The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation

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1. 

   Version published to 10.7554/elife.85241 on eLife

   Apr 21, 2023
2. 

   eLife

   Mar 2, 2023

   Author Response

   Reviewer #1 (Public Review):

   This is a very interesting paper showing that during amino acid starvation of Neurospora, the general amino acid control factors CPC-1 and CPC-3 are crucial to maintaining circadian rhythm at the levels of rhythmic growth and transcription of the FRQ gene. They show that deleting both genes leads to reduced and arrhythmic cell growth and FRQ transcription that can be accounted for by severely reduced occupancy of the FRQ promoter by the key transcription factor WCC. This defect in turn appears to result from diminished H3 acetylation of the FRQ promoter that was observed at least in the cpc-1 mutant, which is mediated by Gcn5. Thus, they show that Gcn5 occupancy at FRQ is rhythmic and impaired by cpc-1 knock-out, that CPC-1 occupies the FRQ promoter, and provide coIP evidence that Cpc-1 …

   Author Response

   Reviewer #1 (Public Review):

   This is a very interesting paper showing that during amino acid starvation of Neurospora, the general amino acid control factors CPC-1 and CPC-3 are crucial to maintaining circadian rhythm at the levels of rhythmic growth and transcription of the FRQ gene. They show that deleting both genes leads to reduced and arrhythmic cell growth and FRQ transcription that can be accounted for by severely reduced occupancy of the FRQ promoter by the key transcription factor WCC. This defect in turn appears to result from diminished H3 acetylation of the FRQ promoter that was observed at least in the cpc-1 mutant, which is mediated by Gcn5. Thus, they show that Gcn5 occupancy at FRQ is rhythmic and impaired by cpc-1 knock-out, that CPC-1 occupies the FRQ promoter, and provide coIP evidence that Cpc-1 interacts with Gcn5 and Ada2 and, hence, could act directly to recruit these cofactors to the FRQ promoter. Importantly, they show that knock out of GCN5 eliminates rhythmic cell growth and FRQ expression (although surprisingly not FRQ mRNA abundance), as well as reducing H3ac levels and WCC binding at FRQ. They further show that TSA treatment can reverse the effects of histidine starvation on the circadian period in WT cells, and can partially restore rhythmic growth to histidine-starved cpc-3 cells, and that elimination of HDAC Hda1 increases H3ac at FRQ in WT cells. They provide some evidence that transcriptional activation of certain aa biosynthetic genes by CPC-1 is also rhythmic, although the evidence for this is not strong and it's unclear whether CPC-1 occupancy or its activation function would be periodic. They also did not address whether CPC-1 occupancy at FRQ is rhythmic.

   This work is important in providing convincing evidence that CPC-1-mediated induction of transcription factor CPC-3 in starved Neurospora cells mediates CPC-1-mediated recruitment of Gcn5 and acetylation of the FRQ promoter, which counteracts the function of histone deacetylase HDA1 to maintain high occupancy of the transcription factor WCC and attendant circadian rhythm of FRQ transcription. Although the work does not identify new regulatory circuits, such as rhythmic transcription of FRQ, the role of Gcn5, Hda1, and promoter histone acetylation in supporting transcriptional activation, and the general amino acid control response to amino acid starvation are all well-established mechanisms, the work is significant in showing how these pathways and mechanisms are integrated to maintain circadian rhythm in the face of amino acid limitation.

   There is an abundance of convincing experimental evidence provided to support the key claims just summarized above. However, there are a few instances in which additional experiments might be required to resolve a discrepancy in the data or provide stronger evidence to support a claim.

   Thanks for the comments. We have revised the manuscript as suggested.

   Reviewer #2 (Public Review):

   This study by Liu et al. investigates the mechanism that enables the Neurospora circadian clock to maintain robust molecular and physiological rhythms under conditions of nutrient stress. The authors showed that the nutrient-sensing GCN2 signaling pathway is required to maintain robust circadian clock function and output rhythms under amino acid starvation in the filamentous fungus Neurospora. Specifically, they observed that under amino acid starvation conditions, knocking out GCN2 pathway components GCN4 (CPC-1) and GCN2 (CPC-3) severely disrupts rhythmic transcription of core clock gene frequency (frq) and clock-regulated conidiation rhythm. They provided data to indicate that the observed disruptions are due to reduced binding of the White Collar (WC) complex to the frq promoter stemming from lower histone H3 acetylation levels. This prompted the authors to propose a model in which GCN2 (CPC-3) and GCN4 (CPC-1) are activated upon sensing amino acid starvation, recruit GCN-5 containing SAGA acetyltransferase complex to maintain robust histone acetylation rhythm at the frq promoter. They then performed a battery of assays to show that both GCN-5 and ADA-2 are necessary for maintaining robust H3ac, frq mRNA, and conidiation rhythms under normal conditions. To support that low H3ac level at the frq promoter is the cause for impaired WC binding and frq transcription, they demonstrated they can partially rescue the observed rhythm defects of the knockout mutants under amino acid starvation using an HDAC inhibitor. Finally, the authors used RNA-seq to identify genes and pathways that are differentially activated by GCN4 (CPC-1) under amino acid starvation conditions. Many of these genes are involved in amino acid metabolism and they showed that 3 of them exhibit rhythmic expression in WT but low and non-rhythmic expression in the CPC-1 KO strain.

   Strength: The 24-hour period length of the circadian clock is known to be stable over a range of environmental and metabolic conditions because of circadian compensation mechanisms. Whereas temperature compensation (maintenance of circadian period length over a physiological range of temperature) has been studied extensively in multiple model organisms, the phenomenon of nutritional compensation and its underlying mechanisms are poorly understood. This study provides new insights into this important yet understudied area of research in chronobiology. In addition to advancing our understanding of fundamental mechanisms governing clock compensation mechanisms, this study also adds to our understanding of metabolic regulation of rhythmic biology and the relationship between nutrition and healthy biological rhythms. Given that the GCN2 nutrient-sensing pathway is broadly conserved beyond Neurospora, findings from this study will likely be relevant to other eukaryotic systems.

   The authors provided strong evidence supporting their claims that the GCN2 signaling pathway is important for maintaining the robustness of the Neurospora clock under conditions of amino acid starvation. The authors performed parallel experiments in normal (no 3-AT) vs amino acid-starved conditions (+3-AT). Their observations of relatively minor disruptions of molecular and conidiation rhythms in cpc-3 and cpc-1 KO strains in normal nutrient conditions compared to starvation conditions support their model that sensing of amino acid starvation by GCN2 pathway-induced changes at the chromatin and transcriptional level that are necessary to maintain a robust frq oscillator. Without the comparison between normal vs amino acid starved conditions, this part of their model will not be as strong.

   Previously Karki et al. (2020) showed that rhythmic activation of GCN2 kinase is regulated by the clock, resulting in clock-control rhythmic translation initiation. This study uncovers an additional mechanism through which GCN2 pathway modulates circadian rhythms by regulating histone acetylation of rhythmic genes. RNA-seq as described in Figure 7 provides some potential targets.

   Thanks for the comments and suggestions. We have revised the manuscript as suggested.

   Weakness:

   (1) The authors propose a model (Figure 8) in which the GCN2 pathway is ,activated by amino acid starvation and recruits the SAGA complex to promote histone acetylation level at the frq promoter. There is however no data in this study showing that the GCN2 pathway is activated in amino acid-starved conditions, only that it is required to maintain robust frq and conidiation rhythms. The authors should clarify how they are defining "activation of the GCN2 pathway" in this study. For example, is it recruitment of GCN-5 and SAGA complex to frq promoter?

   Thanks for the question. CPC-3, the GCN2 homolog in Neurospora, is the only eIF2α kinase responsible for eIF2α phosphorylation at serine 51(Karki S et al. 2020, PMID: 32355000). As shown in the revised Figure 1-figure supplement 1A, the eIF2α phosphorylation and CPC-1 were induced by 3-AT treatment in the WT but not in the cpc-3KO strain. These results demonstrate that the GCN2 pathway is activated by amino acid starvation, and as a result, the CPC-1 expression is activated to recruit the SAGA complex to the frq promoter.

   (2) The experiments to examine the involvement of GCN-5 and ADA-2 were performed in normal conditions (no amino acid starvation). Unlike cpc-1 and cpc-3 KO strains, gcn-5 and ada-2 KO strains showed severely disrupted frq rhythms in normal nutrient conditions, suggesting they are normally required for robust circadian rhythms. If GCN-5 and the SAGA complex are normally involved in regulating H3ac rhythms in the frq loci, how does GCN2 pathway modulates the activity of GCN-5 and SAGA complex in conditions of amino acid starvation? Are the interactions between GCN2/4 with GCN-5 and SAGA complex different in normal vs amino acid starved conditions? The authors should clarify their model.

   As mentioned above, our data suggested that GCN-5 and ADA-2 are required for robust circadian rhythms under normal conditions. As suggested, we did detect dampened rhythmic expression of frq in the gcn-5KO and ada-2KO strains under amino acid starvation (Figure 5D and 5E and Figure 5–figure supplement 1E and 1F). We also performed Co-IP to compare the difference of interactions between CPC-1 with ADA-2 and GCN5 with ADA-2 under normal and amino acid starved conditions. The results showed that although the Myc.GCN-5, MYC.CPC-1 or Flag.ADA-2 protein level was repressed by 3 mM 3-AT treatment (likely due to global translational inhibition by induced eIF2α phosphorylation) (Karki S et al. 2020, PMID: 32355000), the interactions between CPC-1 with ADA-2 and GCN-5 with ADA-2 were almost the same under normal and amino acid starved conditions (IP was normalized with Input) (Figure 4B and 4C). These results indicated that amino acid starved conditions had little impact on the protein interactions between CPC-1 with GCN-5 and SAGA complex.

   In our model, we proposed that amino acid starvation resulted in compact chromatin structure (due to decreased H3ac) in the frq promoter in the WT strain (Figure 3B), likely due to activation of histone deacetylases or inhibition of histone acetyltransferases. Amino acid starvation activates GCN2 pathway and induces CPC-1 expression. The induced CPC-1 can recruit GCN5-containing SAGA complex to the frq promoter to loosen the chromatin structure, promoting frq rhythmic transcription under starvation conditions. However, in the cpc-3KO mutants, CPC-1 could not effectively recruit GCN5 containing SAGA complex to frq promoter, resulting in arrhythmic frq transcription. We have now clarified our model in the revised discussion.

   (3) Given that the GCN2 pathway is important for nutrient sensing, the authors should not disregard the alternative hypothesis that the GCN2 pathway may be important for nutrient compensation and plays a role in maintaining the robustness of rhythms in a range of nutrient conditions.

   Thanks for the suggestion. We now discussed the alternative hypothesis in the revised manuscript. “Because GCN2 signaling pathway is important for nutrient sensing, it may be important for nutrient compensation and plays a role in maintaining the robustness of rhythms in a range of nutrient conditions”.

   (4) The authors should use circadian statistics to compute the phase and amplitude of the mRNA, DNA binding of the WC complex, and H3Ac rhythms. This will allow them to compare between rhythms and provide statistical significance values, rather than just providing qualitative descriptions. This will be valuable when comparing rhythms between strains and between nutrient conditions.

   As suggested, we used CircaCompare to analyze our data.

   Reviewer #3 (Public Review):

   This is an important paper anchored by the observation that cultures of Neurospora undergoing amino acid starvation lose circadian rhythmicity if orthologs in the classic GCN2/CPC-3 cross-pathway control system are absent. Data convincingly show that Neurospora orthologs of Saccharomyces GCN2 and GCN4 (CPC-3 and CPC-1 respectively) are needed to promote histone acetylation at the core clock gene frequency to facilitate rhythmicity. While the binding of CPC-1 and thereby GCN-5 are plainly rhythmic, the explanation of exactly where rhythmicity enters the pathway is incomplete.

   Figure 1 shows that inhibition of the HIS-3 activity affected by 3-AT, which should trigger cross-pathway control, is correlated with a graded reduction in the amplitude of the rhythm, and eventually to arrhythmicity at 3 mM 3-AT. While normalized data are shown in Figure 1B, raw data should also be provided in the Supplement as sometimes normalization hides aspects of the data. Ideally, this would be on the same scale in wt and in mutant strains.

   We revised as suggested and added the raw data. The results are now shown in Figure 1–figure supplement 2A and 2B and Figure 5–figure supplement 1B and 1C.

   Figure 2. The logical conclusion from Fig 1 is that circadian frq expression driven by the WCC has been impacted by amino acid starvation in the mutants. If so, either WC-1/WC-2 levels might be low, or else they might not be able to bind to DNA. When this was assessed, ChIP assays showed a loss of DNA binding. Although documented, an interesting result is that WCC protein amounts are sharply increased, especially for WC-1. The authors could comment on possible causes for this.

   Line 176, "hypophosphorylation of WC-1 and WC-2 (which is normally associated with WC activation . . . )". While the authors are correct that this is often the case it is not always the case and this introduces a potentially interesting caveat. That is, the overall phosphorylation status of WCC does not always reflect its activity in driving frq transcription. This was first noticed by Zhou et al., (2018 PLOS Genetics) who reported that even though WCC is always hyperphosphorylated in ∆csp-6, the core clock maintains a normal circadian period with only minor amplitude reduction. This should be noted, cited, and discussed.

   Thanks for the suggestion. We revised the manuscript as suggested, “It should be noted that the overall phosphorylation status of WCC does not always reflect its activity in driving frq transcription, possibly due to the unknown function of multiple key phosphosites on WCC (Wang et al., 2019; X. Zhou et al., 2018)”.

   Figure 2 and Figure 2 Suppl. report different gel conditions and show that the sharply increased WC1/WC-2 levels seen in Fig 2 resulting from 3-AT treatment of the cpc pathway mutants are due to the accumulation of hypophosphorylated WC-1/2. The conclusion would be stronger if the gels in the Supplement showed the same degree of difference between wt and mutants as seen in Fig 2. In any case, these hypophosphorylated WC should be active and able to bind DNA but plainly are not based on Fig 2.

   Thanks for the comments. It’s correct that WC-1/WC-2 were hypo-phosphorylated and their protein levels were increased (Figure 2 and Figure 2-figure supplement 1). However, the reduced binding of WC-1/WC-2 at the frq promoter explains for the reduced frq transcription in the cpc-1KO or cpc-3KO mutants under amino acid starvation.

   Figure 3 correlates the unexpected loss of DNA binding by hypophosphorylated WCC with reduced histone H3 acetylation at frq. The 3 mM 3-AT reported to result in arrhythmicity in cpc mutants in Figures 1 and 2 results in a small (~20%?) and not statistically significant reduction in H3 acetylation in wt, compatible with the sustained rhythms seen in wt in Figure 1, but in a substantial (~5 fold) loss of binding in the ∆cpc-1 background; so CPC-1 is needed for H3 acetylation at frq to sustain the rhythm during amino acid starvation. The simplest explanation here then is that the hypophosphorylated WCC cannot bind to DNA because the chromatin is closed due to decreased AcH3.

   Thanks for the comments.

   Figure 4. Title:" Figure 4. CPC-1 recruits GCN-5 to activate frq transcription in response to amino acid starvation"; the conditions of amino acid starvation should be mentioned here for the reader's benefit. (In the unlikely case that there was no amino acid starvation here then many things about the manuscript need to be reconsidered.)

   Based on the model from yeast where amino acid starvation activates GCN2 (aka CPC-3 in Neurospora) kinase which activates the transcriptional activator GCN4 (aka CPC-1) which recruits the SAGA complex containing the histone acetylase GCN5 to regulated promoters, CPC-1 was tagged and shown by ChIP to bind rhythmically at frq. Co-IP experiments establish the interaction of components of the SAGA complex in Neurospora and Neurospora GCN-5 indeed is bound to frq, likely recruited by CPC-1. This part all follows the Saccharomyces model with the interesting twist that the binding CPC-1 is weakly rhythmic and GCN-5 strongly rhythmic in a CPC-1-dependent manner. Based on the figure legend title, these cultures should always be starved for amino acids (although as noted this should be made explicit in the figure legend). In any case, given this, from where does the rhythmicity in GCN-5-binding arise? This question is developed more below.

   Line 224, "low in the cpc-1KO strain, suggesting that CPC-1 rhythmically recruit GCN-5". Because ChIP was done only for a half circadian cycle (DD10-22), it is hard to conclude "rhythmically". The statement should be modified.

   To address the concern, we performed the ChIP assay using the CPC-1 antibody instead of Myc antibody (revised Figure 4A). Analysis of the ChIP results with CircaCompare showed that CPC-1 binding at the frq promoter was rhythmic without 3-AT (Figure 4A) or with 3 mM 3-AT treatment (Figure 4-figure supplement 1A). Due to the ADA-2-GCN5 and CPC-1-ADA-2 interactions with/without 3-AT treatment (Revised Figure 4B-C), CPC-1 should be able to recruit GCN-5-containing SAGA complex to activate frq transcription in response to amino acid starvation. We have now clarified this model in the revised manuscript. Please also see response to Reviewer 2/point 5.

   It was previously reported that the CPC-3/CPC-1 signaling pathway was rhythmically controlled by circadian clock, as indicated by CPC-3-mediated rhythmic eIF2α phosphorylation at serine 51 (Karki S et al. 2020, PMID: 32355000). Our data showed rhythmic CPC-1 and GCN-5 binding at the frq promoter in the WT strain and decreased GCN-5 binding in the cpc-1KO mutant (Figure 4A and 4D). These results suggested that the circadian clock controlled the CPC-3/CPC-1 signaling pathway rhythmically, which in turn promoted the rhythmic frq transcription through recruiting GCN5 containing SAGA complex under amino acid starvation. We clarified the model and description in the discussion.

   As suggested by the reviewer, we modified the statement "suggesting that CPC-1 recruits GCN-5-containing SAGA complex to the frq promoter".

   Figure 5 shows that rhythmicity in general and of frq/FRQ specifically requires GCN-5 even under conditions of normal amino acid sufficiency, and that normal levels of H3 acetylation and its rhythm at frq require GCN-5. Not surprisingly, high H3 acetylation at frq correlated with high WC-2 DNA binding, and ADA-2 is required for SAGA functions.

   As earlier, raw bioluminescence data corresponding to panel B should be provided in the figure or Supplement.

   Also, if CPC-3 and CPC-1 regulate frq transcription through GCN-5, why is the frq level extremely low in the cpc-3KO or cpc-1KO(Fig.1D) but remains normal in gcn-5KO (Fig. 5D)?

   Raw bioluminescence data are listed in Figure 5–figure supplement 1B and 1C. For frq transcription in the WT and gcn-5KO mutant, please see response to Essential Revisions point 4.

   Figure 6 documents the counter effects of TSA which inhibits histone deacetylation and shortens the period versus 3-AT which decreases (via CPC-3 to CPC-1 to GCN-5) histone acetylation and causes period lengthening or arrhythmicity. HDA-1 is necessary for histone deacetylation at frq.

   Thanks for the comments.

   Figure 7 documents extensive changes in gene expression associated with 3-AT-induced amino acid starvation and the CPC-3 to CPC-1 pathway. How do these results compare with other previously studied systems, particularly Saccharomyces, where similar experiments have been done? Are the same genes regulated to the same extent or are there some interesting differences?

   Thanks for the suggestion. We revised our manuscript by comparing the difference of these genes in Saccharomyces. GCN4/CPC-1 targets are similar. “Similar to Saccharomyces cerevisiae (Natarajan et al., 2001), genes in amino acid biosynthetic pathways, vitamin biosynthetic enzymes, peroxisomal components, and mitochondrial carrier proteins were also identified as CPC-1 targets”.

   Figure 8 provides a model consistent with the role of the CPC-3/GCN2 pathway in regulating genes in response to amino acid starvation. It seems this could be any gene responding to amino acid starvation.

   Not accounted for in the model is the data from Fig 4 which show the rhythmic binding of CPC-1 and stronger rhythmic binding of GCN-5 to frq, both under amino acid starvation. In the presence of 3-AT, amino acid starvation is constant, which should mean that CPC-3 and CPC-1 would always be "on". Why doesn't CPC-1 recruit GCN5 at the same level at all times leading to constant high H3 acetylation rather than rhythmic H3 acetylation as seen in Figure 3? Perhaps, unlike the statement in lines 345-34, it is WCC that regulates rhythmic GCN-5 binding and facilitates rhythmic histone acetylation at frq. Or perhaps the clock introduces rhythmicity upstream from GCN5. Without an answer to the question of where rhythmicity comes into the pathway, the story is only about how the CPC-3/GCN2 pathway in regulating genes in response to amino acid starvation; without explaining the rhythmicity the story seems incomplete.

   It was previously reported that the CPC-3/CPC-1 signaling pathway was rhythmically controlled by circadian clock, as indicated by CPC-3-mediated rhythmic eIF2α phosphorylation at serine 51 (Karki S et al. 2020, PMID: 32355000). Our data showed rhythmic CPC-1 and GCN-5 binding at the frq promoter in the WT strain and decreased GCN-5 binding in the cpc-1KO mutant (Figure 4A and 4D). These results suggested that the circadian clock controlled the CPC-3/CPC-1 signaling pathway rhythmically, which in turn promoted the rhythmic frq transcription through recruiting GCN5 containing SAGA complex under amino acid starvation. We clarified the model and description in the discussion.

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3. 

   eLife

   Jan 9, 2023

   eLife assessment

   This important study provides evidence for CPC-3 mediating induction of the transcription factor CPC-1 in starved Neurospora cells, with CPC-1-mediated recruitment of Gcn5 and acetylation of the FRQ promoter counteracting the function of histone deacetylase HDA1, which in turn maintains high occupancy of the transcription factor WCC and attendant circadian rhythm of FRQ expression. The findings are significant in showing how the well-established pathways for circadian rhythm centered on FRQ gene expression and cross-pathway control centered on CPC-1 induction are integrated to maintain rhythmic cell growth in the face of amino acid limitation. However, the evidence for these claims is incomplete in certain respects and additional statistical analyses and experimental evidence are needed to better support the claims of …

   eLife assessment

   This important study provides evidence for CPC-3 mediating induction of the transcription factor CPC-1 in starved Neurospora cells, with CPC-1-mediated recruitment of Gcn5 and acetylation of the FRQ promoter counteracting the function of histone deacetylase HDA1, which in turn maintains high occupancy of the transcription factor WCC and attendant circadian rhythm of FRQ expression. The findings are significant in showing how the well-established pathways for circadian rhythm centered on FRQ gene expression and cross-pathway control centered on CPC-1 induction are integrated to maintain rhythmic cell growth in the face of amino acid limitation. However, the evidence for these claims is incomplete in certain respects and additional statistical analyses and experimental evidence are needed to better support the claims of rhythmic CPC-1 binding at FRQ, of the role of GCN-5 in rhythmic FRQ transcription in starvation conditions, and of rhythmic transcription of CPC-1-regulated amino acid biosynthetic genes.

   Read the original source
4. 

   eLife

   Jan 9, 2023

   Reviewer #1 (Public Review):

   This is a very interesting paper showing that during amino acid starvation of Neurospora, the general amino acid control factors CPC-1 and CPC-3 are crucial to maintaining circadian rhythm at the levels of rhythmic growth and transcription of the FRQ gene. They show that deleting both genes leads to reduced and arrhythmic cell growth and FRQ transcription that can be accounted for by severely reduced occupancy of the FRQ promoter by the key transcription factor WCC. This defect in turn appears to result from diminished H3 acetylation of the FRQ promoter that was observed at least in the cpc-1 mutant, which is mediated by Gcn5. Thus, they show that Gcn5 occupancy at FRQ is rhythmic and impaired by cpc-1 knock-out, that CPC-1 occupies the FRQ promoter, and provide coIP evidence that Cpc-1 interacts with Gcn5 …

   Reviewer #1 (Public Review):

   This is a very interesting paper showing that during amino acid starvation of Neurospora, the general amino acid control factors CPC-1 and CPC-3 are crucial to maintaining circadian rhythm at the levels of rhythmic growth and transcription of the FRQ gene. They show that deleting both genes leads to reduced and arrhythmic cell growth and FRQ transcription that can be accounted for by severely reduced occupancy of the FRQ promoter by the key transcription factor WCC. This defect in turn appears to result from diminished H3 acetylation of the FRQ promoter that was observed at least in the cpc-1 mutant, which is mediated by Gcn5. Thus, they show that Gcn5 occupancy at FRQ is rhythmic and impaired by cpc-1 knock-out, that CPC-1 occupies the FRQ promoter, and provide coIP evidence that Cpc-1 interacts with Gcn5 and Ada2 and, hence, could act directly to recruit these cofactors to the FRQ promoter. Importantly, they show that knock out of GCN5 eliminates rhythmic cell growth and FRQ expression (although surprisingly not FRQ mRNA abundance), as well as reducing H3ac levels and WCC binding at FRQ. They further show that TSA treatment can reverse the effects of histidine starvation on the circadian period in WT cells, and can partially restore rhythmic growth to histidine-starved cpc-3 cells, and that elimination of HDAC Hda1 increases H3ac at FRQ in WT cells. They provide some evidence that transcriptional activation of certain aa biosynthetic genes by CPC-1 is also rhythmic, although the evidence for this is not strong and it's unclear whether CPC-1 occupancy or its activation function would be periodic. They also did not address whether CPC-1 occupancy at FRQ is rhythmic.

   This work is important in providing convincing evidence that CPC-1-mediated induction of transcription factor CPC-3 in starved Neurospora cells mediates CPC-1-mediated recruitment of Gcn5 and acetylation of the FRQ promoter, which counteracts the function of histone deacetylase HDA1 to maintain high occupancy of the transcription factor WCC and attendant circadian rhythm of FRQ transcription. Although the work does not identify new regulatory circuits, such as rhythmic transcription of FRQ, the role of Gcn5, Hda1, and promoter histone acetylation in supporting transcriptional activation, and the general amino acid control response to amino acid starvation are all well-established mechanisms, the work is significant in showing how these pathways and mechanisms are integrated to maintain circadian rhythm in the face of amino acid limitation.

   There is an abundance of convincing experimental evidence provided to support the key claims just summarized above. However, there are a few instances in which additional experiments might be required to resolve a discrepancy in the data or provide stronger evidence to support a claim.

   Read the original source
5. 

   eLife

   Jan 9, 2023

   Reviewer #2 (Public Review):

   This study by Liu et al. investigates the mechanism that enables the Neurospora circadian clock to maintain robust molecular and physiological rhythms under conditions of nutrient stress. The authors showed that the nutrient-sensing GCN2 signaling pathway is required to maintain robust circadian clock function and output rhythms under amino acid starvation in the filamentous fungus Neurospora. Specifically, they observed that under amino acid starvation conditions, knocking out GCN2 pathway components GCN4 (CPC-1) and GCN2 (CPC-3) severely disrupts rhythmic transcription of core clock gene frequency (frq) and clock-regulated conidiation rhythm. They provided data to indicate that the observed disruptions are due to reduced binding of the White Collar (WC) complex to the frq promoter stemming from lower …

   Reviewer #2 (Public Review):

   This study by Liu et al. investigates the mechanism that enables the Neurospora circadian clock to maintain robust molecular and physiological rhythms under conditions of nutrient stress. The authors showed that the nutrient-sensing GCN2 signaling pathway is required to maintain robust circadian clock function and output rhythms under amino acid starvation in the filamentous fungus Neurospora. Specifically, they observed that under amino acid starvation conditions, knocking out GCN2 pathway components GCN4 (CPC-1) and GCN2 (CPC-3) severely disrupts rhythmic transcription of core clock gene frequency (frq) and clock-regulated conidiation rhythm. They provided data to indicate that the observed disruptions are due to reduced binding of the White Collar (WC) complex to the frq promoter stemming from lower histone H3 acetylation levels. This prompted the authors to propose a model in which GCN2 (CPC-3) and GCN4 (CPC-1) are activated upon sensing amino acid starvation, recruit GCN-5 containing SAGA acetyltransferase complex to maintain robust histone acetylation rhythm at the frq promoter. They then performed a battery of assays to show that both GCN-5 and ADA-2 are necessary for maintaining robust H3ac, frq mRNA, and conidiation rhythms under normal conditions. To support that low H3ac level at the frq promoter is the cause for impaired WC binding and frq transcription, they demonstrated they can partially rescue the observed rhythm defects of the knockout mutants under amino acid starvation using an HDAC inhibitor. Finally, the authors used RNA-seq to identify genes and pathways that are differentially activated by GCN4 (CPC-1) under amino acid starvation conditions. Many of these genes are involved in amino acid metabolism and they showed that 3 of them exhibit rhythmic expression in WT but low and non-rhythmic expression in the CPC-1 KO strain.

   Strength: The 24-hour period length of the circadian clock is known to be stable over a range of environmental and metabolic conditions because of circadian compensation mechanisms. Whereas temperature compensation (maintenance of circadian period length over a physiological range of temperature) has been studied extensively in multiple model organisms, the phenomenon of nutritional compensation and its underlying mechanisms are poorly understood. This study provides new insights into this important yet understudied area of research in chronobiology. In addition to advancing our understanding of fundamental mechanisms governing clock compensation mechanisms, this study also adds to our understanding of metabolic regulation of rhythmic biology and the relationship between nutrition and healthy biological rhythms. Given that the GCN2 nutrient-sensing pathway is broadly conserved beyond Neurospora, findings from this study will likely be relevant to other eukaryotic systems.

   The authors provided strong evidence supporting their claims that the GCN2 signaling pathway is important for maintaining the robustness of the Neurospora clock under conditions of amino acid starvation. The authors performed parallel experiments in normal (no 3-AT) vs amino acid-starved conditions (+3-AT). Their observations of relatively minor disruptions of molecular and conidiation rhythms in cpc-3 and cpc-1 KO strains in normal nutrient conditions compared to starvation conditions support their model that sensing of amino acid starvation by GCN2 pathway-induced changes at the chromatin and transcriptional level that are necessary to maintain a robust frq oscillator. Without the comparison between normal vs amino acid starved conditions, this part of their model will not be as strong.

   Previously Karki et al. (2020) showed that rhythmic activation of GCN2 kinase is regulated by the clock, resulting in clock-control rhythmic translation initiation. This study uncovers an additional mechanism through which GCN2 pathway modulates circadian rhythms by regulating histone acetylation of rhythmic genes. RNA-seq as described in Figure 7 provides some potential targets.

   Weakness:
   (1) The authors propose a model (Figure 8) in which the GCN2 pathway is activated by amino acid starvation and recruits the SAGA complex to promote histone acetylation level at the frq promoter. There is however no data in this study showing that the GCN2 pathway is activated in amino acid-starved conditions, only that it is required to maintain robust frq and conidiation rhythms. The authors should clarify how they are defining "activation of the GCN2 pathway" in this study. For example, is it recruitment of GCN-5 and SAGA complex to frq promoter?

   (2) The experiments to examine the involvement of GCN-5 and ADA-2 were performed in normal conditions (no amino acid starvation). Unlike cpc-1 and cpc-3 KO strains, gcn-5 and ada-2 KO strains showed severely disrupted frq rhythms in normal nutrient conditions, suggesting they are normally required for robust circadian rhythms. If GCN-5 and the SAGA complex are normally involved in regulating H3ac rhythms in the frq loci, how does GCN2 pathway modulates the activity of GCN-5 and SAGA complex in conditions of amino acid starvation? Are the interactions between GCN2/4 with GCN-5 and SAGA complex different in normal vs amino acid starved conditions? The authors should clarify their model.

   (3) Given that the GCN2 pathway is important for nutrient sensing, the authors should not disregard the alternative hypothesis that the GCN2 pathway may be important for nutrient compensation and plays a role in maintaining the robustness of rhythms in a range of nutrient conditions.

   (4) The authors should use circadian statistics to compute the phase and amplitude of the mRNA, DNA binding of the WC complex, and H3Ac rhythms. This will allow them to compare between rhythms and provide statistical significance values, rather than just providing qualitative descriptions. This will be valuable when comparing rhythms between strains and between nutrient conditions.

   Read the original source
6. 

   eLife

   Jan 9, 2023

   Reviewer #3 (Public Review):

   This is an important paper anchored by the observation that cultures of Neurospora undergoing amino acid starvation lose circadian rhythmicity if orthologs in the classic GCN2/CPC-3 cross-pathway control system are absent. Data convincingly show that Neurospora orthologs of Saccharomyces GCN2 and GCN4 (CPC-3 and CPC-1 respectively) are needed to promote histone acetylation at the core clock gene frequency to facilitate rhythmicity. While the binding of CPC-1 and thereby GCN-5 are plainly rhythmic, the explanation of exactly where rhythmicity enters the pathway is incomplete.

   Figure 1 shows that inhibition of the HIS-3 activity affected by 3-AT, which should trigger cross-pathway control, is correlated with a graded reduction in the amplitude of the rhythm, and eventually to arrhythmicity at 3 mM 3-AT. While …

   Reviewer #3 (Public Review):

   This is an important paper anchored by the observation that cultures of Neurospora undergoing amino acid starvation lose circadian rhythmicity if orthologs in the classic GCN2/CPC-3 cross-pathway control system are absent. Data convincingly show that Neurospora orthologs of Saccharomyces GCN2 and GCN4 (CPC-3 and CPC-1 respectively) are needed to promote histone acetylation at the core clock gene frequency to facilitate rhythmicity. While the binding of CPC-1 and thereby GCN-5 are plainly rhythmic, the explanation of exactly where rhythmicity enters the pathway is incomplete.

   Figure 1 shows that inhibition of the HIS-3 activity affected by 3-AT, which should trigger cross-pathway control, is correlated with a graded reduction in the amplitude of the rhythm, and eventually to arrhythmicity at 3 mM 3-AT. While normalized data are shown in Figure 1B, raw data should also be provided in the Supplement as sometimes normalization hides aspects of the data. Ideally, this would be on the same scale in wt and in mutant strains.

   Figure 2. The logical conclusion from Fig 1 is that circadian frq expression driven by the WCC has been impacted by amino acid starvation in the mutants. If so, either WC-1/WC-2 levels might be low, or else they might not be able to bind to DNA. When this was assessed, ChIP assays showed a loss of DNA binding. Although documented, an interesting result is that WCC protein amounts are sharply increased, especially for WC-1. The authors could comment on possible causes for this.

   Line 176, "hypophosphorylation of WC-1 and WC-2 (which is normally associated with WC activation . . . )". While the authors are correct that this is often the case it is not always the case and this introduces a potentially interesting caveat. That is, the overall phosphorylation status of WCC does not always reflect its activity in driving frq transcription. This was first noticed by Zhou et al., (2018 PLOS Genetics) who reported that even though WCC is always hyperphosphorylated in ∆csp-6, the core clock maintains a normal circadian period with only minor amplitude reduction. This should be noted, cited, and discussed.

   Figure 2 and Figure 2 Suppl. report different gel conditions and show that the sharply increased WC1/WC-2 levels seen in Fig 2 resulting from 3-AT treatment of the cpc pathway mutants are due to the accumulation of hypophosphorylated WC-1/2. The conclusion would be stronger if the gels in the Supplement showed the same degree of difference between wt and mutants as seen in Fig 2. In any case, these hypophosphorylated WC should be active and able to bind DNA but plainly are not based on Fig 2.

   Figure 3 correlates the unexpected loss of DNA binding by hypophosphorylated WCC with reduced histone H3 acetylation at frq. The 3 mM 3-AT reported to result in arrhythmicity in cpc mutants in Figures 1 and 2 results in a small (~20%?) and not statistically significant reduction in H3 acetylation in wt, compatible with the sustained rhythms seen in wt in Figure 1, but in a substantial (~5 fold) loss of binding in the ∆cpc-1 background; so CPC-1 is needed for H3 acetylation at frq to sustain the rhythm during amino acid starvation. The simplest explanation here then is that the hypophosphorylated WCC cannot bind to DNA because the chromatin is closed due to decreased AcH3.

   Figure 4. Title:" Figure 4. CPC-1 recruits GCN-5 to activate frq transcription in response to amino acid starvation"; the conditions of amino acid starvation should be mentioned here for the reader's benefit. (In the unlikely case that there was no amino acid starvation here then many things about the manuscript need to be reconsidered.)
   Based on the model from yeast where amino acid starvation activates GCN2 (aka CPC-3 in Neurospora) kinase which activates the transcriptional activator GCN4 (aka CPC-1) which recruits the SAGA complex containing the histone acetylase GCN5 to regulated promoters, CPC-1 was tagged and shown by ChIP to bind rhythmically at frq. Co-IP experiments establish the interaction of components of the SAGA complex in Neurospora and Neurospora GCN-5 indeed is bound to frq, likely recruited by CPC-1. This part all follows the Saccharomyces model with the interesting twist that the binding CPC-1 is weakly rhythmic and GCN-5 strongly rhythmic in a CPC-1-dependent manner. Based on the figure legend title, these cultures should always be starved for amino acids (although as noted this should be made explicit in the figure legend). In any case, given this, from where does the rhythmicity in GCN-5-binding arise? This question is developed more below.
   Line 224, "low in the cpc-1KO strain, suggesting that CPC-1 rhythmically recruit GCN-5".
   Because ChIP was done only for a half circadian cycle (DD10-22), it is hard to conclude "rhythmically". The statement should be modified.

   Figure 5 shows that rhythmicity in general and of frq/FRQ specifically requires GCN-5 even under conditions of normal amino acid sufficiency, and that normal levels of H3 acetylation and its rhythm at frq require GCN-5. Not surprisingly, high H3 acetylation at frq correlated with high WC-2 DNA binding, and ADA-2 is required for SAGA functions.
   As earlier, raw bioluminescence data corresponding to panel B should be provided in the figure or Supplement.
   Also, if CPC-3 and CPC-1 regulate frq transcription through GCN-5, why is the frq level extremely low in the cpc-3KO or cpc-1KO(Fig.1D) but remains normal in gcn-5KO (Fig. 5D)?

   Figure 6 documents the counter effects of TSA which inhibits histone deacetylation and shortens the period versus 3-AT which decreases (via CPC-3 to CPC-1 to GCN-5) histone acetylation and causes period lengthening or arrhythmicity. HDA-1 is necessary for histone deacetylation at frq.

   Figure 7 documents extensive changes in gene expression associated with 3-AT-induced amino acid starvation and the CPC-3 to CPC-1 pathway. How do these results compare with other previously studied systems, particularly Saccharomyces, where similar experiments have been done? Are the same genes regulated to the same extent or are there some interesting differences?

   Figure 8 provides a model consistent with the role of the CPC-3/GCN2 pathway in regulating genes in response to amino acid starvation. It seems this could be any gene responding to amino acid starvation.
   Not accounted for in the model is the data from Fig 4 which show the rhythmic binding of CPC-1 and stronger rhythmic binding of GCN-5 to frq, both under amino acid starvation. In the presence of 3-AT, amino acid starvation is constant, which should mean that CPC-3 and CPC-1 would always be "on". Why doesn't CPC-1 recruit GCN5 at the same level at all times leading to constant high H3 acetylation rather than rhythmic H3 acetylation as seen in Figure 3? Perhaps, unlike the statement in lines 345-34, it is WCC that regulates rhythmic GCN-5 binding and facilitates rhythmic histone acetylation at frq. Or perhaps the clock introduces rhythmicity upstream from GCN5. Without an answer to the question of where rhythmicity comes into the pathway, the story is only about how the CPC-3/GCN2 pathway in regulating genes in response to amino acid starvation; without explaining the rhythmicity the story seems incomplete.

   Read the original source
7. 

   Version published to 10.1101/2022.12.08.519602v1 on bioRxiv

   Dec 12, 2022