical model of the N. crassa clock. This includes the two genes frq and wc-1 and both the long and short forms of the FRQ protein. WC1* represents light-activated WC-1. Download figure Download PowerPoint A key ingredient of the model is the shape of the curve chosen for the translation rates of s-FRQ and l-FRQ as a function of temperature. These are different for the WT and the four mutant strains discussed above. The forms used in the model are shown in Figure 2 and further details are given in the Supplementary information (section 4). The simulated FRQ protein profiles generated are in qualitative agreement with experimental data (Liu et al, 1997; Diernfellner et al, 2005). Figure 2.Temperature-dependent changes in s-FRQ and l-FRQ translation rates. Open symbols: translation of s-FRQ; closed symbols: translation of I-FRQ. Left panel: WT. Middle panel: mutant strains producing mainly s-FRQ. Inverted triangles: strain A; triangles: strain A with asymmetric FRQ pathways; squares: strain C. Right panel: mutant strains producing mainly l-FRQ. Diamonds: strain B; triangles: strain B with asymmetric FRQ pathways; squares: strain D. Download figure Download PowerPoint Simulations of temperature dependence and arrhythmicity in WT and mutant strains Simulated variations of period with temperature for the WT and strains A-D are shown in Figure 3. For the basic near-symmetric model, all parameters of the l-FRQ and s-FRQ pathways except for those controlling translation rates are approximately equal. This reproduced the main features of the WT and strains C and D including the dependence of period upon temperature. However, to reproduce the temperature-dependent increase in period that has been observed experimentally for strain A (Diernfellner et al, 2005, 2007), it was necessary to allow some parameters of the l-FRQ pathway to vary significantly from those of the s-FRQ pathway (see Supplementary information, section 5). Figure 3.Dependence of period on temperature for the Neurospora model. Circles denote the WT. Left panel: mutant strains obtained through optimisation or suppression of splicing (Diernfellner et al, 2005, 2007). Inverted triangles: strain A; triangles: strain A with divergent FRQ pathways; diamonds: strain B; squares: strain B with divergent FRQ pathways. For the simulations obtained assuming FRQ pathway asymmetry, strain A has an increasing period–temperature profile, while strain B has a decreasing one with the period of strain A greater than that of strain B, as observed experimentally (Diernfellner et al, 2007). Right panel: mutant strains obtained through modification of the l-FRQ AUG or s-FRQ coding region (Liu et al, 1997). Triangles: strain C; squares: strain D. Strain is compensated at lower temperatures with a period greater than that of the wild-type, becoming arrhythmic at the upper end of the range. Strain D is compensated at higher temperatures with a period smaller than that of the WT, becoming arrhythmic at the lower end of the range. This is in agreement with experimental data (Liu et al, 1997). Download figure Download PowerPoint Liu et al (1997) reported that expression of either the short or the long form of FRQ significantly reduced the range of temperatures where the clock was rhythmic. The strain expressing l-FRQ alone (strain D) only supports rhythmicity at high temperatures (above 22°C), whereas that expressing s-FRQ alone (strain C) only allows rhythmicity at low temperatures (below 25°C; Liu et al, 1997). The right panel of Figure 3 shows that our model accounts qualitatively for this behaviour, with the simulations of strains C and D becoming arrhythmic close to their experimental values. By contrast, in both the experiments and model, the strains A and B of Diernfellner et al (2005, 2007) maintained rhythmicity across the temperature range even though they effectively only express one form of FRQ. Brunner and Diernfellner (2006) suggested that the arrhythmicity of strains C and D was due to the reduced overall amount of FRQ protein in these strains. The model supports this explanation and throws some new light on it. Figure 4 shows how total FRQ translation rate varies with temperature in the different strains, resulting in temperature-dependent loss of rhythmicity in strains C and D as total FRQ protein levels fall below critical levels. Insets within Figure 4 show that, as the overall amount of FRQ translation is decreased below a critical value in these strains, the limit cycle shrinks down to an equilibrium with the amplitude of FRQ oscillations converging to zero as this occurs. On the other hand, the model shows that high FRQ protein levels are not necessarily sufficient to ensure clock functionality. If FRQ translation levels are increased too much then the system become arrhythmic again, as excessive FRQ levels turn frq transcription off too quickly (see Figure 4). Thus the optimisation of functionality is not straightforward and FRQ translation rates have to be tuned. It is shown below how this can be achieved and how it is related to temperature compensation and the control of period. Figure 4.Simulated changes in total FRQ translation rate with temperature. Filled circles denote the WT. Filled triangles: strain A; filled squares: strain B; open triangles: strain C; open squares: strain D. Thick solid lines denote the net translation rates at which rhythmicity is lost, derived as described in the Supplementary information (section 6). The system is rhythmic for net rates lying between the thick curves, becoming arrhythmic if there is insufficient or excessive translation of FRQ. Inset figures: bifurcation diagrams showing the loss of rhythmicity of strains C and D. For each temperature value on the x-axis, the corresponding value on the y-axis denotes the minimum and maximum levels of FRQ. Solid lines denote stable attractors and broken lines unstable attractors. The solid circles indicate Hopf bifurcations at which the attractor changes from a fixed point (corresponding to arrhythmicity) to a limit cycle (corresponding to rhythmicity). Download figure Download PowerPoint Characterising period control based on balancing opposite reactions Hastings and Sweeney (1957) proposed that a balance of opposing reactions could allow temperature compensation (slope of period change with respect to temperature dp/dT approximately 0, denoted dp/dT≈0). This hypothesis was first tested by Ruoff using a simple model for an oscillatory feed-back loop (Ruoff, 1992, 1994; Ruoff et al, 2000). He assumed that the temperature dependence of each of the model parameters k1,…,km was similar to that for rate constants of chemical reactions and was described by the Arrhenius equation. The Arrhenius equation expresses the dependence of the rate constant kj on the temperature T and activation energy Ej as kj=Aj exp (−Ej/RT), where Aj is a constant specific to the individual parameter and R is the gas constant (8.314472 J K−1 mol−1). This assumption allowed Ruoff to deduce an expression for the local period slope dp/dT in terms of the activation energies Ej and control coefficients cj for each of the parameters: Each term in this expression corresponds to one of the parameters kj. The product (p/RT2)cjEj combines the sensitivity cj of the period to a change in the parameter kj with the sensitivity of the parameter kj to temperature. Because we are using the Arrhenius relation, the latter is (1/RT2)Ej. The control coefficient cj is defined mathematically by cj=∂ log p/∂ log kj, that is, the ratio of the relative change δp/p in period to the relative change in the parameter δkj/kj, for a given small change δkj in kj with all other parameters fixed. In our model, the temperature dependence of the s-FRQ and l-FRQ translation rates rS and rL are distinct from the other parameters k1,…,km−2, because their dependence on temperature is determined by the thermosensitive post-transcriptional regulation described above and not by the Arrhenius equation. Thus Ruoff's equation is modified to (units: h K−1). Here, and are the control coefficients for the parameters rS and rL. The quantities rS′(T) and rL′(T) are the derivatives of the curves rS=rS(T) and rL=rL(T), indicating the local change in s-FRQ and l-FRQ translation rates at the temperature T (see Figure 2). For a given model, all the quantities in equation (1) can be easily calculated using the methods of (Rand et al, 2006). Based on values given in (Ruoff and Rensing, 1996; Ruoff et al, 2005), we expect that a significant proportion of control coefficients will have magnitudes between 0.1 and 1 (cf. Table 6 of the Supplementary information), and that the Ejs will be in the region of 1–150 kJ mol−1. The Arrhenius formula predicts activation energies of at least 50 for a parameter kj with a Q10 of 2 or more, which is the generally accepted value for biological reactions. It follows that a number of the individual terms in equation (1) should each contribute a few hours change in period per degree. For an arbitrary oscillator without any imposed period control mechanism, there is no reason that the positive and negative terms in this sum should balance each other, suggesting that for such an oscillator, we expect a variation of period of several hours over a 10°C temperature range. To be able to obtain temperature compensation (dp/dT≈0) at a given temperature T=T0, it is necessary that the more significant terms in equation (1) balance each other out and therefore have different signs. This can be achieved by adjusting either the control coefficients cj, the activation energies Ej or the FRQ translation rates rS and rL. Adjusting the activation energies Ej would be equivalent to altering the components of the oscillator to change binding constants for example, whereas altering the control coefficients cj (which are functions of the parameters kj) could be achieved through changes in the activities of regulatory proteins that modulate the rate of post-translational modification or proteolytic degradation of these oscillator components. One approach to temperature compensation (Ruoff, 1992, 1994; Ruoff et al, 2000) involves balancing equation (1) to get dp/dT≈0 at a single temperature T0. Such single-point balancing, referred to as 'static compensation' in (Ruoff et al, 2007), can achieve local compensation even for simple negative feedback loop models. However, the period p as a function of temperature near T0 will be a parabolic curve of the form p(T)=a+b(T−T0)2+c(T−T0)3+d(T−T0)4+…. Formulas for the parameters b, c, d etc can be derived. The formula for b is given in section 4 of the Supplementary information. Using this formula with typical values for the variables it depends on, one sees that b may well be of order 0.1 or more for a system where balance has been achieved by this local method, resulting in significant quadratic variations of the period about the balance point. Thus to ensure good compensation over a significant interval of temperatures, it is necessary that the terms b, c, d etc are made small, meaning that many parameters have to be independently tuned. An example of a locally compensated system obtained through the single-point technique is shown in Figure 5, demonstrating the expected parabolic change in period with temperature. Interestingly, the clock becomes arrhythmic as temperature changes away from T0. Testing of a large number of parameter sets for which our model is locally compensated indicated that this behaviour was typical of such systems (data not shown). Figure 5.Left panel: comparison of isoform switching and single-point methods for temperature compensation of WT strains. Open circles: globally compensated system obtained through isoform s