Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa
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Evaluation Summary:
This manuscript will be of interest to researchers working in chronobiology and metabolism. The authors have found evidence that starvation decreases the abundance of the fungal circadian clock protein white collar complex (WCC), even though WC-1 is required for responses to starvation. This observation is interesting, but the authors should consider that WCC has several other functions (as a light receptor, in transcriptional regulation) that are not necessarily clock connected. As such the most interesting result from this paper is that the standard model for the molecular mechanism of the fungal circadian clock does not explain the persistence of normal rhythms under extreme starvation conditions, where the levels of clock proteins are drastically altered.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.
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Author Response
Reviewer #1 (Public Review):
The stated goal of this research was to look for interactions between metabolism, (manipulated by glucose starvation) and the circadian clock. This is a hot topic currently, as bi-directional links between metabolism and rhythmicity are found in several organisms and this connection has important implications for human health. The authors work with the model organism Neurospora crassa, a filamentous fungus that has many advantages for this type of research.
The authors' first approach was to assay the effects of glucose starvation on the levels of the RNA and protein products of the key clock genes frq, wc-1, and wc-2. The WC-1 and WC-2 proteins form a complex, WCC, that activates frq transcription. The surprising finding was that WC-1 and WC-2 protein levels and WCC transcriptional …
Author Response
Reviewer #1 (Public Review):
The stated goal of this research was to look for interactions between metabolism, (manipulated by glucose starvation) and the circadian clock. This is a hot topic currently, as bi-directional links between metabolism and rhythmicity are found in several organisms and this connection has important implications for human health. The authors work with the model organism Neurospora crassa, a filamentous fungus that has many advantages for this type of research.
The authors' first approach was to assay the effects of glucose starvation on the levels of the RNA and protein products of the key clock genes frq, wc-1, and wc-2. The WC-1 and WC-2 proteins form a complex, WCC, that activates frq transcription. The surprising finding was that WC-1 and WC-2 protein levels and WCC transcriptional activity were drastically reduced but frq RNA and protein levels remained the same. Under conditions where rhythmicity is expressed, the rhythms of frq RNA, FRQ protein, and expression of clock-driven "output" genes were also unaffected by starvation. The standard model for the molecular clock is a transcription/translation feedback loop dependent on the levels and activity of these clock gene products, so this disconnect between the starvation-induced changes in the stoichiometry of the loop components and the lack of effects of starvation on rhythmicity calls into question our understanding of the molecular mechanism of the clock. This is yet another example of the inadequacy of the TTFL model to explain rhythmicity. For me, the most significant sentence in the paper was this: "...an unknown mechanism must recalibrate the central clockwork to keep frq transcript levels and oscillation glucose-compensated despite the decline in WCC levels."
The author's second approach was to try to identify mechanisms for the response to starvation by focussing on frq and its regulators, using mutations in the frq gene and strains with alterations in the activity of kinases and phosphatases known to modify FRQ protein. The finding that all of these manipulations have some effect on the starvation-induced changes in WC protein level is taken by the authors to indicate a role for FRQ itself in the response to starvation. This conclusion is subject to the caveat that manipulations of the activity of multifunctional kinases and phosphatases will certainly have pleiotropic effects on many cellular processes beyond FRQ protein activity.
Because of the sometimes-speculative nature of our conclusions and based on the suggestion of the editor, we restructured the Discussion and discuss now the mechanism addressed by the Reviewer in the subsection "Ideas and Speculation". We added a sentence to the section about the possible pleiotropic effects of the tested signaling pathways: "Starvation triggers characteristic changes in the activity of signaling routes that affect basic components of the circadian clock. Although the multifunctional pathways might act via pleiotropic mechanisms as well, based on their earlier characterized role in the control of the Neurospora clock, their action can be inserted into a model describing the glucose-dependent reorganization of the oscillator."
The third section of the paper is a major transcriptomic study of the effects of starvation on global gene expression. Two strains are compared under two conditions: wc wild-type and the wc-1 knockout strain, under fed and starved conditions. The hypothesis is that WCC has a role in the starvation response. The results of starvation on the wild-type are unsurprising and predictable: the expression of many genes involved in metabolic processes is affected. There are no new insights that come from these results and no new testable hypotheses are generated by the data.
We agree with the reviewer that it is not surprising that glucose depletion strongly affects genes involved in metabolic processes and monosaccharide transport. These data obtained in wt served rather as a control for our experimental conditions. As a new aspect, our analysis focused on the differences between wt and wc-1 in the transcriptomic response to altered glucose availability.
The authors refer to the wc-1 mutant strain as "clockless" and discuss its effects on the transcriptome only in terms of WC-1's function in the clock mechanism. However, WCC is known to be a major transcriptional regulator, controlling a number of genes beyond the TTFL. As acknowledged earlier in the paper, WC-1 is also the major light receptor in Neurospora. The transcriptomics experiments were carried out in a light/dark cycle, with cultures harvested at the end of the light period, when "an adapted state for light-dependent genes can be expected" according to the authors. However, wc-1 mutants are essentially blind, and so those samples are equivalent to being harvested in the dark. The multifunctional nature of WCC complicates the interpretation of the transcriptomics data. The differences in the transcriptome between wild-type and wc-1 may not be due to loss of clock function, but rather the loss of a major multifunctional transcription factor, or the difference between light and "dark".
The reviewer is right, when we discussed the difference between wt and wc-1 in the transcriptional response to glucose, we did not emphasize the possible contribution of the photoreceptor function of the WCC. We added the following sentence to the revised version of the discussion: "Further investigations could differentiate between the clock and photoreceptor functions of the WCC in the glucose-dependent control of the transcriptome." Furthermore, we more specifically indicate that in wc-1 the lack of the WCC (and not the lack of a functional clock) results in the altered transcriptomic response to starvation when compared to wt (P15 L14-17).
In the final set of experiments, the authors tested the hypothesis that the changes in the transcriptome between wild type and wc-1 might make wc-1 less competent to recover growth after starvation. They also test the recovery of frq9, a "clockless" mutant. The very surprising result is that the growth rates of these two mutants are slower than the wild type after transfer from starvation media to high glucose. This is surprising because there will be several generations of nuclear division and doublings of mass within a few hours and the transcriptome should have recovered fully fairly rapidly. A mechanism for this apparent "after-effect" is suggested with evidence concerning differences in expression of a glucose transporter, but it is not clear why this expression should not change rapidly with re-feeding on high glucose. As with previous experiments, the cultures were grown in light/dark cycles, which results in different conditions for the mutants, both of which have very low or absent WC-1 and are therefore blind to light. The potential effects of light have been disregarded.
The reviewer is right that several generations of nuclear divisions occur within a few hours and lead to a number of doublings of the biomass. However, when the first phase of regeneration is delayed in one or more strains compared to the control, until the stationary phase a substantial difference in the biomass can be expected.
To the expression change of the glucose transporter: In order to emphasize the different tendency of how glt-1 levels respond to glucose in the different strains, in the previous version of the manuscript we normalized the expression levels to the beginning of recovery (time point of glucose addition). Thus, expression differences between the strains were not shown. To give a more comprehensive picture, in the revised version of the manuscript expression levels without normalization are depicted (Fig 5F). The mutants did not adapt efficiently to changes in the glucose levels, i.e. expression of the transporter was relatively high in both wc-1 and frq10 during starvation and did not further increase upon glucose addition. On the other hand, 24 hours after glucose resupply, glt-1 levels were similar in all strains which might contribute to the similar growth rates observed under steady-state conditions in the standard medium.
To the photoreceptor-independent function of the WCC during growth recovery: In the revised version of the manuscript we present additional data suggesting the importance of the photoreceptor-independent function of the WCC for efficient recovery from starvation. Fig. 5C and Fig. 5D show now that upon resupply of glucose, wt grows faster than the clock-deficient strains Δwc-1 and frq10 in both LD cycles and constant darkness, indicating that the role of the WCC in growth regeneration is at least partially independent of its photoreceptor function. To the function of the WCC in frq10: frq10 can not be considered blind. Although both Δwc-1 and frq10 lack a functional clock and WC levels are reduced in frq10, these strains show significant differences in WCC activity. While Δwc-1 is considered blind, in frq10 lack of the negative feedback results in high activity of the WCC in both DD and LL and expression levels of all examined, light-sensitive or light-dependent genes were found comparable in wt and in frq-less mutants (Schafmeier et al., 2005; Hunt et al., 2007; own unpublished data).
The title of the paper refers to a "flexible circadian clock" but this concept of flexibility is not developed in the paper. I would substitute "the White Collar Complex" for this phrase: "Adaptation to starvation requires a functional White Collar Complex in Neurospora crassa" would be more accurate. Some experiments are also conducted using an frq null "clockless" strain, but because WC expression is very low in frq null mutants, any effects of frq null could also be attributed to WC depletion.
As detailed above, low level of the WCC in the frq-less mutant does not mean low transcriptional activity and accordingly, the two clock mutants, wc-1 and frq10 show important functional differences. We used the word "flexible" to indicate that the molecular clock is able to operate under critical nutrient conditions and with a significantly changed stoichiometry of its key components. Results of our new experiments performed in DD (mentioned above) indicate that growth regeneration is rather independent of the photoreceptor function of the WCC. Nevertheless, we accepted the criticism of the reviewer and changed the title to "Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa".
The major conclusion I took away from this paper is the multifunctional nature of the WCC as a transcription factor complex. It has been known for a long time that WCC controls the expression of many genes beyond the frq gene at the core of the circadian transcription/translation feedback loop. WC-1 is also the major blue light photoreceptor in Neurospora, controlling the expression of light-regulated genes, and this fact is barely touched on in the paper. These new data now extend the role of WCC in the regulation of metabolic networks as well.
Reviewer #2 (Public Review):
The authors have performed an interesting study addressing a topical question in considering how circadian oscillators remain accurate in changing environmental conditions and these circadian oscillators contribute to responses to environmental changes. The authors have performed their studies in Neurospora crassa. The authors have made a very interesting finding that starvation causes a profound decrease in white collar 1 WC-1 abundance, yet the circadian system continues to run despite this decrease in the abundance of a core oscillator component. The study of chronic glucose starvation in a Δwc-1 mutant is interesting and provides the opportunity to investigate the role of the WHITE COLLAR COMPLEX (WCC) and the clock system in adaption to starvation.
Strengths:
The authors have used a range of techniques to measure clock behaviour, including qPCR, phosphorylation, protein abundance, and subcellular localisation studies.
An frq9 mutant was used to test the effects of FRQ on WC1 abundance since WC1 decreased during starvation. This is elegant, though it is not quite clear the logic of this experiment because FRQ did not change abundance during starvation, so why did the author think this experiment was needed?
We regret that the examination of frq9 was not clearly justified in the previous version of the manuscript. It is true that FRQ levels did not change during starvation, only phosphorylation of the protein was affected, i.e. FRQ became more phosphorylated (displayed by an electrophoretic mobility shift on the Western blot (Garceau N, Liu Y, Loros J J, Dunlap J C. Cell. 1997;89:469–476.)) under low glucose conditions. We tested the starvation response in the FRQ-less strain because WCC level changed significantly in wt upon glucose depletion and expression of WC proteins is known to be controlled by FRQ. In the revised version of the manuscript we tried to introduce and explain the experiments performed with frq9 more thoroughly (P7 L22-P8 L14; P16 L21 – P17 L6).
An interesting experiment was performed to test whether CK1a-dependent phosphorylation and inactivation of the WCC are involved in the starvation response. An FRQΔFCD1-2 mutant is used in which FRQ cannot interact with CK1a and therefore CK1a cannot phosphorylate and inactivate WC. This experiment suggested that CK1a is not involved in the response to starvation, again leading to the conclusion that FRQ is not involved in the starvation regulation of WC.
The referee is right, effect of FRQ-bound CK-1a seems to be minor on the adaptation of the molecular clock to starvation, and this is also our conclusion in the manuscript. The major message of this experiment was that FRQ became phosphorylated in response to starvation without stably interacting with CK1a, probably via another mechanism. We agree with the notion that the behavior of WCC levels upon starvation was similar to that in the FRQ-less mutant.
PKA is shown to be involved in the starvation-induced reduction of WC because the starvation-induced reduction in abundances of WC-1 was absent in the mcb strain in which the regulatory subunit of PKA is defective and hence, PKA is constitutively active.
The authors have found an interesting potential link between glucose levels and WCC phosphorylation, they demonstrated that starvation reduces PP2A activity and that in a regulatory mutant of PP2A, which has reduced PP2A activity, there is little effect of starvation on WCC levels, suggesting the hypothesis that glucose-dependent PP2A dephosphorylation stabilises WCC.
Analysis of starvation-regulated transcriptome in Δwc-1 and wild type found strong evidence that the transcriptomic response to starvation is in part dependent on WCC. Much of the misregulated transcriptome appears to be associated with metabolism.
In a series of growth studies in wild-type frq and wc-1 mutants the authors provide strong evidence that FRQ and WC are involved in growth and survival following starvation, and recovery from starvation.
Weaknesses:
The authors describe Neurospora crassa as a model for circadian biology and apparently make the assumption that the findings are indicative of the behaviour of clock systems in other kingdoms. This is not the case. Neurospora crassa is a wonderful model for studying fungal clocks and is a great tool for studying basic circadian dynamics, but the interesting findings here are of a detailed molecular nature and therefore are applicable for fungal clocks, but not other kingdoms.
We agree that we still do not know whether the described mechanism is specific for only fungal clocks. However, besides the basic feedback loop, overlapping mechanisms (controlled by e.g. casein kinases, glycogen synthase kinase, PKA, PP2A) are involved in the regulation of circadian timekeeping in different eukaryotic systems (reviewed in Reischl and Kramer, 2011, FEBS Lett; Brenna and Albrecht, 2020, Front Physiol). Our results suggest that some of these common factors (PKA, GSK, PP2A) are involved in the reorganization of the Neurospora clock in response to changes in glucose availability. Therefore, it is possible that analogous changes occur in the time keeping mechanisms of other eukaryotic systems when they face serious environmental challenges.
We included a short section into the Discussion which gives a short overview about known interactions between glucose availability and circadian timekeeping at different levels of the phylogenetic hierarchy (P15 L18 – P16 L7).
The authors assume that the reader is intimate with the intricacies of Neurospora crassa circadian studies and the significance of differences between LL and DD investigations. More background on the logic of the experiments would be helpful for readers from other fields.
Thank you for the comment. In the revised version of the manuscript we tried to introduce the molecular clock of Neurospora more thoroughly and completed the description of the experimental conditions with detailed explanations.
The data in Figure 2 are essential for the interpretation of the findings, demonstrating the presence of free-running rhythms. However, the data are entirely qualitative, making it hard to fully assess the authors' interpretations, a more quantitative assessment of the data would improve clarity.
We quantified the Western blot signals and show the results in Fig 1E in the new version of the manuscript (according to the reviewer's suggestion Fig 2 of the old version is now part of Fig 1). Our data indicate that oscillation of FRQ levels is similar under both nutrient conditions.
The conclusion that FRQ contributes to the regulation of WC1 abundance in response to starvation does not seem to be supported by the data because FRQ RNA does not change upon starvation. Furthermore, the authors conclude that the starvation-induced decrease in WC-1 and WC-2 protein levels are due to FRQ because a lack of reduction in an frq9 mutant is open to misinterpretation because this mutant makes WC levels low and therefore starvation might not lower already low levels of WC. Indeed WC-1 is lower in the frq9 mutant under any condition than in the WT under starvation and WC-2 does decrease in abundance in the frq9 mutant in starvation. The data strongly suggest to this reader that FRQ does not participate in the regulation of WC abundance in response to starvation.
After rereading the criticized section, we admit that the text was not well structured and we carried out several modifications. We intended to emphasize that upon drastic changes of the glucose availability frq RNA levels remained compensated in wt, but this compensation was affected when functional FRQ was not present. We agree with the reviewer's opinion that the low expression of the WCC in frq9 makes it difficult to compare the glucose-dependence of WCC expression in frq9 and wt. We modified the conclusion by adding this information and now mainly focus on the strain-dependent difference in the changes of frq RNA expression. (P7 L22-P8 L14)
The discussion accurately summarises the results and provides an interpretation but lacking is a comparison to other circadian systems in other kingdoms. How do the data compare with the effects of glucose and other sugars on the mammalian, plant, and insect clocks?
We included a short section into the Discussion which gives a short overview about known interactions between glucose availability and circadian timekeeping in different organisms (P15 L18-P16 L7).
How changes in WCC might result in changes in transcription is not explained. This might be very obvious to the authors but to the reader, it is not. Are the transcriptional outputs direct targets of WCC? Has WCC CHIPseq been performed by the authors or others, are the regulated transcripts directly bound by WCC? What are the enriched promoter sequences in the regulated genes, is it possible to identify the network by which these changes in transcription occur?
We now show the list of genes (Figure 4 – Figure supplement 2) that changed in a strain-specific manner in response to glucose starvation and, based on Chip-Seq results, were earlier described as direct targets of the WCC (Smith et al., 2010; Hurley et al., 2014). Based on the literature data showing that the WCC affects the expression of several other transcription factors and controls basic cellular functions which might affect the expression of further genes, it was not surprising that only 90 out of the 1377 genes were reported to be direct targets of the WCC.
Whilst the authors claim it is the circadian clock that is involved in the starvation response, in my view a more precise interpretation of the data is that WCC is involved in the response. Since WCC is a photoreceptor with dual function in the clock, is it yet possible to conclude that the effects discovered are due to the clock role of WCC? Or do the data support the role of light signalling in regulating the starvation response through WCC?
We thank you for the comment. In the revised version of the manuscript we more specifically indicate that in wc-1 the lack of the WCC (and not the lack of a functional clock) results in the altered transcriptomic response to starvation compared to wt. In addition, in the revised version we present a new experiment (Fig. 5D.) which shows that upon resupply of glucose wt grows faster also in constant darkness than the clock-deficient strains wc-1 and frq10 do. This indicates that the role of the WCC in growth regeneration is largely independent of its photoreceptor function.
The authors do not apparently reconcile that the effect of starvation is to hugely decreases WCC levels, but they find the transcriptional and growth response to starvation requires WCC?
We agree with the reviewer that the problem of how low levels of WCC could sufficiently support the transcription of frq and different output genes under starvation conditions was not discussed properly. Our results suggest a model in which the maintained level of nuclear WCC and the weakened inhibition by both FRQ (the hyperphosphorylated form is less active in the negative feedback) and PKA (its activity lowered upon glucose depletion) together might ensure that transcriptional activity of the WCC is preserved upon glucose withdrawal in both DD and LL despite the decrease of the overall level of the complex. In the revised version these aspects are discussed more thoroughly (P16-18).
This study contributes to the increased focus of the circadian community on the regulation of outputs by circadian oscillators. The manuscript will be of interest to many in the field. There needs to be less assumption of knowledge about the N. Crassa circadian system, and better discussion in a broader context of clocks in other kingdoms.
We added a new section to the Discussion with data concerning interrelationships between glucose availability and the circadian clock in other organisms.
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Evaluation Summary:
This manuscript will be of interest to researchers working in chronobiology and metabolism. The authors have found evidence that starvation decreases the abundance of the fungal circadian clock protein white collar complex (WCC), even though WC-1 is required for responses to starvation. This observation is interesting, but the authors should consider that WCC has several other functions (as a light receptor, in transcriptional regulation) that are not necessarily clock connected. As such the most interesting result from this paper is that the standard model for the molecular mechanism of the fungal circadian clock does not explain the persistence of normal rhythms under extreme starvation conditions, where the levels of clock proteins are drastically altered.
(This preprint has been reviewed by eLife. We include the …
Evaluation Summary:
This manuscript will be of interest to researchers working in chronobiology and metabolism. The authors have found evidence that starvation decreases the abundance of the fungal circadian clock protein white collar complex (WCC), even though WC-1 is required for responses to starvation. This observation is interesting, but the authors should consider that WCC has several other functions (as a light receptor, in transcriptional regulation) that are not necessarily clock connected. As such the most interesting result from this paper is that the standard model for the molecular mechanism of the fungal circadian clock does not explain the persistence of normal rhythms under extreme starvation conditions, where the levels of clock proteins are drastically altered.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The stated goal of this research was to look for interactions between metabolism, (manipulated by glucose starvation) and the circadian clock. This is a hot topic currently, as bi-directional links between metabolism and rhythmicity are found in several organisms and this connection has important implications for human health. The authors work with the model organism Neurospora crassa, a filamentous fungus that has many advantages for this type of research.
The authors' first approach was to assay the effects of glucose starvation on the levels of the RNA and protein products of the key clock genes frq, wc-1, and wc-2. The WC-1 and WC-2 proteins form a complex, WCC, that activates frq transcription. The surprising finding was that WC-1 and WC-2 protein levels and WCC transcriptional activity were drastically …
Reviewer #1 (Public Review):
The stated goal of this research was to look for interactions between metabolism, (manipulated by glucose starvation) and the circadian clock. This is a hot topic currently, as bi-directional links between metabolism and rhythmicity are found in several organisms and this connection has important implications for human health. The authors work with the model organism Neurospora crassa, a filamentous fungus that has many advantages for this type of research.
The authors' first approach was to assay the effects of glucose starvation on the levels of the RNA and protein products of the key clock genes frq, wc-1, and wc-2. The WC-1 and WC-2 proteins form a complex, WCC, that activates frq transcription. The surprising finding was that WC-1 and WC-2 protein levels and WCC transcriptional activity were drastically reduced but frq RNA and protein levels remained the same. Under conditions where rhythmicity is expressed, the rhythms of frq RNA, FRQ protein, and expression of clock-driven "output" genes were also unaffected by starvation. The standard model for the molecular clock is a transcription/translation feedback loop dependent on the levels and activity of these clock gene products, so this disconnect between the starvation-induced changes in the stoichiometry of the loop components and the lack of effects of starvation on rhythmicity calls into question our understanding of the molecular mechanism of the clock. This is yet another example of the inadequacy of the TTFL model to explain rhythmicity. For me, the most significant sentence in the paper was this: "...an unknown mechanism must recalibrate the central clockwork to keep frq transcript levels and oscillation glucose-compensated despite the decline in WCC levels."
The author's second approach was to try to identify mechanisms for the response to starvation by focussing on frq and its regulators, using mutations in the frq gene and strains with alterations in the activity of kinases and phosphatases known to modify FRQ protein. The finding that all of these manipulations have some effect on the starvation-induced changes in WC protein level is taken by the authors to indicate a role for FRQ itself in the response to starvation. This conclusion is subject to the caveat that manipulations of the activity of multifunctional kinases and phosphatases will certainly have pleiotropic effects on many cellular processes beyond FRQ protein activity.
The third section of the paper is a major transcriptomic study of the effects of starvation on global gene expression. Two strains are compared under two conditions: wc wild-type and the wc-1 knockout strain, under fed and starved conditions. The hypothesis is that WCC has a role in the starvation response. The results of starvation on the wild-type are unsurprising and predictable: the expression of many genes involved in metabolic processes is affected. There are no new insights that come from these results and no new testable hypotheses are generated by the data.
The authors refer to the wc-1 mutant strain as "clockless" and discuss its effects on the transcriptome only in terms of WC-1's function in the clock mechanism. However, WCC is known to be a major transcriptional regulator, controlling a number of genes beyond the TTFL. As acknowledged earlier in the paper, WC-1 is also the major light receptor in Neurospora. The transcriptomics experiments were carried out in a light/dark cycle, with cultures harvested at the end of the light period, when "an adapted state for light-dependent genes can be expected" according to the authors. However, wc-1 mutants are essentially blind, and so those samples are equivalent to being harvested in the dark. The multifunctional nature of WCC complicates the interpretation of the transcriptomics data. The differences in the transcriptome between wild-type and wc-1 may not be due to loss of clock function, but rather the loss of a major multifunctional transcription factor, or the difference between light and "dark".
In the final set of experiments, the authors tested the hypothesis that the changes in the transcriptome between wild type and wc-1 might make wc-1 less competent to recover growth after starvation. They also test the recovery of frq9, a "clockless" mutant. The very surprising result is that the growth rates of these two mutants are slower than the wild type after transfer from starvation media to high glucose. This is surprising because there will be several generations of nuclear division and doublings of mass within a few hours and the transcriptome should have recovered fully fairly rapidly. A mechanism for this apparent "after-effect" is suggested with evidence concerning differences in expression of a glucose transporter, but it is not clear why this expression should not change rapidly with re-feeding on high glucose. As with previous experiments, the cultures were grown in light/dark cycles, which results in different conditions for the mutants, both of which have very low or absent WC-1 and are therefore blind to light. The potential effects of light have been disregarded.
The title of the paper refers to a "flexible circadian clock" but this concept of flexibility is not developed in the paper. I would substitute "the White Collar Complex" for this phrase: "Adaptation to starvation requires a functional White Collar Complex in Neurospora crassa" would be more accurate. Some experiments are also conducted using an frq null "clockless" strain, but because WC expression is very low in frq null mutants, any effects of frq null could also be attributed to WC depletion.
The major conclusion I took away from this paper is the multifunctional nature of the WCC as a transcription factor complex. It has been known for a long time that WCC controls the expression of many genes beyond the frq gene at the core of the circadian transcription/translation feedback loop. WC-1 is also the major blue light photoreceptor in Neurospora, controlling the expression of light-regulated genes, and this fact is barely touched on in the paper. These new data now extend the role of WCC in the regulation of metabolic networks as well.
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Reviewer #2 (Public Review):
The authors have performed an interesting study addressing a topical question in considering how circadian oscillators remain accurate in changing environmental conditions and these circadian oscillators contribute to responses to environmental changes. The authors have performed their studies in Neurospora crassa. The authors have made a very interesting finding that starvation causes a profound decrease in white collar 1 WC-1 abundance, yet the circadian system continues to run despite this decrease in the abundance of a core oscillator component. The study of chronic glucose starvation in a Δwc-1 mutant is interesting and provides the opportunity to investigate the role of the WHITE COLLAR COMPLEX (WCC) and the clock system in adaption to starvation.
Strengths:
The authors have used a range of techniques …
Reviewer #2 (Public Review):
The authors have performed an interesting study addressing a topical question in considering how circadian oscillators remain accurate in changing environmental conditions and these circadian oscillators contribute to responses to environmental changes. The authors have performed their studies in Neurospora crassa. The authors have made a very interesting finding that starvation causes a profound decrease in white collar 1 WC-1 abundance, yet the circadian system continues to run despite this decrease in the abundance of a core oscillator component. The study of chronic glucose starvation in a Δwc-1 mutant is interesting and provides the opportunity to investigate the role of the WHITE COLLAR COMPLEX (WCC) and the clock system in adaption to starvation.
Strengths:
The authors have used a range of techniques to measure clock behaviour, including qPCR, phosphorylation, protein abundance, and subcellular localisation studies.
An frq9 mutant was used to test the effects of FRQ on WC1 abundance since WC1 decreased during starvation. This is elegant, though it is not quite clear the logic of this experiment because FRQ did not change abundance during starvation, so why did the author think this experiment was needed?
An interesting experiment was performed to test whether CK1a-dependent phosphorylation and inactivation of the WCC are involved in the starvation response. An FRQΔFCD1-2 mutant is used in which FRQ cannot interact with CK1a and therefore CK1a cannot phosphorylate and inactivate WC. This experiment suggested that CK1a is not involved in the response to starvation, again leading to the conclusion that FRQ is not involved in the starvation regulation of WC.
PKA is shown to be involved in the starvation-induced reduction of WC because the starvation-induced reduction in abundances of WC-1 was absent in the mcb strain in which the regulatory subunit of PKA is defective and hence, PKA is constitutively active.
The authors have found an interesting potential link between glucose levels and WCC phosphorylation, they demonstrated that starvation reduces PP2A activity and that in a regulatory mutant of PP2A, which has reduced PP2A activity, there is little effect of starvation on WCC levels, suggesting the hypothesis that glucose-dependent PP2A dephosphorylation stabilises WCC.
Analysis of starvation-regulated transcriptome in Δwc-1 and wild type found strong evidence that the transcriptomic response to starvation is in part dependent on WCC. Much of the misregulated transcriptome appears to be associated with metabolism.
In a series of growth studies in wild-type frq and wc-1 mutants the authors provide strong evidence that FRQ and WC are involved in growth and survival following starvation, and recovery from starvation.
Weaknesses:
The authors describe Neurospora crassa as a model for circadian biology and apparently make the assumption that the findings are indicative of the behaviour of clock systems in other kingdoms. This is not the case. Neurospora crassa is a wonderful model for studying fungal clocks and is a great tool for studying basic circadian dynamics, but the interesting findings here are of a detailed molecular nature and therefore are applicable for fungal clocks, but not other kingdoms.
The authors assume that the reader is intimate with the intricacies of Neurospora crassa circadian studies and the significance of differences between LL and DD investigations. More background on the logic of the experiments would be helpful for readers from other fields.
The data in Figure 2 are essential for the interpretation of the findings, demonstrating the presence of free-running rhythms. However, the data are entirely qualitative, making it hard to fully assess the authors' interpretations, a more quantitative assessment of the data would improve clarity.
The conclusion that FRQ contributes to the regulation of WC1 abundance in response to starvation does not seem to be supported by the data because FRQ RNA does not change upon starvation. Furthermore, the authors conclude that the starvation-induced decrease in WC-1 and WC-2 protein levels are due to FRQ because a lack of reduction in an frq9 mutant is open to misinterpretation because this mutant makes WC levels low and therefore starvation might not lower already low levels of WC. Indeed WC-1 is lower in the frq9 mutant under any condition than in the WT under starvation and WC-2 does decrease in abundance in the frq9 mutant in starvation. The data strongly suggest to this reader that FRQ does not participate in the regulation of WC abundance in response to starvation.
The discussion accurately summarises the results and provides an interpretation but lacking is a comparison to other circadian systems in other kingdoms. How do the data compare with the effects of glucose and other sugars on the mammalian, plant, and insect clocks?
How changes in WCC might result in changes in transcription is not explained. This might be very obvious to the authors but to the reader, it is not. Are the transcriptional outputs direct targets of WCC? Has WCC CHIPseq been performed by the authors or others, are the regulated transcripts directly bound by WCC? What are the enriched promoter sequences in the regulated genes, is it possible to identify the network by which these changes in transcription occur?
Whilst the authors claim it is the circadian clock that is involved in the starvation response, in my view a more precise interpretation of the data is that WCC is involved in the response. Since WCC is a photoreceptor with dual function in the clock, is it yet possible to conclude that the effects discovered are due to the clock role of WCC? Or do the data support the role of light signalling in regulating the starvation response through WCC?
The authors do not apparently reconcile that the effect of starvation is to hugely decreases WCC levels, but they find the transcriptional and growth response to starvation requires WCC?
This study contributes to the increased focus of the circadian community on the regulation of outputs by circadian oscillators. The manuscript will be of interest to many in the field. There needs to be less assumption of knowledge about the N. Crassa circadian system, and better discussion in a broader context of clocks in other kingdoms.
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