Neurospora. The deletion of GCN2 pathway components disrupts rhythmic transcription of clock gene frq by suppressing WC complex binding at the frq promoter due to its reduced histone H3 acetylation levels. Under amino acid starvation, the activation of GCN2 kinase and its downstream transcription factor CPC-1 establish a proper chromatin state at the frq promoter by recruiting the histone acetyltransferase GCN-5. The arrhythmic phenotype of the GCN2 kinase mutants under amino acid starvation can be rescued by inhibiting histone deacetylation. Finally, genome-wide transcriptional analysis indicates that the GCN2 signaling pathway maintains robust rhythmic expression of metabolic genes under amino acid starvation. Together, these results uncover an essential role of the GCN2 signaling pathway in maintaining the robust circadian clock function in response to amino acid starvation, and demonstrate the importance of histone acetylation at the frq locus in rhythmic gene expression.Circadian clocks are evolved to adapt to the daily environmental changes under different conditions. The ability to maintain circadian clock functions in response to various stresses and perturbations is important for organismal fitness. Here, we show that the nutrient-sensing GCN2 signaling pathway is required for robust circadian clock function under amino acid starvation in  Neurospora and the ATM signaling pathway in mammalian cells  in urospora . Generald sensor . GCN2 isd sensor . Activatd sensor . Meanwhipathways . Recentlurospora . AlthougNeurospora crassa has been established as one of the best studied model systems for analyzing the molecular mechanism of eukaryotic circadian clocks  and WC-2 form a complex (WCC) and bind to the C-box of the frq promoter to activate its transcription , and the Clock ATPase (CATP) (frq transcription by regulating rhythmic histone compositions and modifications at the frq locus (Chromatin structure and histone modification changes play important roles in regulating the transcription of circadian clock genes . In mamme (CATP) . Histonerq locus . Howeverfrq promoter by decreasing its histone H3 acetylation levels. Amino acid starvation activated CPC-3 and CPC-1, which re-established a proper chromatin state at the frq promoter by recruiting the histone acetyltransferase GCN-5 to allow rhythmic frq expression. Furthermore, the inhibition of histone deacetylases could rescue the impaired circadian rhythm phenotypes under amino acid starvation, demonstrating the importance of rhythmic histone acetylation at the frq gene promoter for maintaining robust circadian rhythms of gene expression.In this study, we discovered that the disruption of the GCN2 (CPC-3) signaling pathway abolished robust circadian rhythms under amino acid starvation, which was important for rhythmic expression of metabolic genes. In the GCN2 signaling pathway mutants, amino acid starvation abolished the rhythmic binding of WC-2 at the cpc-3 and cpc-1 knockout mutants . In Neurospora, cpc-3 and cpc-1 encode for the GCN2 and GCN4 homolog, respectively. As expected, the CPC-3-mediated eIF2α phosphorylation and CPC-1 induction by 3-aminotriazole (3-AT) treatment were completely abolished in the KOcpc-3 strain , cpc-3 deletion had no effect on the circadian period but cpc-1 deletion led to the period lengthening of ~1.7 hr . Although addition of the same amount of histidine could rescue the growth of the KO his-3−cpc-3 strain, it could not rescue its circadian conidiation rhythm , n rhythm , indicatfrq promoter-driven luciferase reporter into the KOcpc-3 and KOcpc-1 strains. As shown in KOcpc-3 and KOcpc-1 strains upon 3 mM 3-AT treatment. Consistent with these results, western blot analysis showed that, after the initial light/dark transition, rhythms of FRQ levels and its phosphorylation were dampened in the KOcpc-3 and KOcpc-1 strains in the presence of 3-AT   and the levels of frq mRNA were constantly low in DD  . The sta 0.0004)  . However, 3-AT treatment resulted in constant low levels of WC-2 binding to the frq C-box during a circadian cycle in both KOcpc-3 and KOcpc-1 strains   . These rfrq locus, we examined the occupancy of CPC-1 at the frq promoter by ChIP assays using CPC-1 antibody. As shown in frq promoter in the WT strain but not in the KOcpc-1 strain under normal  or amino acid starvation (WT: p = 7.64E−05) conditions in DD, peaking at ~DD14, a time point corresponding to the peak of frq mRNA levels. Co-immunoprecipitation (Co-IP) assay showed that CPC-1 did not associate with WC-1 or WC-2   SAGA complex to selective gene promoters, likely through its physical interaction with the ADA2 subunit . To testylation) , the intylation) , suggestyc.GCN-5 . These rfrq promoter, we performed ChIP assay to examine the occupancy of GCN-5 at the frq promoter. As shown in frq promoter in DD in the WT strain but its binding was constantly low and arrhythmic in the KOcpc-1 strain , suggesting that CPC-1 recruits the GCN-5 containing SAGA complex to the frq promoter to allow rhythmic histone acetylation levels, which maintain rhythmic WC-2 binding and thus rhythmic frq transcription.To further confirm if CPC-1 can recruit GCN5 to the Neurospora   or with 3 mM 3-AT   . Reverse > 0.05) . Togethefrq promoter, and their rhythmic occupancies were severely dampened in the KOgcn-5 strain compared with the WT strain   (frq promoter to drive rhythmic frq transcription.ChIP assay results showed that the H3ac levels were significantly decreased at the  > 0.05) . Further 0.0459) . These rNeurospora ada-2 knockout mutant and examined the function of ADA-2 in the circadian clock. As expected, we found that the circadian phenotypes and frq expression of the KOada-2 strain were very similar to those of the KOgcn-5 strain  is a histone deacetylase (HDACs) inhibitor, which can inhibit HDACs activity and increase the histone acetylation levels in Neurospora . As shown in KOcpc-1 strain after 3-AT treatment, suggesting that these genes were regulated by CPC-1 under amino acid starvation (Saccharomyces cerevisiae (his-3 (NCU03139), arg-1 (NCU02639), trp-3 (NCU08409), and ser-2 (NCU01439) were upregulated in the WT strain, but unchanged in the KOcpc-1 strain under amino acid starvation , trp-3 (NCU08409), and ser-2 (NCU01439) genes (WT (0 mM 3-AT): p > 0.05, WT (3 mM 3-AT): p > 0.05, KOcpc-1 (0 mM 3-AT): p > 0.05), and the enrichment was enhanced by 3-AT treatment. Because the CPC-1 binding was not rhythmic, we performed RT-qPCR experiments to re-analyze whether those genes were rhythmically transcribed in DD. Consistent with the published RNA-seq analysis results, we found that frq and the amino acid synthetic genes such as his-3 (NCU03139) , arg-1 (NCU02639) , and trp-3 (NCU08409)  were rhythmically expressed in the WT but not KOcpc-1 strain under amino acid starvation (ser-2 (NCU01439)  exhibited rhythmic expression in the WT but not in the KOcpc-1 strain under amino acid starvation (arg-1 (NCU02639) (WT: p = 0.0353), trp-3 (NCU08409) (WT: p = 0.0436), and ser-2 (NCU01439) (WT: p = 0.0008) genes, but not the his-3 (NCU03139) (WT: p > 0.05) gene, were rhythmic in the WT strain in DD  in the frq promoter in the WT strain  and 301-6  strain was used as the wild-type (WT) strain in this study. Knockout mutants were all generated based on the bdras-1 background (KOcpc-3 (NCU01187), KOcpc-1 (NCU04050), KOgcn-5 (NCU10847), KOada-2 (NCU04459), and KOhda-1 (NCU01525) strains were obtained from the Fungal Genetic Stock Center (FGSC) and were crossed with a bdras-1 strain to create the bd;cpc-3KOras-1, bd;cpc-1KOras-1, bd;gcn-5KOras-1, bd;ada-2KOras-1, and bd;hda-1KOras-1 strains (cpc-1 promoter driving expression of Myc.CPC-1 were introduced into the KOcpc-1 strains at the his-3 (NCU03139) locus by homologous recombination. Constructs with the cfp promoter driving expression of Flag.ADA-2 were introduced into the 301-6, cfp-Myc.CPC-1 or 301-6, cfp-Myc.GCN-5 strains by random insertion with nourseothricin selection . For rhythmic experiments, Neurospora strain could be calculated according to the ratio between the distance of marked conidia band positions and the distance of conidiation bands.The medium for race tube assay contained 1× Vogel’s salts, 0.1% glucose, 0.17% arginine, 50 ng/ml biotin, and 1.5% agar. After entrainment of 24 hr in the constant light condition, race tubes were transferred to constant darkness conditions and marked every 24 hr. The circadian period of the frq-luc) containing the luciferase gene under the control of the frq promoter, was introduced into 301-6, KOcpc-3 or KOcpc-1 strains by transformation. The luciferase reporter construct (FRQ-LUC) containing a luciferase fused to the C terminus of the FRQ protein, was introduced into KOgcn-5 or KOada-2 strains by crossing. Firefly luciferin  was added to autoclaved FGS-Vogel’s medium containing 1× FGS , 1× Vogel’s medium, 50 μg/l biotin, and 1.8% agar. Conidia suspension was placed on autoclaved FGS-Vogel’s medium and grown in constant light overnight. The cultures were then transferred to constant darkness, and luminescence was recorded in real time using a LumiCycle after 1 day in DD. The data were then normalized with LumiCycle analysis software by subtracting the baseline luciferase signal, which increases as cell grows.The luciferase reporter assay was performed as reported previously . The lucProtein extraction, quantification, and western blot analyses were performed as previously described . Briefly4P2O7·10H2O, 2 mM Na3VO4, 1 mM ethylenediaminetetraacetic acid EDTA) were made fresh and added to the protein extraction buffer. Proteins were loaded in each protein lane of 7.5% SDS–PAGE gels containing a ratio of 149:1 acrylamide/bisacrylamide.To detect the phosphorylation levels of WC-1 and WC-2, PPase inhibitors -labeled RNA probes specific for frq. RNA probes were transcribed in vitro from PCR products by T7 RNA polymerase  with the manufacturer’s protocol. The frq primers used for the template amplification are shown in Key Resources Table.RNA was extracted with Trizol and further purified with 2.5 M LiCl as described previously . For NorKOcpc-1 strains were collected at the indicated time points in constant darkness in liquid growth medium . RT-qPCR were performed as previously described (frq (NCU02265), his-3 (NCU03139), ser-2 (NCU01439), trp-3 (NCU08409), his-4 (NCU06974), arg-1 (NCU02639), and arg-10 (NCU08162) were designed, and the β-tubulin (NCU04054) was used as an internal control. The primers used for RT-qPCR are shown in Key Resources Table.For RT-qPCR, the cultures of WT and escribed . Each RNTwo CPC-1 peptides (SELDLLDFATFDGG and RDKPLPPIIVEDPS) were synthesized and used as the antigens to generate rabbit polyclonal antisera  as described previously .Neurospora proteins were extracted as described above. For each immunoprecipitation reaction, 2 mg protein and 2 μl c-Myc , 2 μl Flag , or 2 μl WC-2 antibody . After sequencing, the raw data were treated and mapped to the genome of Neurospora crassa and transformed into expression value. The gene expression levels were scored by fragments per kb per million (FPKM) method. The differences in gene expression between samples was compared by comparing FPKM values, and those with fold change more than 1  were thought to be differentially expressed genes. The functional category enrichments, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes KEGG terms, were analyzed. The KEGG pathway enrichment was evaluated based on hypergeometric distribution, and the R package ‘ggplot2’ version 3.3.6 was used to visualize the enrichment results.The WT and t test. Statistical tests for the presence of rhythmicity and differences between two rhythms in parameters, including amplitude, phase, and mesor (the midline estimating statistic of rhythms) were analyzed using CircaSingle and CircaCompare (https://rwparsons.shinyapps.io/circacompare/)  . CircaCoAll data generated or analyzed during this study are included in the manuscript and supporting files. Our generated RNA sequencing data have been deposited in GEO under accession code GSE220169. RNA sequencing data from  This fundamental work is important in demonstrating that the general amino acid control response to amino acid limitation in Neurospora, which includes the key nutrient-controlled protein kinase Gcn2, is crucial to maintain circadian rhythmic cell growth and transcription of the FRQ gene, the master regulator of rhythmicity. There is an abundance of compelling evidence supporting the conclusions, with rigorous molecular and genetic assays of key mutants impaired for general amino acid control or transcriptional cofactors. The work will be of broad interest to geneticists and molecular biologists, and will be particularly valuable to researchers interested in circadian rhythm or nutrient control of gene expression. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Joanna Chiu (Reviewer #2); Bin Wang and Jay C Dunlap .Thank you for submitting your article "The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor Alan Hinnebusch has drafted this to help you prepare a revised submission.Essential revisions:1) Conduct statistical analysis to assess the significance of the circadian rhythms claimed for FRQ mRNA abundance, and for WC complex binding, H3Ac levels, GCN-5 binding, and CPC-1 binding at the FRQ promoter.2) The data are not compelling that CPC-1 occupancy at FRQ is rhythmic. If the statistically significant rhythmic binding of CPC-1 is not demonstrated, then the molecular basis of the rhythmic recruitment of Gcn5 and H3Ac will be unclear. In that event, the authors may need to consider the model that CPC-1 assists constitutively in recruiting Gcn5 and attendant H3 acetylation, and that some other factor is involved in producing the circadian rhythms of these events, and in turn, the rhythmic binding of WC complex at the FRQ promoter.3) It appears that the experiments to examine the involvement of GCN-5 and ADA-2 in the rhythmic transcription of FRQ were performed only in non-starvation conditions. If this was indeed the case, then at a minimum, key conclusions would have to be qualified. More likely, the experiments would have to be extended to include starvation conditions.4) Provide clarification about why knock out of GCN5 reduces FRQ protein expression and diminishes its rhythm without corresponding effects on FRQ mRNA abundance . Are the data lacking in precision or accuracy, or is a more complex model required to explain these results?5) The data provided to support the claim of rhythmic transcription of the four candidate amino acid biosynthetic genes in Figure 7F is not very compelling. If the statistical analysis fails to confirm their rhythmic transcription in WT, it might be possible to bolster their claim by evaluating published RNA-seq data  for known CPC-1 target genes. Because these published experiments were not carried out in amino acid starvation conditions however, the measured expression of the biosynthetic gene transcripts might be dominated by transcription factors that bind constitutively rather than by CPC-1. In the absence of published data supporting rhythmic transcription of amino acid biosynthetic genes regulated by CPC-1, the authors should consider conducting additional RNA-Seq experiments at different time points in amino acid-starved cells. Alternatively, it might be possible to provide RT-qPCR data on additional candidate genes, coupled with statistical analysis of rhythmic transcription. Failing that, the authors would likely need to soften or eliminate claims of rhythmic transcription of these genes.6) The proposed rhythmic transcription of amino acid biosynthetic genes would also predict rhythmic binding by CPC-1 in their promoters under starvation conditions. Conducting ChIP analysis of CPC-1 binding at a few candidate biosynthetic genes that show convincing rhythmic transcription would be highly desirable, and if observed, might compensate for less compelling transcript data. Such measurements would also be relevant to the question noted above of whether CPC-1 binding at the FRQ promoter itself is truly rhythmic.7) It is necessary to add new text to address queries raised by Referees #2 and #3 about various findings that require additional clarification or qualification.8) It is important to provide raw source data for all measurements.Reviewer #1 (Recommendations for the authors):i) Explain why knock out of GCN5 eliminates rhythmic FRQ expression but does not have a corresponding effect on FRQ mRNA abundance.ii) Determine whether elimination of HDA1 restores H3ac and WCC binding at FRQ, and rhythmic growth, in 3AT-treated cpc-1 and gcn5 mutants.iii) Provide stronger evidence for rhythmic transcription of aa biosynthetic genes.iv) Possibly attempt to determine whether CPC-1 occupancy at aa biosynthetic genes (and at FRQ itself) is rhythmic.Reviewer #2 (Recommendations for the authors):1. The authors should use circadian statistics  to compute the phase and amplitude of the mRNA, DNA binding of WC complex, and H3Ac rhythms. This will allow them to compare between rhythms and provide statistical significance values, rather than just providing qualitative descriptions, e.g. "slightly decreased" in lines 189-190. This will be valuable when comparing rhythms between strains and between nutrient conditions.2. Line 119: The authors indicate that the rhythmicity of histone acetylation at the frq gene promoter is lost in many of the mutants they tested, e.g. CPC-3 and CPC-1. However, it is not clear whether these mutants simply have a lower level of H3ac , resulting in a lower level of WC binding to frq promoter , and consequently lower level of frq mRNA . When the levels of these measures are low, it is difficult to observe any rhythms. Furthermore, is it really necessary to emphasize the maintenance of rhythms? When clock mRNA levels are too low, clocks are normally disrupted anyway.3. The authors propose that GCN5 works downstream of CPC-1 and CPC-3. If that is the case, why does frq mRNA in gcn-5 KO strain show dampened but consistently high level , rather than a consistently low level as in the case of cpc-1 and cpc-3 ko strains? Can the authors comment on this? Also, it appears that H3ac and WC binding to frq promoter is lower compared to WT . Can the authors explain why frq mRNA level is higher given H3ac and WC binding to frq promoter is lower?4. There is 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. Can the authors 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?5. 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, 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 the 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.6. 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.eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor, Alan Hinnebusch.Thank you for resubmitting your work entitled "The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation" for further consideration by It appears that you have satisfied some, but not all, of the major issues raised about the previous version of the paper. It was recognized that you provided new evidence indicating that knockout of GCN5 eliminates rhythmic FRQ expression with a corresponding effect on FRQ mRNA abundance. While you also provided new ChIP evidence suggesting that CPC-1 occupancy at FRQ is rhythmic; it was provided only to the referees. You performed statistical analysis of rhythmicity using the CircaCompare tool; however, the application of tests and the resulting statistics have not been presented in a transparent or comprehensive manner. It also appears that you incorrectly cited published evidence regarding the rhythmicity of amino acid biosynthetic gene expression. These and a few other issues are fully described below:1. The description of the CircaCompare statistical tests of rhythmicity is inadequate and needs to be better explained, providing a brief "layman's" description in the Methods of the meaning of the statistical parameters and how the tests were applied to their data. In addition, the results need to be documented completely for all experiments in which rhythmicity was concluded to exist for any parameter. While the authors added time-series statistics in certain figure legends, they did not indicate the p-values for rhythmicity, and the asterisks in figure panels seem to refer to t-tests rather than to RAIN/CircaCompare statistics. To increase transparency, the results of the CircaCompare statistical tests could be summarized in an additional supplementary table.2. Discrepancies remain regarding whether transcription of amino acid (AA) biosynthetic genes is rhythmic as, contrary to the statement in the Response to Reviewers, the publication by Hurley et al. 2014 does not show ser-2 (NCU01439), trp-3 (NCU08409) or arg-1 (NCU02639) to be rhythmic. The authors should note that the original data were re-analyzed using better statistical tools, described in Suppl Dataset S1 of Hurley et al. (2018), and in the re-analyzed data, ser-2(NCU01439) is not rhythmic at the RNA level, although it is rhythmic at the protein level; trp-3(NCU08409) is rhythmic at the RNA level, and arg-1 (NCU02639)- is rhythmic at the RNA level. The authors need to carefully examine the results of Hurley et al. (2018) and provide an accurate summary of the presence or absence of rhythmicity in the expression of all known CPC-1-induced target genes, possibly with an additional supplementary table that lists the key statistical criteria for the assessments. They should also explain to the reviewers why they incorrectly cited the findings of Hurley et al. (2014) in the revised manuscript.3. The new ChIP data for CPC-1 binding at the AA genes should be included as a supplementary figure with the addition of the appropriate explanatory text in Results and Discussion, including the suggestion made in their Response to Reviewers of rhythmic binding of WCC or WCC-controlled transcription factors, at the AA genes and whether any evidence exists in the literature for the latter.4. Clarify whether the authors are interpreting their data to indicate that GCN5/SAGA perform the same functions at FRQ promoter in both non-starved and starved cells and are simply being additionally mobilized by CPC-1 in starved cells, possibly by altering the model in Figure 8.Reviewer #1 (Recommendations for the authors):The authors appear to have satisfied some, but not all, of the major issues raised about the previous version of the paper. They have provided new evidence indicating that knock out of GCN5 eliminates rhythmic FRQ expression with a corresponding effect on FRQ mRNA abundance. They also provided new ChIP evidence suggesting that CPC-1 occupancy at FRQ is rhythmic. However, the following issues seem to require additional attention and adequate responses from the authors.1. To support the conclusions of rhythmic FRQ mRNA abundance, WCC complex binding, H3Ac levels, GCN-5 binding, and CPC-1 binding at the FRQ promoter, and of expression levels of amino acid biosynthetic genes in WT cells, they claim to have conducted a statistical analysis using CircaCompare. However, I could not find even a cursory explanation of this tool and the meaning of the different parameters it evaluates in Methods, nor a description of how it was being applied in comparing results for WT and mutants. In addition, it is generally unclear what data have been analyzed with CircaCompare and what the outcomes were. In particular, the legends often state that data were judged to be arhythmic in a cpc mutant without indicating whether it was significantly rhythmic in WT. What is needed is a table that lists clearly all the CircaCompare tests that were conducted and the P-values for all of the parameters that were analyzed, with a final column indicating whether the data are rhythmic or arhythmic and from what results this assessment was made. In short, for each experiment, it is necessary to provide justification using CircaCompare that the data are significantly rhythmic in WT, not only that they are arhythmic in mutants, and to make the CircaCompare tests and results completely transparent, accessible, and understandable to a general audience.2. The aforementioned shortcoming is particularly notable regarding the expression of amino acid (AA) biosynthetic genes in WT cells. It is unclear whether the CircaCompare analysis actually confirms rhythmic mRNA expression for these genes in WT cells, both for the group of genes they examined by RT-PCR and those interrogated using published RNA-seq data. It is also unclear why they are showing the published RNA-seq results for only 4 genes out of a presumably much larger number of AA biosynthetic genes known to be induced by CPC-1 in starved cells. Did many other AA genes not show rhythmic transcription? It would useful to have a table summarizing the CircaCompare analysis of the RNA-seq data for all known CPC-1 target genes.3. This last issue of whether AA biosynthetic gene transcription is truly rhythmic was exacerbated by their new CPC-1 ChIP data (presented only for referees) indicating constitutive CPC-1 occupancy at all four AA biosynthetic genes they tested, in contrast to its apparently rhythmic binding at the FRQ gene. They suggest that the AA biosynthetic genes might exhibit rhythmic binding of WCC, or some other transcriptional activator; however, is there any evidence that WCC binds to these AA genes, or that any other activator besides WCC binds to promoters rhythmically? I remain skeptical of the claim of rhythmic transcription of AA biosynthetic genes that are induced by CPC-1 in starved cells.Reviewer #2 (Recommendations for the authors):The authors have adequately addressed my comments and concerns from the previous manuscript version, in some cases with additional experiments. I greatly appreciate their effort. I think this study is a nice contribution to the field and will inform on the metabolic regulation of circadian rhythms at the chromatin level. One minor comment that will help clarify this complex model for readers.In the previous version, I commented:"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 already 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? "Based on the additional experiments and their text edit, there appears to be no difference in the activity of GCN-5 and SAGA complex in normal vs amino acid-starved conditions. It's doing what it normally does in normal conditions even when it is in amino acid-starved conditions. Its activity is however more necessary in amino acid-starved conditions because the chromatin at frq promoter is constitutively compacted in amino acid-starved conditions. So since CPC-3 and CPC-1 are necessary to activate the GCN-5/ADA-2 complex and since an amino acid-starved condition activates the GCN2 pathway and induced CPC-1 expression, the arrhythmic phenotype in circadian rhythm is more severe in CPC-3 and CPC-1 KO mutants in amino acid starved condition when compared to normal condition.I wonder if the model figure  would be more clear to readers if it includes the normal scenario instead of just having the amino acid-starved conditions. Besides that, I think the manuscript is much approved.Reviewer #3 (Recommendations for the authors):There are frequent and numerous issues with English usage that should be corrected. There are too many of these to mark each one, and they begin with problems in the Abstract.Here are a few examples just from the Introduction:"Circadian clocks are evolved to adapt to the daily environmental changes caused by the earth rotation". Should be earth's rotation "The ability to maintain circadian clock.54 function in response to various stress and perturbations is an important property of living systems.55 . Although gene expression is sensitive to temperature.56 changes, temperature compensation is a key feature of circadian clocks that maintain circadian.57 period length".Should read "in response to various stresses" and "compensation is a key feature of circadian clocks that maintains".Lines 128 and 136, the same sentence appears twice: "3-AT is an inhibitor…".Lines 168, 170, 171, and 177, cpc-1 KO to cpc-1KO; line 364, "related echanisms found a synthetic lethality between ∆chd1 and rasIn the model , formation of the CPC-1 oligomer was not confirmed experimentally, and this could be mentioned.eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled "The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1. In the new Supplementary file S1. The authors have provided lists of 148 cpc-1 up-regulated genes and 127 cpc-1 down-regulated genes, of which 79 up-regulated genes apparently show rhythmic expression and 67 down-regulated genes apparently show rhythmic expression. They have not however provided the statistics of the CircaCompare tests of rhythmicity for the WT strain, which needs to be added for each gene listed in the new file S1. In addition, they should provide new text in the RESULTS explaining that ~53% of both the up- and down-regulated genes exhibit rhythmicity and indicate that this represents a highly significant enrichment of all cpc-1 regulated genes as judged by the hypergeometric distribution test, providing the P-value. The file S1 should also include a notes sheet fully explaining the data provided there.2. A notes sheet should also be added to the new Supplementary file S2 in which the results in each sheet should be linked to data in the corresponding figures, and also stipulating the difference between results listed in different sheets for FRQ versus frq (Protein vs. mRNA?). In short, each Supplementary file should be understandable as a stand-alone document.3. There are also some grammatical errors in the revised text that should be corrected. Essential Revisions:1) Conduct statistical analysis to assess the significance of the circadian rhythms claimed for FRQ mRNA abundance, and for WC complex binding, H3Ac levels, GCN-5 binding, and CPC-1 binding at the FRQ promoter.We revised as suggested by reviewer 2 using CircaCompare.2) The data are not compelling that CPC-1 occupancy at FRQ is rhythmic. If the statistically significant rhythmic binding of CPC-1 is not demonstrated, then the molecular basis of the rhythmic recruitment of Gcn5 and H3Ac will be unclear. In that event, the authors may need to consider the model that CPC-1 assists constitutively in recruiting Gcn5 and attendant H3 acetylation, and that some other factor is involved in producing the circadian rhythms of these events, and in turn, the rhythmic binding of WC complex at the FRQ promoter.frq promoter with CircaCompare, which showed that the CPC-1 occupancy at the frq promoter was rhythmic.To address this concern, we now performed ChIP assays using the CPC-1 antibody instead of Myc antibody. As shown in the revised Figure 4A, the CPC-1 enrichment at DD14/DD18 was significantly higher than that of DD10/DD22. In addition, we also analyzed the circadian rhythm of CPC-1 binding at the 3) It appears that the experiments to examine the involvement of GCN-5 and ADA-2 in the rhythmic transcription of FRQ were performed only in non-starvation conditions. If this was indeed the case, then at a minimum, key conclusions would have to be qualified. More likely, the experiments would have to be extended to include starvation conditions.frq in both amino acid non-starvation and starvation conditions in the KOgcn-5  and KOada-2 mutants . The results were found to be similar with/without amino acid starvation in the KOgcn-5 and KOada-2 mutants.Thanks for the suggestion. We have now examined the rhythmic transcription of 4) Provide clarification about why knock out of GCN5 reduces FRQ protein expression and diminishes its rhythm without corresponding effects on FRQ mRNA abundance . Are the data lacking in precision or accuracy, or is a more complex model required to explain these results?frq mRNA in the WT and KOgcn-5 mutant. The results showed that the frq mRNA were decreased in the KOgcn-5 mutant with or without 3-AT, especially at DD16h and DD40h, when frq mRNA peaked in WT . Thus, the frq mRNA and FRQ protein results are consistent with each other.To address this concern, we performed RT-qPCR analysis to compare the rhythms of 5) The data provided to support the claim of rhythmic transcription of the four candidate amino acid biosynthetic genes in Figure 7F is not very compelling. If the statistical analysis fails to confirm their rhythmic transcription in WT, it might be possible to bolster their claim by evaluating published RNA-seq data  for known CPC-1 target genes. Because these published experiments were not carried out in amino acid starvation conditions however, the measured expression of the biosynthetic gene transcripts might be dominated by transcription factors that bind constitutively rather than by CPC-1. In the absence of published data supporting rhythmic transcription of amino acid biosynthetic genes regulated by CPC-1, the authors should consider conducting additional RNA-Seq experiments at different time points in amino acid-starved cells. Alternatively, it might be possible to provide RT-qPCR data on additional candidate genes, coupled with statistical analysis of rhythmic transcription. Failing that, the authors would likely need to soften or eliminate claims of rhythmic transcription of these genes.et al., 2014, PMID: 25362047) and found that these amino acid biosynthetic gene  were expressed rhythmically. Our analysis data is now presented in Figure 7—figure supplement 1C. Second, we also performed RT-qPCR analysis to examine the relative mRNA levels of his-3, ser-2, trp-3, and three additional amino acid biosynthetic genes  at different time points (sampled every 6 hrs over 30 hrs). We found that these amino acid biosynthetic genes were rhythmic expressed in the WT but not in the cpc-1KO strain with/without 3-AT treatment .The rhythmic expression of these candidate amino acid biosynthetic genes are supported by the following evidence. First, we analyzed the published rhythmic RNA-seq data from Dr. Jay Dunlap’s lab (Hurley JM 6) The proposed rhythmic transcription of amino acid biosynthetic genes would also predict rhythmic binding by CPC-1 in their promoters under starvation conditions. Conducting ChIP analysis of CPC-1 binding at a few candidate biosynthetic genes that show convincing rhythmic transcription would be highly desirable, and if observed, might compensate for less compelling transcript data. Such measurements would also be relevant to the question noted above of whether CPC-1 binding at the FRQ promoter itself is truly rhythmic.frq promoter and a few amino acid biosynthetic genes including his-3, ser-2 and trp-3. As the results shown in Figure 4A, CPC-1 binding at the frq promoter was rhythmic (measured with CircaCompare). However, the CPC-1 occupancy at the his-3, ser-2 and trp-3 promoters was not rhythmic (measured with CircaCompare). These results suggested that CPC-1 binding is rhythmic at the frq promoter but not at the amino acid biosynthetic genes. Thus, the rhythmic transcription of amino acid biosynthetic genes might be controlled by rhythmic binding of WCC or WCC-controlled transcription factors but not by rhythmic CPC-1 binding.To address this concern, we performed ChIP using our CPC-1 antibody to analyze CPC-1 occupancy at the 7) It is necessary to add new text to address queries raised by Referees #2 and #3 about various findings that require additional clarification or qualification.Revised as suggested. We have edited the text and revised the figures in the revised manuscript.8) It is important to provide raw source data for all measurements.Revised as suggested. Raw source data of measurements are now shown in the revised folder of source data.Reviewer #1 (Recommendations for the authors):i) Explain why knock out of GCN5 eliminates rhythmic FRQ expression but does not have a corresponding effect on FRQ mRNA abundance.Please see response to the Essential revisions point 4.ii) Determine whether elimination of HDA1 restores H3ac and WCC binding at FRQ, and rhythmic growth, in 3AT-treated cpc-1 and gcn5 mutants.KOcpc-3 strain after 3-AT treatment , we created KOcpc-3KOhda-1 double mutant and performed race tube assay with addition of different concentrations of 3-AT. The results showed that the KOcpc-3KOhda-1 mutant was sensitive to 3-AT, becoming arhythmic with 2 or 3 mM 3-AT, which is similar to KOcpc-3 strain  Provide stronger evidence for rhythmic transcription of aa biosynthetic genes.Please see response to Essential revisions point 5.iv) Possibly attempt to determine whether CPC-1 occupancy at aa biosynthetic genes (and at FRQ itself) is rhythmic.Please see response to Essential revisions point 6.Reviewer #2 (Recommendations for the authors):1. The authors should use circadian statistics  to compute the phase and amplitude of the mRNA, DNA binding of WC complex, and H3Ac rhythms. This will allow them to compare between rhythms and provide statistical significance values, rather than just providing qualitative descriptions, e.g. "slightly decreased" in lines 189-190. This will be valuable when comparing rhythms between strains and between nutrient conditions.As suggested, we used CircaCompare to analyze our data.2. Line 119: The authors indicate that the rhythmicity of histone acetylation at the frq gene promoter is lost in many of the mutants they tested, e.g. CPC-3 and CPC-1. However, it is not clear whether these mutants simply have a lower level of H3ac , resulting in a lower level of WC binding to frq promoter , and consequently lower level of frq mRNA . When the levels of these measures are low, it is difficult to observe any rhythms. Furthermore, is it really necessary to emphasize the maintenance of rhythms? When clock mRNA levels are too low, clocks are normally disrupted anyway.Thanks for the suggestions. We re-analyzed the data using CircaCompare and revised the manuscript.3. The authors propose that GCN5 works downstream of CPC-1 and CPC-3. If that is the case, why does frq mRNA in gcn-5 KO strain show dampened but consistently high level , rather than a consistently low level as in the case of cpc-1 and cpc-3 ko strains? Can the authors comment on this? Also, it appears that H3ac and WC binding to frq promoter is lower compared to WT . Can the authors explain why frq mRNA level is higher given H3ac and WC binding to frq promoter is lower?Please see response to Essential revisions point 4.4. There is 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. Can the authors 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?Neurospora, is the only eIF2α kinase responsible for eIF2α phosphorylation at serine 51. 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.Thanks for the question. CPC-3, the GCN2 homolog in 5. 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, 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 the 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.frq in the KOgcn-5 and KOada-2 strains under amino acid starvation . 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  , 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  . These results indicated that amino acid starved conditions had little impact on the protein interactions between CPC-1 with GCN-5 and SAGA complex.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 promoter in the WT strain , 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 KOcpc-3 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.In our model, we proposed that amino acid starvation resulted in compact chromatin structure (due to decreased H3ac) in the 6. 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”.Reviewer #3 (Recommendations for the authors):There are frequent and numerous issues with English usage that should be corrected. There are too many of these to mark each one, and they begin with problems in the Abstract.Here are a few examples just from the Introduction:"Circadian clocks are evolved to adapt to the daily environmental changes caused by the earth rotation". Should be earth's rotation "The ability to maintain circadian clock.54 function in response to various stress and perturbations is an important property of living systems.55 . Although gene expression is sensitive to temperature.56 changes, temperature compensation is a key feature of circadian clocks that maintain circadian.57 period length".Should read "in response to various stresses" and "compensation is a key feature of circadian clocks that maintains".Thanks for the suggestions. The manuscript is revised as suggested.Lines 128 and 136, the same sentence appears twice: "3-AT is an inhibitor…".Lines 168, 170, 171, and 177, cpc-1 KO to cpc-1KO; line 364, "related echanisms found a synthetic lethality between ∆chd1 and ras-1 bdras background was revised in the Materials and methods, and stated in the beginning of the main text “we created cpc-3 and cpc-1 knockout mutants ”.Thanks for the suggestions. The manuscript is revised as suggested. The In the model , formation of the CPC-1 oligomer was not confirmed experimentally, and this could be mentioned.et al., 1987, EMBO J, PMID: 3678204). We revised our model in Figure 8 and edited the description in the text, “the GCN2 kinase responds to nutrient deprivation, whereas it phosphorylates eIF2α and upregulates the master transcription factors GCN4 or CPC-1, respectively, which binds target DNA as a dimer to activate amino acid biosynthetic genes”.GCN4/CPC-1 was previously reported to bind DNA in the form of dimers  biosynthetic genes is rhythmic as, contrary to the statement in the Response to Reviewers, the publication by Hurley et al. 2014 does not show ser-2 (NCU01439), trp-3 (NCU08409) or arg-1 (NCU02639) to be rhythmic. The authors should note that the original data were re-analyzed using better statistical tools, described in Suppl Dataset S1 of Hurley et al. (2018), and in the re-analyzed data, ser-2(NCU01439) is not rhythmic at the RNA level, although it is rhythmic at the protein level; trp-3(NCU08409) is rhythmic at the RNA level, and arg-1 (NCU02639)- is rhythmic at the RNA level. The authors need to carefully examine the results of Hurley et al. (2018) and provide an accurate summary of the presence or absence of rhythmicity in the expression of all known CPC-1-induced target genes, possibly with an additional supplementary table that lists the key statistical criteria for the assessments. They should also explain to the reviewers why they incorrectly cited the findings of Hurley et al. (2014) in the revised manuscript.Thanks for the correction and suggestions. We have revised the manuscript as suggested. Please see our response to the Reviewer #1.3. The new ChIP data for CPC-1 binding at the AA genes should be included as a supplementary figure with the addition of the appropriate explanatory text in Results and Discussion, including the suggestion made in their Response to Reviewers of rhythmic binding of WCC or WCC-controlled transcription factors, at the AA genes and whether any evidence exists in the literature for the latter.Thanks for the suggestions. We have revised the manuscript as suggested. Please see our response to the Reviewer #1.4. Clarify whether the authors are interpreting their data to indicate that GCN5/SAGA perform the same functions at FRQ promoter in both non-starved and starved cells and are simply being additionally mobilized by CPC-1 in starved cells, possibly by altering the model in Figure 8.We have revised the Figure 8 and manuscript as suggested. Please see our response to the Reviewer #2.Reviewer #1 (Recommendations for the authors):The authors appear to have satisfied some, but not all, of the major issues raised about the previous version of the paper. They have provided new evidence indicating that knock out of GCN5 eliminates rhythmic FRQ expression with a corresponding effect on FRQ mRNA abundance. They also provided new ChIP evidence suggesting that CPC-1 occupancy at FRQ is rhythmic. However, the following issues seem to require additional attention and adequate responses from the authors.1. To support the conclusions of rhythmic FRQ mRNA abundance, WCC complex binding, H3Ac levels, GCN-5 binding, and CPC-1 binding at the FRQ promoter, and of expression levels of amino acid biosynthetic genes in WT cells, they claim to have conducted a statistical analysis using CircaCompare. However, I could not find even a cursory explanation of this tool and the meaning of the different parameters it evaluates in Methods, nor a description of how it was being applied in comparing results for WT and mutants. In addition, it is generally unclear what data have been analyzed with CircaCompare and what the outcomes were. In particular, the legends often state that data were judged to be arhythmic in a cpc mutant without indicating whether it was significantly rhythmic in WT. What is needed is a table that lists clearly all the CircaCompare tests that were conducted and the P-values for all of the parameters that were analyzed, with a final column indicating whether the data are rhythmic or arhythmic and from what results this assessment was made. In short, for each experiment, it is necessary to provide justification using CircaCompare that the data are significantly rhythmic in WT, not only that they are arhythmic in mutants, and to make the CircaCompare tests and results completely transparent, accessible, and understandable to a general audience.Thanks for the comments and suggestions. We have now revised the manuscript as suggested. We have added in the Methods details of the CircaCompare statistical analysis described in previously published papers . The results of the CircaCompare statistical tests were summarized in the Supplementary file 2.2. The aforementioned shortcoming is particularly notable regarding the expression of amino acid (AA) biosynthetic genes in WT cells. It is unclear whether the CircaCompare analysis actually confirms rhythmic mRNA expression for these genes in WT cells, both for the group of genes they examined by RT-PCR and those interrogated using published RNA-seq data. It is also unclear why they are showing the published RNA-seq results for only 4 genes out of a presumably much larger number of AA biosynthetic genes known to be induced by CPC-1 in starved cells. Did many other AA genes not show rhythmic transcription? It would useful to have a table summarizing the CircaCompare analysis of the RNA-seq data for all known CPC-1 target genes.Thanks for the suggestions. In the previous manuscript, we only cited the Hurley et al. (2014) paper, because we downloaded and re-analyzed the RNA-seq data in that study. However, the data were re-analyzed using improved statistical methods described in the Supplemental Dataset 1 (https://doi.org/10.17632/r68j3rnxhw.1) of Hurley et al. (2018). We have now cited both papers in our manuscript.KOcpc-1 strain , suggesting that these genes were regulated by CPC-1 under amino acid starvation. As shown in the Supplementary file 1, we summarized the rhythmic expression of 79 up-regulated and 67 down-regulated CPC-1 target genes based on the results of Hurley et al. (2018) . We also performed RT-qPCR experiments to re-analyze the expression rhythms for some of those genes and found that the rhythmic expression of his-3 (NCU03139), trp-3 (NCU08409) and arg-1 (NCU02639) in the WT was abolished in the KOcpc-1 strain under amino acid starvation, which is consistent with the published RNA-seq analysis results. In addition, we found that ser-2 (NCU01439) was also rhythmic in the WT but not in the KOcpc-1 strain under amino acid starvation, even though it was not previously shown to be rhythmic in the published RNA-seq analysis study. These results suggest that many CPC-1 activated metabolic genes under amino acid starvation are regulated by circadian clock.In our study, there were 148 up-regulated and 127 down-regulated genes found in the WT strain after 3-AT treatment but their regulation were abolished in the 3. This last issue of whether AA biosynthetic gene transcription is truly rhythmic was exacerbated by their new CPC-1 ChIP data (presented only for referees) indicating constitutive CPC-1 occupancy at all four AA biosynthetic genes they tested, in contrast to its apparently rhythmic binding at the FRQ gene. They suggest that the AA biosynthetic genes might exhibit rhythmic binding of WCC, or some other transcriptional activator; however, is there any evidence that WCC binds to these AA genes, or that any other activator besides WCC binds to promoters rhythmically? I remain skeptical of the claim of rhythmic transcription of AA biosynthetic genes that are induced by CPC-1 in starved cells.arg-1 (NCU02639) (https://www.pnas.org/doi/suppl/10.1073/pnas.1418963111/suppl_file/pnas.1418963111.sd11.xlsx). Third, although the binding of CPC-1 is arrhythmic, its activity in activating transcription can still be rhythmic, which could be due to rhythmic posttranslational modification/association of other factors.Thanks for the concern of this review. Our results and those of Hurley et al. (2018) demonstrated that CPC-1 is required for the rhythmic expression of many CPC-1 targeted metabolic genes. We agree with this reviewer that the exact mechanism for how this regulation is achieved is still unclear since our ChIP experiments showed that the CPC-1 binding at several selected amino acid biosynthetic genes was not rhythmic . There are several possibilities. First, the rhythms of CPC-1 binding at these amino acid biosynthetic genes have very low amplitude and could not be detected by our ChIP assays. Second, the rhythmic transcription of these genes could be controlled by rhythmic binding of WCC or WCC-controlled transcription factors . Indeed, the ChIP-seq analysis by Hurley et al. (2014) showed that WC-2 is associated at the promoter of his-3 (NCU03139), trp-3 (NCU08409) and ser-2 (NCU01439) genes, and the enrichment was enhanced by 3-AT treatment”. In the discussion, we added that “Although CPC-1 rhythmically binds at the frq promoter, ChIP experiments showed that CPC-1 binding at several selected amino acid biosynthetic genes did not appear to be rhythmic . However, our RT-qPCR results  and the previous RNA-seq data showed that many CPC-1 targeted metabolic genes were rhythmic expressed . It is possible that the binding of CPC-1 on these promoters are still rhythmic but with low amplitudes. As a result, the limited sensitivity of our ChIP assays failed to detected these rhythms. Alternatively, the rhythmic transcription of these genes might be controlled by rhythmic transcriptional activation activity of CPC-1 rather than its binding. In addition, the rhythmic binding of WCC or WCC-controlled transcription factors  might also contribute to their rhythmic transcription.”We have now revised the manuscript to address this concern. In the results, we described “We performed ChIP experiments to examine whether CPC-1 directly activates the expression of amino acid synthetic genes. As shown in Figure 7—figure supplement 1C, CPC-1 was found to be constitutively enriched at the promoters of Reviewer #2 (Recommendations for the authors):The authors have adequately addressed my comments and concerns from the previous manuscript version, in some cases with additional experiments. I greatly appreciate their effort. I think this study is a nice contribution to the field and will inform on the metabolic regulation of circadian rhythms at the chromatin level. One minor comment that will help clarify this complex model for readers.In the previous version, I commented:"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 already 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? "Based on the additional experiments and their text edit, there appears to be no difference in the activity of GCN-5 and SAGA complex in normal vs amino acid-starved conditions. It's doing what it normally does in normal conditions even when it is in amino acid-starved conditions. Its activity is however more necessary in amino acid-starved conditions because the chromatin at frq promoter is constitutively compacted in amino acid-starved conditions. So since CPC-3 and CPC-1 are necessary to activate the GCN-5/ADA-2 complex and since an amino acid-starved condition activates the GCN2 pathway and induced CPC-1 expression, the arrhythmic phenotype in circadian rhythm is more severe in CPC-3 and CPC-1 KO mutants in amino acid starved condition when compared to normal condition.I wonder if the model figure  would be more clear to readers if it includes the normal scenario instead of just having the amino acid-starved conditions. Besides that, I think the manuscript is much approved.KOcpc-3 and KOcpc-1 mutants compared to the WT strain under amino acid starved conditions. Because the chromatin structure at the frq promoter is constitutively compacted by amino acid starvation, CPC-1 protein level is a limiting factor for efficiently recruiting GCN-5 and SAGA complex to loosen the chromatin to drive rhythmic frq expression under amino acid starved conditions. As suggested, we have now revised the model in Figure 8 and edited the figure legend and discussion to make it more clear to readers.Thanks for the comments and suggestions. It’s correct that the interaction between CPC-1 with ADA-2 and GCN5 is not changed by amino acid starvation. However, the activity of GCN-5 and SAGA complex is more important in the The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1. In the new Supplementary file S1. The authors have provided lists of 148 cpc-1 up-regulated genes and 127 cpc-1 down-regulated genes, of which 79 up-regulated genes apparently show rhythmic expression and 67 down-regulated genes apparently show rhythmic expression. They have not however provided the statistics of the CircaCompare tests of rhythmicity for the WT strain, which needs to be added for each gene listed in the new file S1. In addition, they should provide new text in the RESULTS explaining that ~53% of both the up- and down-regulated genes exhibit rhythmicity and indicate that this represents a highly significant enrichment of all cpc-1 regulated genes as judged by the hypergeometric distribution test, providing the P-value. The file S1 should also include a notes sheet fully explaining the data provided there.Thanks for the comments to our revised manuscript. We have now revised the manuscript to address the concerns.First, it should be noted that the published RNA-seq data were first generated by Hurley et al. in 2014 and were re-analyzed by them using the improved statistical tool eJTK Cycle in 2018. The P-values of eJTK Cycle analysis  has now been added in our revised Supplementary file 1. CircaCompare is used to compare the differences between two rhythmic datasets and CircaSingle  is used to analyze the rhythmic parameters of a single group. As suggested by the reviewer, we re-analyzed the rhythmicity of CPC-1 targeted genes (148 up-regulated and 127 down-regulated) using CircaSingle and added the P-values in the revised Supplementary file 1. There were 146 rhythmic genes based on the eJTK Cycle and 132 rhythmic genes based on the CircaSingle. There were 106 overlapping genes between the two sets of data, confirming the results using eJTK Cycle. Thus, we performed further analysis based on the data from eJTK Cycle.Second, we have now added the description of the rhythmicity of CPC-1 targeted genes in the Results “There were 146/275 (53%) of the CPC-1 up- and down-regulated genes under amino acid starvation exhibiting rhythmicity, indicating a highly significant enrichment of CPC-1 regulated genes as clock-controlled genes ”. The rhythmic genes and all detected genes of RNA-seq data from Hurley et al. 2018 were added in the revised Supplementary file 1, which was used for the hypergeometric distribution test.Third, we have now revised the Supplementary file 1 by adding a notes sheet.2. A notes sheet should also be added to the new Supplementary file S2 in which the results in each sheet should be linked to data in the corresponding figures, and also stipulating the difference between results listed in different sheets for FRQ versus frq (Protein vs. mRNA?). In short, each Supplementary file should be understandable as a stand-alone document.frq mRNA.Thanks for the suggestions. We have now added a notes sheet for Supplementary file 2 and stipulated the information for FRQ protein and 3. There are also some grammatical errors in the revised text that should be corrected.Thanks for the suggestion. We have carefully checked and improved the English in the revised manuscript.


This review discusses chromatin-based regulation mechanisms as determined in the fungal plant pathogen Verticillium dahliae and relates the importance of epigenetic transcriptional regulation and other nuclear processes more broadly in fungal plant pathogens.Through the association of protein complexes to DNA, the eukaryotic nuclear genome is broadly organized into open euchromatin that is accessible for enzymes acting on DNA and condensed heterochromatin that is inaccessible. Chemical and physical alterations to chromatin may impact its organization and functionality and are therefore important regulators of nuclear processes. Studies in various fungal plant pathogens have uncovered an association between chromatin organization and expression of in planta Arguably, one of the most important transition towards eukaryotic evolution has been cell compartmentalization, which allowed physical separation of diverse cellular processes . One of Regulation of DNA-templated processes involves the histone code, made up of posttranslational chemical modifications to DNA-interacting histone proteins that help regulate genome functionality ,3. EukarNeurospora crassa . Ad. AdV. datromeres . The crutromeres . However and 5mC ,157, sugfunction ,157. TheIn this review, we have highlighted the importance of epigenetics in the regulation of nuclear processes in fungal plant pathogens. Moreover, we describe experimental evidence that genomic regions containing effector genes are characterized by the presence of heterochromatic features, but it remains to be seen if in planta transcriptional activation requires chromatin de-condensation or epigenome remodeling. As we show that differential gene expression in vitro for only a subset of genes located in H3K27me3 domains involves local H3K27me3 depletion, we postulate that gene expression in planta may display local H3K27me3 depletion, but that it is not strictly required. To demonstrate this, future studies will require a functional and reliable procedure to perform in planta chromatin immunoprecipitation assays or other types of nuclear capture or single-cell interrogation.Histone proteins are not only found in eukaryotes, but are also common in archaea ,159, ind


In the ongoing arms race between virus and host, fine-tuned gene expression plays a critical role in antiviral signaling. However, viruses have evolved to disrupt this process and promote their own replication by targeting host restriction factors. Polymerase-associated factor 1 complex (PAF1C) is a key player in this relationship, recruiting other host factors to regulate transcription and modulate innate immune gene expression. Consequently, PAF1C is consistently targeted by a diverse range of viruses, either to suppress its antiviral functions or co-opt them for their own benefit. In this review, we delve into the current mechanisms through which PAF1C restricts viruses by activating interferon and inflammatory responses at the transcriptional level. We also highlight how the ubiquity of these mechanisms makes PAF1C especially vulnerable to viral hijacking and antagonism. Indeed, as often as PAF1C is revealed to be a restriction factor, viruses are found to have targeted the complex in reply. As the first line of host defense, pathogen sensing by the innate immune response culminates in the reconfiguration of host gene expression to restrict virus replication. Polymerase-associated factor 1 complex (PAF1C) plays a critical role in this response. Its diverse molecular functions may bring about death by a thousand cuts for viruses, stemming from PAF1C’s ability to regulate transcription elongation, chromatin remodeling, histone modification, and more.PAF1C was initially characterized in yeast ,3. ConcePAF1C has been classically viewed as a positive regulator of gene expression, wherein its occupancy at a TSS creates a transcriptional environment conducive to RNAPII release and productive transcription elongation ,14,15,16Beyond the direct modulation of transcription, PAF1C can affect gene expression indirectly via epigenetic and structural modifications to chromatin, altering accessibility and activity at various TSS. As with transcription, PAF1C relies on several interacting partners to alter histone methylation and ubiquitination ,29,30,31Since the innate immune recognition of viral infections often culminates in an antiviral gene expression program , in thisSeveral omics-based screens have implicated PAF1C in both interferon-related and inflammatory innate immune responses. Both responses are central to antiviral signaling and the ultimate restriction of virus replication. Although work on PAF1C and the immune response is diverse, sometimes incongruous, and complex, these pleiotropic effects may be advantageous to the host under different contexts .Upon pathogen detection, type I interferons (IFN-I) initiate broad interferon-stimulated gene (ISG) expression. This contributes to the restriction of viruses in infected cells, inflammation, antigen presentation, and the eventual activation of adaptive immunity . SeveralTwo independent studies have shown drastic decreases in interferon-related gene expression in the absence of PAF1. In one of these studies, the treatment of cells with both poly(I:C) and IFN-β following PAF1 knockdown resulted in a significant reduction in mRNA expression by the RNA-sequencing of known ISGs, including IFITs and OASs . Our ownHowever, the disruption of PAF1C by removing individual subunits does not necessarily translate to the full abrogation of an immune response. For example, the overexpression of the CDC73 subunit is sufficient to trigger a IFN-II response via STAT1 activation and full PAF1C complex formation is not required for this to occur . Taken tPAF1C subunits were identified as positive regulators of TNF expression—a bellwether of the inflammatory response—through a genome-wide CRISPR screen of primary immune cells following LPS stimulation . PAF1C dIn another study exploring the role of PAF1C in the inflammatory response, PAF1C subunits displayed increased expression in mice upon LPS treatment . Using tThe same research group also investigated PAF1C’s role in IL-1β-mediated inflammatory responses . SilenciThese results add further complexity to PAF1C-mediated inflammatory responses and emphasize the importance of investigating its precise mechanisms in different contexts. While PAF1C regulates IL-6 and IL-1β responses through inverted mechanisms B, this mWhile our understanding of PAF1C mechanisms continues to mature, the significance of the complex in eliciting an immune response is self-evident. It is no surprise, then, that PAF1C has been repeatedly identified as a restriction factor for several viruses in separate studies. The first of these studies was a whole genome siRNA screen of human immunodeficiency virus 1 (HIV-1) restriction factors . The knoPAF1C also restricts other viruses. The knockdown of PAF1C subunits increased virus replication for influenza A virus (IAV) , vesiculThe means by which this restriction occurs could be through several mechanisms and targets of gene expression. The activation of interferon and inflammatory responses—as previously discussed—is a definite contributor to canonical antiviral signaling, yet there are likely nontraditional pathways of viral restriction given the positive and negative regulatory modes of PAF1C ,18. We fWe broadly captured previously identified PAF1-mediated responses to reinforce several key conclusions. First, PAF1 knockout resulted in up- and down-regulated gene expression, suggesting that PAF1C likely engages in immune activation and suppression across diverse innate immune responses. Secondly, the transcriptional motifs and regulatory elements of these gene groups were distinct. For instance, the genes likely induced by PAF1C were strongly associated with promoter motifs connected to Nuclear factor-κB (NF-κB) signaling and related immune pathways. In contrast, genes expected to be suppressed were weakly associated with promoters and were more enriched for enhancer elements. Since super enhancers are linked to the PAF1C pausing of RNAPII ,19, thisIn that same vein, recent work connecting PAF1C-mediated transcription and mitophagy—a form of selective autophagy for mitochondria—provides yet another avenue by which PAF1C can restrict viruses . The stuPAF1C can facilitate an antiviral response across several axes, sometimes leveraging multiple functions at once. As a positive regulator of transcription elongation, PAF1C can recruit various transcription factors and histone-modifying enzymes to modulate RNAPII pausing, release, and elongation, resulting in the increase or decrease of interferon or inflammatory gene expression, depending on the genetic landscape. All these processes drive both canonical and non-canonical pathways that restrict virus replication A. PAF1C’Though not the first study to associate PAF1C with viruses, antagonism by IAV has brought the complex to the forefront of innate immunity in the virus–host arms race. In a seminal study, the NS1 protein of IAV H3N2 was found to possess a histone mimic “tail” that interacted with the PAF1 subunit in a virus strain-specific manner . This inInterestingly, recent studies have correlated PAF1C activity with IAV-induced SUMOylation, an epigenetic regulator of transcription. It has been previously demonstrated that IAV infection increases the rate of SUMOylation . A subseThe NS5 protein of flaviviruses is also well-characterized in its ability to interfere with PAF1C-mediated immune responses. Global virus–host proteomics for DENV and ZIKV first revealed that NS5 interacted with several PAF1C subunits , with thRecent work by us and others has addressed this need to characterize the NS5–PAF1C interaction. Our own work established that DENV NS5 relied on nuclear localization to facilitate an interaction with PAF1C . AdditioConnecting these two studies would require discerning whether disruption of the pivot region we isolated may be sufficient to break the competitive binding between NS5, PAF1C, and chromatin. It is also relevant to elucidate whether NS5 antagonism is sufficient to displace PAF1C from paused sites of expression, as current work has focused predominantly on active transcript elongation. Regardless, both studies effectively highlight how flavivirus NS5 quite literally blocks antiviral signaling at PAF1C-mediated sites of expression, boosting virus replication and exacerbating pathogenesis. Aside from the antiviral signaling outcomes of PAF1C transcriptional regulation, the actual, direct functionality of the complex is especially relevant for the expression of integrated viruses. Following the integration of HIV-1, the transactivator protein, Tat, binds the viral promoter and phosphorylates RNAPII and associated factors to drive the productive elongation of viral transcripts . P-TEFb—However, this model for PAF1C-mediated HIV transcription does not readily align with the maintenance of latency and viral restriction correlated with PAF1C subunits ,50,51. HThe same study articulated how PAF1C is possibly displaced following HIV reactivation. A cascade underlies proviral transcriptional activation during HIV latency reversal: LEDGF/p75 associates and recruits the MLL1 branch of COMPASS to catalyze H3K4 trimethylation . This epTat may contribute to PAF1C displacement in this way, yet other HIV proteins are implicated as well. Recent work found that Vpr expression reduced histone H1.2 ubiquitination, and when H1.2 was affinity purified, proteomics showed Vpr expression enhanced PAF1C binding to H1.2  Figure C. HistonBeing able to reconfigure this balance to benefit the virus or host could have powerful implications, and the contemporary identification of a small molecule inhibitor, referred to as iPAF1C, makes this a current reality. iPAF1C targets a key binding interface between CTR9 and PAF1 subunits, and the drug disrupted PAF1C chromatin localization and induced RNAPII release . MoreoveSimilar complexities extend to DNA viruses, such as HSV-1. During HSV-1 infection, the transcription of immediate early (IE) genes is a critical rate-limiting step. This step relies on the SEC, including pTEFb, as the inhibition of SEC led to the reduced occupancy of viral IE promoters, less viral transcription, and suppressed viral reactivation as a result . As withAnother IE gene, ICP22, further complicates PAF1C antagonism. As an inhibitor of pTEFb activity , ICP22 iPAF1C continues to appear in virus–host interaction studies. Most recently, SARS-CoV-2 nucleocapsid (N) has been suggested to interact with PAF1C subunits, yet these interactions were weak and inconsistent across several proteomics-based studies ,85,86,87Despite being able to activate several distinct antiviral regulatory programs through transcriptional and epigenetic controls, PAF1C is the anti-hero of its own story, typically being antagonized or hijacked by the same viruses it restricts. Considering the broad scope of the studies we have discussed, and the sheer evolutionary diversity of viruses implicated, repeated association with PAF1C is far from a coincidence. It is more likely to be an invisible string tying together viruses that evolutionarily converged on targeting such a pleiotropic conserved host factor. Then again, our understanding of the context-based effects of PAF1C, especially as it relates to viruses, is lacking, and new virus–PAF1C interactions continue to be identified contemporarily. Since PAF1C fulfills several functions simultaneously, recognizing which viruses hijack which functions will be crucial to navigating the labyrinth of PAF1C and antiviral immunity.


The chicken genome is one-third the size of the human genome and has a similarity of sixty percent when it comes to gene content. Harboring similar genome sequences, chickens’ gene arrangement is closer to the human genomic organization than it is to rodents. Chickens have been used as model organisms to study evolution, epigenome, and diseases. The chicken nucleated erythrocyte’s physiological function is to carry oxygen to the tissues and remove carbon dioxide. The erythrocyte also supports the innate immune response in protecting the chicken from pathogens. Among the highly studied aspects in the field of epigenetics are modifications of DNA, histones, and their variants. In understanding the organization of transcriptionally active chromatin, studies on the chicken nucleated erythrocyte have been important. Through the application of a variety of epigenomic approaches, we and others have determined the chromatin structure of expressed/poised genes involved in the physiological functions of the erythrocyte. As the chicken erythrocyte has a nucleus and is readily isolated from the animal, the chicken erythrocyte epigenome has been studied as a biomarker of an animal’s long-term exposure to stress. In this review, epigenomic features that allow erythroid gene expression in a highly repressive chromatin background are presented. Gallus gallus domesticus), a subspecies of the red jungle fowl , has a karyotype consisting of 78 chromosomes. Chromosomes 1–10, Z, and W are classified as macrochromosomes and 11–38 are microchromosomes. The domesticated chicken (poultry) diverged from their ancestor more than 8000 years ago  lysine for one hour to label newly synthesized histones . Wi. WiHBBA TAL1, CTCF, NF1A, VEZF1, Sp3, CEBPG (CCAAT/enhancer binding protein)). However, the role of these and other transcription factors in regulating the expression of genes involved in the chicken hemoglobin synthesis pathway requires further study. For some transcription factor coding genes such as GATA1 that play critical roles in erythroid gene expression, the genomic location of the gene remains to be identified.Many transcription factors regulating the expression of the erythroid genes are among the genes with the highest level of transcripts in polychromatic erythrocytes  chromatin structure [Among the cells participating in the innate immune response in chickens is the erythrocyte . The inntructure .TLR3), TLR21, TRAF3, TRAM1, TICAM1, TIRAP, MYD88, IRF7, and IKBKB (inhibitor of nuclear factor kappa B kinase subunit beta), and NFKB2. Chicken erythroleukemic 6C2 cells, but not polychromatic erythrocytes, have transcripts for TLR4 and TLR21; however, the chromatin of both genes has active marks associated with the 5′ gene, suggesting that these genes are poised for transcription in the polychromatic erythrocyte.The chicken polychromatic erythrocyte and 6C2 cells express genes that respond to viral RNA as part of the innate immunity machinery  Figure . Both ceTLR3 gene responds to double-stranded RNA produced by viral infection. Incubation of chicken polychromatic erythrocytes with the double-stranded RNA mimic, poly(I:C), results in increased TLR3 transcript levels [TLR3 gene has F1 Seq peaks indicative of enhanced chromatin solubility at physiological ionic strength and nucleosomes that are multiply modified . At the 3′end of the gene in the intergenic region is a CpG island that has a FAIRE Seq peak (nucleosome-free region), surrounded by nucleosomes that are modified at H3R2me2s, K27ac, and H4R3me2a), which is a signature associated with enhancers. Whether this is an enhancer for the TLR3 gene remains to be determined.The t levels . In the IFNA3 genes and INFW1. Transcripts for these IFN genes were not detected in our RNA Seq data for chicken polychromatic erythrocyte and 6C2 cells. However, the chromatin structure of this region in chicken polychromatic erythrocytes appears as a gene region poised for transcription. The 5′ end of the IFN cluster has F1 Seq peaks and a salt-soluble chromatin structure, and the nucleosomes throughout the cluster have multiple modifications for H3  and H4R3me2a. This chromatin region is unstable as indicated by the FAIRE Seq peaks (nucleosome-free regions) throughout the cluster. It is noted that the cluster has many CpG islands.Chromosome Z has a cluster of four IFNA3 and INFW1 genes [IRF1 gene is immersed in F1 Seq broad peaks , and the gene promoter region and body have nucleosomes that are multiply modified. In addition to IRF1, the chicken polychromatic erythrocyte, and 6C2 cells express several other interferon regulatory factors including IRF2, IRF8, and IRF9 . All expressed IRF genes, except IRF8, are associated with CpG islands.IRF1 (interferon regulatory factor) and NF-κB are involved in the expression of the W1 genes . The chiIL-15, but not other interleukins. The chromatin structure of the IL-15 gene in polychromatic erythrocytes is an enigma as the gene and surrounding regions do not have any distinguishing features that are associated with gene transcription. In contrast, IL1B transcripts were not detected in chicken polychromatic erythrocytes but the gene has chromatin features of a transcribed gene. Similarly, transcripts from the cytokine CCL4 gene were not detected in polychromatic erythrocytes but the gene chromatin has many features of a transcribed gene. It appears that both the IL1B and CCL4 genes are poised for expression. Consistent with this observation, IL1B transcripts were observed for 3 h following stimulation of chicken polychromatic erythrocytes with poly(I:C) [Among the interleukins, 6C2 and polychromatic erythrocytes have transcripts for oly(I:C) .The nucleated erythrocyte in chickens has a critical role in carrying oxygen to tissues and the removal of carbon dioxide. The erythrocyte also contributes to protecting the host from various pathogens. The configuration of the erythrocyte epigenome plays a major role in the organization and function of genes involved in these red blood cell functions. Transcription factors, chromatin modifying enzymes, and histone PTMs play a critical role in maintaining a transcription-permissive decondensed chromatin state in a background of highly condensed chromatin.The erythrocyte epigenome is responsive to both internal (metabolism) and external  conditions that impact chromatin organization and gene expression. The order of genes in the chicken genome is like that of the human genome but the chicken has a smaller genome. Thus, the chicken red blood cell is an excellent model system to study the consequences of the environment on the epigenome and translate these insights to human health.There is still much to be done in the characterization of the chicken erythrocyte epigenome and the proteins and chromatin-modifying enzymes involved in the writing, reading, and removal of the epigenomic marks. The chicken erythrocyte chromatin fractionation protocol can be applied to study novel histone PTMs and obtain insights as to whether the PTM contributes to gene expression or repression when antibodies to that histone PTM are not available. For example, we know very little about the acid labile H4His18ph  and hist