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

To investigate whether the nutrient-sensing GCN2 pathway is involved in regulating circadian clock function under amino acid starvation, we created the cpc-3 and cpc-1 knockout mutants (see Materials and methods). 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 cpc-3^KO strain (Figure 1—figure supplement 1A). 3-AT is an inhibitor of the histidine synthesis enzyme encoded by his-3 which triggers the amino acid starvation response (Natarajan et al., 2001). On the other hand, CPC-1 expression and its induction by 3-AT were eliminated in the cpc-1^KO mutant (Figure 1—figure supplement 1B). The circadian conidiation rhythms of the cpc-3^KO and cpc-1^KO mutants were examined by race tube assays. Under a normal growth condition (0 mM 3-AT), cpc-3 deletion had no effect on the circadian period but cpc-1 deletion led to the period lengthening of ~1.7 hr (Figure 1A). The long period of cpc-1^KO strain could be rescued by the expression of Myc.CPC-1 (Figure 1—figure supplement 1C).

To investigate how amino acid starvation affects circadian clock, we treated the wild-type (WT), cpc-3^KO, and cpc-1^KO strains with different concentrations of 3-AT. As shown in Figure 1A, although treatment with 3 or 4 mM 3-AT resulted in modest inhibition of the WT growth rate, the robust circadian conidiation rhythms were maintained. In the cpc-3^KO and cpc-1^KO strains, however, addition of 3 or 4 mM 3-AT resulted in severe inhibition of growth rates and the loss of circadian conidiation rhythms. To exclude the effect of 3-AT on other target genes, we examined the circadian rhythm of the his-3⁻ strain, which contained a single mutation in the his-3 gene required for histidine synthesis and could not grow in the medium without histidine. Race tube assays showed that the his-3⁻ strain grew normally and exhibited a robust circadian conidiation rhythm in the presence of histidine (1.0 × 10⁻² mg/ml). Although addition of the same amount of histidine could rescue the growth of the cpc-3^KO his-3⁻ strain, it could not rescue its circadian conidiation rhythm (Figure 1—figure supplement 1D), indicating that CPC-3 is required for robust circadian rhythms under histidine starvation stress.

To confirm the loss of circadian rhythms at the molecular level, we introduced a frq promoter-driven luciferase reporter into the cpc-3^KO and cpc-1^KO strains. As shown in Figure 1B and Figure 1—figure supplement 2A, B, the robust circadian rhythms of luciferase activity seen in the WT strain were severely dampened or arrhythmic in the cpc-3^KO and cpc-1^KO 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 cpc-3^KO and cpc-1^KO strains in the presence of 3-AT (WT: p = 5.00E−08, cpc-3^KO: p = 0.0016, cpc-1^KO: p = 0.0004) (Figure 1C and Figure 1—figure supplement 2C). The statistical tests of circadian rhythms were performed using a circadian statistical analysis tool CircaCompare (Parsons et al., 2020) (see Materials and methods). Northern blot analysis showed that the circadian rhythms of frq mRNA in the cpc-3^KO and cpc-1^KO strains were also dampened in the presence of 3-AT (WT: p = 6.56E−05, cpc-3^KO: p = 0.0039, cpc-1^KO: p > 0.05) and the levels of frq mRNA were constantly low in DD (Figure 1D and Figure 1—figure supplement 2D). Together, these results suggest that the GCN2 pathway is required for a functional clock by regulating rhythmic frq transcription in response to amino acid starvation.

Since 3-AT treatment resulted in low frq mRNA levels in the cpc-3^KO and cpc-1^KO strains (Figure 1D), we first examined the protein levels of WC-1 and WC-2 and found that their levels were higher in the mutants than those in the WT strain at different time points (Figure 2A, B). We then performed WC-2 chromatin immunoprecipitation (ChIP) assays to examine whether the WCC binding to the frq promoter was affected. As shown in Figure 2—figure supplement 1A, WC-2 rhythmically bound to the frq C-box in the WT, cpc-3^KO, and cpc-1^KO strains without 3-AT treatment (WT: p = 2.60E−07, cpc-3^KO: p = 0.0016, cpc-1^KO: p = 1.06E−05). However, 3-AT treatment resulted in constant low levels of WC-2 binding to the frq C-box during a circadian cycle in both cpc-3^KO and cpc-1^KO strains (Figure 2C, D). These results indicate that the loss of circadian rhythms in the cpc-3^KO and cpc-1^KO strains under amino acid starvation is caused by loss of rhythmic frq transcription, due to impaired WCC binding at the frq promoter.

Because WCC phosphorylation inhibited its transcriptional activation activity (He et al., 2006; He and Liu, 2005; Lee et al., 2000; Schafmeier et al., 2005; Wang et al., 2019), we also examined WCC phosphorylation profiles and found that 3-AT treatment resulted in hypophosphorylation of WC-1 and WC-2 (which is usually associated with WCC activation) in the cpc-3^KO and cpc-1^KO strains (Figure 2—figure supplement 1B, C). Thus, their reduced WCC binding at the frq promoter is not caused by WCC hyperphosphorylation. It should be noted that the overall phosphorylation status of WCC does not always reflect its activity in driving frq transcription, which is possibly due to the unknown function of multiple key phosphosites on WCC (Wang et al., 2019; Zhou et al., 2018).

The low WC-2 binding at the frq promoter prompted us to examine the chromatin structure of the frq promoter. We first performed ChIP assay to examine the histone and its acetylation levels at the frq promoter in the WT strain. Although amino acid starvation had little effect on the histone H2B levels (Figure 3A), it led to significantly decreased histone H3 acetylation levels (H3 acetylated at the N-terminus) at the frq promoter at high concentrations of 3-AT (Figure 3B). These results suggest that amino acid starvation can affect frq transcription by reducing the histone acetylation levels at the frq promoter.

We then examined whether the CPC-3 and CPC-1 signaling pathway was involved in regulating chromatin structure at the frq promoter in response to amino acid starvation. Histone H2B and H3ac ChIP assays at different circadian time points showed that the relative histone H3ac levels were slightly decreased in the cpc-1^KO strain compared to the WT strain under normal condition (Figure 2—figure supplement 1D). H2B levels were not markedly different between the WT and cpc-1^KO strains in the presence of 3 mM 3-AT (Figure 3C). However, the relative histone H3ac levels were very different in these two strains in the presence of 3 mM 3-AT: it was rhythmic with a peak at DD18 in the WT strain but was dramatically reduced and arrhythmic in the cpc-1^KO strain at different time points in DD (WT: p = 0.0168, cpc-1^KO: p > 0.05) (Figure 3D). These results indicate that CPC-1 is required for maintaining the proper histone acetylation status at the frq promoter under amino acid starvation. The low H3ac levels at the frq promoter, which is critical for transcription activation, results in constant low WCC binding and arrhythmic frq transcription in the cpc-1^KO strain.

To determine how CPC-1 is involved in regulating histone acetylation levels at the frq locus, we examined the occupancy of CPC-1 at the frq promoter by ChIP assays using CPC-1 antibody. As shown in Figure 4A and Figure 4—figure supplement 1A, CPC-1 was found to be rhythmically enriched at the frq promoter in the WT strain but not in the cpc-1^KO strain under normal (WT: p = 8.37E−06, cpc-1^KO: p > 0.05) 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 (Figure 4—figure supplement 1B), suggesting that CPC-1 and WCC bind independently to the frq promoter.

How does the GCN2 signaling pathway regulate histone acetylation in response to amino acid starvation? The yeast GCN4 was previously shown to recruit the histone acetyltransferase GCN5 containing (Spt-Ada-Gcn5 acetyltransferase) SAGA complex to selective gene promoters, likely through its physical interaction with the ADA2 subunit (Barlev et al., 1995; Drysdale et al., 1998; Kuo et al., 2000). To test this possibility, we performed Co-IP assay to check the interaction between CPC-1 and the SAGA complex in Neurospora in strains expressing the epitope-tagged Neurospora SAGA homologs. As shown in Figure 4B, Myc-tagged GCN-5 was efficiently immunoprecipitated by the Flag-tagged ADA-2, indicating the existence of an SAGA complex in Neurospora. Although the Myc.GCN-5, MYC.CPC-1 or Flag.ADA-2 protein levels were repressed by 3 mM 3-AT treatment (potentially due to global translational inhibition by eIF2α phosphorylation) (Karki et al., 2020), the interaction between GCN-5 and ADA-2 was almost the same under either normal or amino acid starved conditions (IP was normalized with Input). Importantly, Myc.CPC-1 was also found to associate specifically with Flag.ADA-2 with/without 3-AT treatment (Figure 4C), suggesting that CPC-1 can recruit the SAGA complex to the frq promoter through its ADA-2 subunit to regulate histone acetylation levels at the frq locus under normal or amino acid starvation conditions. Furthermore, immunoprecipitation assays showed that WC-1 and WC-2 also interacted with Myc.GCN-5 (Figure 4—figure supplement 1C). These results suggest that CPC-1 can regulate histone acetylation by recruiting the SAGA complex to the frq promoter.

To further confirm if CPC-1 can recruit GCN5 to the frq promoter, we performed ChIP assay to examine the occupancy of GCN-5 at the frq promoter. As shown in Figure 4D, Myc-tagged GCN-5 rhythmically bound at the frq promoter in DD in the WT strain but its binding was constantly low and arrhythmic in the cpc-1^KO strain (WT: p = 0.0006, cpc-1^KO: p > 0.05), 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.

GCN-5 has been shown to regulate light induction and oxidative stress response in Neurospora (Grimaldi et al., 2006; Qi et al., 2018), but its role in the circadian clock remains unclear. To determine the function of GCN-5 in the circadian clock, we created the gcn-5 knockout mutant, and found that it exhibited slow growth and lacked a conidiation rhythm (Figure 5A). To determine its circadian clock at the molecular level, we introduced the FRQ-LUC reporter (luciferase fused at the C terminus of the FRQ protein) into the gcn-5^KO strain (Larrondo et al., 2015). As shown in Figure 5B and Figure 5—figure supplement 1B, a robust circadian rhythm of luciferase activity was observed in the WT strain, but it was quickly dampened after 1 day in DD and became arrhythmic in the gcn-5^KO strain. Western blot analysis showed that, after the initial light/dark transition, the rhythmic FRQ abundance and phosphorylation were significantly dampened in the gcn-5^KO mutant (WT: p = 5.00E−8, gcn-5^KO: p = 0.0016) (Figure 5C). Reverse Transcription Quantitative PCR RT-qPCR analysis showed that the circadian rhythms of frq mRNA were also severely dampened in the gcn-5^KO strain without (WT: p = 8.37E−06, gcn-5^KO: p > 0.05) or with 3 mM 3-AT (WT: p = 5.39E−12, gcn-5^KO: p > 0.05) (Figure 5D, E). Together, these results indicate that GCN-5 is critical for maintaining the function of circadian clock.

ChIP assay results showed that the H3ac levels were significantly decreased at the frq promoter, and their rhythmic occupancies were severely dampened in the gcn-5^KO strain compared with the WT strain (WT: p = 8.10E−05, gcn-5^KO: p > 0.05) (Figure 5F). Furthermore, the rhythmic WC-2 binding at the frq C-box of the WT strain was dramatically reduced in the gcn-5^KO strain in DD (WT: p = 0.0003, gcn-5^KO: p = 0.0459) (Figure 5G). These results indicate that GCN-5 is critical for circadian clock function by regulating rhythmic chromatin structure changes to allow rhythmic WC-2 binding at the frq promoter to drive rhythmic frq transcription.

Since ADA-2 interacts with GCN-5 and it is a subunit of the SAGA complex, we also created the Neurospora 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 ada-2^KO strain were very similar to those of the gcn-5^KO strain (Figure 5—figure supplement 1A–F). Together, these results demonstrate the importance of GCN-5 and ADA-2 in the Neurospora circadian clock function.

Our results above suggest that amino acid starvation results in low histone acetylation levels at the frq promoter, which impairs the WC-2 binding and rhythmic frq transcription in the cpc-3^KO and cpc-1^KO mutants. To further confirm this conclusion, we hypothesized that the circadian clock defects under amino acid starvation should be rescued by increasing the histone acetylation levels. Trichostatin A (TSA) is a histone deacetylase (HDACs) inhibitor, which can inhibit HDACs activity and increase the histone acetylation levels in Neurospora (Selker, 1998). We treated the WT strain with different concentrations of 3-AT and TSA, and found that high concentrations of 3-AT lengthened the circadian period of the WT strain to ~24 hr (Figure 6A), but high concentrations of TSA resulted in a slight period shortening to 21 hr (Figure 6B). When TSA was used together with a high concentration of 3-AT (7.5 mM), the long period phenotype can be gradually rescued by increasing TSA concentrations (Figure 6C), which is consistent with our conclusion that the histone acetylation levels changes are responsible for the circadian clock defects caused by amino acid starvation.

GCN-5 is the major histone acetyltransferase responsible for histone acetylation at the frq locus in response to amino acid starvation. On the other hand, the histone deacetylase HDA-1 was previously reported as a major histone deacetylase that can antagonize and compete with GCN-5 for recruitment to promoters to deacetylate H3 (Islam et al., 2011; Vogelauer et al., 2000). ChIP assays showed that H3ac levels were indeed significantly decreased in the gcn-5^KO strain and were markedly increased in the hda-1^KO strain at the frq promoter region (Figure 6D), indicating that HDA-1 is responsible for histone deacetylation at the frq locus. Race tube assays showed that in contrast to period lengthening in the WT strain by 3-AT (Figure 6A), the hda-1^KO strain exhibited nearly normal circadian period even in the presence of high 3-AT concentrations (Figure 6E), suggesting that reduced histone deacetylation can partially rescue the circadian clock defects in response to amino acid starvation. To further confirm our conclusion, we treated the cpc-3^KO strains with different concentrations of TSA and found that the arrhythmic circadian conidiation rhythm caused by 3-AT treatment could be partially rescued by TSA treatments (Figure 6F). Together, these results strongly suggest that the amino acid starvation induced clock defects in the GCN2 signaling pathway mutants are largely due to the decreased histone acetylation at the frq promoter, which prevents efficient WC-2 binding and disrupts the rhythmic frq transcription.

Circadian clock has been shown to control metabolic processes and rhythmic transcription of metabolic genes (Baek et al., 2019; Hurley et al., 2014; Hurley et al., 2018). To determine the role of the GCN2 pathway in controlling gene expression under amino acid starvation, we performed RNA-seq experiments to analyze the genome-wide mRNA levels in the WT and cpc-1^KO strains in the presence of 3-AT (12 mM). As shown in Figure 7A, 22.1% of genes were found to be upregulated and 11.2% of genes were downregulated in the WT strain after 3-AT treatment compared with normal condition. Specifically, those genes involved in the regulation of oxidoreductase and amino acid metabolism were particularly enriched and were mostly upregulated during the amino acid starvation (Figure 7B). However, the differential expression of the 148 upregulated and 127 downregulated genes found in the WT strain was abolished in the cpc-1^KO strain after 3-AT treatment, suggesting that these genes were regulated by CPC-1 under amino acid starvation (Figure 7D and Figure 7—figure supplement 1A, B). Similar to those in Saccharomyces cerevisiae (Natarajan et al., 2001), genes of amino acid biosynthetic pathways, vitamin biosynthetic enzymes, peroxisomal components, and mitochondrial carrier proteins were also identified as CPC-1 targets. Among them, the genes involved in amino acid and vitamin metabolism were particularly enriched and were mostly upregulated during the amino acid starvation (Figure 7E). For example, amino acid synthesis genes his-3 (NCU03139), arg-1 (NCU02639), trp-3 (NCU08409), and ser-2 (NCU01439) were upregulated in the WT strain, but unchanged in the cpc-1^KO strain under amino acid starvation (Figure 7A, C).

To examine whether these CPC-1 activated genes were controlled by circadian clock, we re-analyzed and compared our identified CPC-1 target genes with previously published RNA-seq data of rhythm samples (Hurley et al., 2014; Hurley et al., 2018). As shown in Supplementary file 1, we summarized the rhythmic expression of 79 upregulated and 67 downregulated CPC-1 target genes based on the eJTK Cycle results of Hurley et al., 2018 (its Supplemental Datasets 1 and 2). We re-analyzed the rhythmicity of CPC-1-targeted genes (148 upregulated and 127 downregulated) using CircaSingle (Parsons et al., 2020), and added the p-values in Supplementary file 1. There were 146 rhythmic genes based on the eJTK Cycle, and 132 rhythmic genes based on the CircaSingle. As expected, there were 106 overlapping genes between these two sets of data, confirming the results using eJTK Cycle. Thus, we performed further analysis based on the data from eJTK Cycle. There were about 146/275 (53%) of the CPC-1 up- and downregulated genes under amino acid starvation exhibiting rhythmicity, indicating a highly significant enrichment of CPC-1 regulated genes as clock-controlled genes (p = 3.341905e−06, hypergeometric distribution test). Furthermore, 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 his-3 (NCU03139), trp-3 (NCU08409), and ser-2 (NCU01439) genes (WT (0 mM 3-AT): p > 0.05, WT (3 mM 3-AT): p > 0.05, cpc-1^KO (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) (WT: p = 2.21E−05, cpc-1^KO: p > 0.05), arg-1 (NCU02639) (WT: p = 0.0097, cpc-1^KO: p > 0.05), and trp-3 (NCU08409) (WT: p = 0.0009, cpc-1^KO: p > 0.05) were rhythmically expressed in the WT but not cpc-1^KO strain under amino acid starvation (Figure 7F). In addition, we also found that ser-2 (NCU01439) (WT: p = 2.25E−05, cpc-1^KO: p > 0.05) exhibited rhythmic expression in the WT but not in the cpc-1^KO strain under amino acid starvation (Figure 7—figure supplement 1D), 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. Next, we performed RT-qPCR experiments to detect the mRNA levels of amino acid synthesis genes under normal condition (0 mM 3-AT), and found that 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 (Figure 7—figure supplement 1E). Together, these results suggest that the GCN2 signaling pathway functions to maintain the robust circadian clock and rhythmic expression of metabolic genes under amino acid starvation.

The 87-3 (ras-1^bd, a) and 301-6 (ras-1^bd, his-3⁻, A) strain was used as the wild-type (WT) strain in this study. Knockout mutants were all generated based on the ras-1^bd background (Belden et al., 2007). cpc-3^KO (NCU01187), cpc-1^KO (NCU04050), gcn-5^KO (NCU10847), ada-2^KO (NCU04459), and hda-1^KO (NCU01525) strains were obtained from the Fungal Genetic Stock Center (FGSC) and were crossed with a ras-1^bd strain to create the ras-1^bd;cpc-3^KO, ras-1^bd;cpc-1^KO, ras-1^bd;gcn-5^KO, ras-1^bd;ada-2^KO, and ras-1^bd;hda-1^KO strains (Colot et al., 2006). Constructs with the cpc-1 promoter driving expression of Myc.CPC-1 were introduced into the cpc-1^KO 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 (He et al., 2020). Positive transformants were identified by western blot analyses, and homokaryon strains were isolated by microconidia purification with 5 µm filters.

Liquid cultures were grown in minimal media (1× Vogel’s, 2% glucose). For rhythmic experiments, Neurospora was cultured in petri dishes in liquid medium for 2 days. The Neurospora mats were cut into discs and transferred into medium-containing flasks and were harvested at the indicated time points.

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 Neurospora strain could be calculated according to the ratio between the distance of marked conidia band positions and the distance of conidiation bands.

The luciferase reporter assay was performed as reported previously (Gooch et al., 2008; Larrondo et al., 2015; Liu et al., 2017). The luciferase reporter construct (frq-luc) containing the luciferase gene under the control of the frq promoter, was introduced into 301-6, cpc-3^KO or cpc-1^KO strains by transformation. The luciferase reporter construct (FRQ-LUC) containing a luciferase fused to the C terminus of the FRQ protein, was introduced into gcn-5^KO or ada-2^KO strains by crossing. Firefly luciferin (final concentration of 50 μM) was added to autoclaved FGS-Vogel’s medium containing 1× FGS (0.05% fructose, 0.05% glucose, 2% sorbose), 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.

Protein extraction, quantification, and western blot analyses were performed as previously described (Liu et al., 2017). Briefly, tissues were ground in liquid nitrogen with a mortar and pestle and suspended in ice-cold extraction buffer (50 mM Hydroxyethylpiperazine Ethane Sulfonic Acid HEPES (pH 7.4), 137 mM NaCl, 10% glycerol) with protease inhibitors (1 μg/ml Pepstatin A, 1 μg/ml Leupeptin, and 1 mM phenylmethylsulfonyl fluoride PMSF). After centrifugation, protein concentrations were measured using protein assay dye (Bio-Rad). For western blot analyses, equal amounts of total protein (40 μg) were loaded in each protein lane of 7.5% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels containing a ratio of 37.5:1 acrylamide/bisacrylamide. After electrophoresis, proteins were transferred onto PVDF membranes, and western blot analyses using FRQ, WC-1, WC-2, P-eIF2α (Abcam, ab32157), and CPC-1 antibodies were performed. Western blot signals were detected by X-ray films and scanned for quantification.

To detect the phosphorylation levels of WC-1 and WC-2, PPase inhibitors (25 mM NaF, 10 mM Na₄P₂O₇·10H₂O, 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.

RNA was extracted with Trizol and further purified with 2.5 M LiCl as described previously (Liu et al., 2017). For Northern blot analysis, equal amounts of total RNA (20 μg) were loaded onto agarose gels. After electrophoresis, the RNA was transferred onto nitrocellulose membrane. The membrane was probed with [³²P] UTP (PerkinElmer)-labeled RNA probes specific for frq. RNA probes were transcribed in vitro from PCR products by T7 RNA polymerase (Invitrogen, AM1314M) with the manufacturer’s protocol. The frq primers used for the template amplification are shown in Key Resources Table.

For RT-qPCR, the cultures of WT and cpc-1^KO strains were collected at the indicated time points in constant darkness in liquid growth medium (1× Vogel’s, 2% glucose). RT-qPCR were performed as previously described (Cui et al., 2020). Each RNA sample (1 μg) was subjected to reverse transcription with HiScript II reverse transcriptase (Vazyme, R223), and then amplified by real-time PCR (Bio-Rad, CFX96). For RT-qPCR, primers target the coding genes of 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.

Two CPC-1 peptides (SELDLLDFATFDGG and RDKPLPPIIVEDPS) were synthesized and used as the antigens to generate rabbit polyclonal antisera (ABclonal) as described previously (Cui et al., 2020; Zhou et al., 2013).

Immunoprecipitation analyses were performed as previously described (Cao et al., 2018; Cheng et al., 2001b). Briefly, Neurospora proteins were extracted as described above. For each immunoprecipitation reaction, 2 mg protein and 2 μl c-Myc (TransGen, HT101), 2 μl Flag (Sigma, F1804), or 2 μl WC-2 antibody (Cheng et al., 2001a) were used. After incubation with antibody for 3 hr, 40 μl GammaBind G Sepharose beads (GE Healthcare, 17061801) were added, and samples were incubated for 1 hr. Immunoprecipitated proteins were washed three times using extraction buffer before western blot analysis. IP experiments were performed using cultures harvested in constant light.

ChIP assays were performed as previously described (Cui et al., 2020; Zhou et al., 2013) with 1 mg protein used for each immunoprecipitation reaction. The ChIP reaction was carried out with 2 μl WC-2 (Cheng et al., 2001a), CPC-1, H2B (Abcam, ab1790), or H3ac (Millipore, 06-599) antibody. Immunoprecipitated DNA was quantified by real-time qPCR. Occupancies were normalized by the ratio of ChIP to Input DNA. ChIP was performed using 2 μl c-Myc monoclonal antibody (TransGen, HT101) to examine occupancy of Myc.GCN-5. Occupancies of ChIP were normalized using IgG. The primers used for ChIP-qPCR are shown in Key Resources Table.

The WT and cpc-1^KO strains were cultured with or without 12 mM 3-AT treatment in constant light. Total RNAs were extracted using Trizol reagents. Libraries were prepared according to the manufacturer’s instructions and analyzed using 150 bp paired-end Illumina sequencing (Annoroad Gene Technology, Beijing). 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 (False Discovery Rate FDR <0.1) 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.

Quantification of western blot data were performed using Image J software. Error bars are standard deviations for ChIP assays from at least three independent technical experiments, and standard error of means for race tube assays from at least three independent biological experiments, unless otherwise indicated. Statistical significance was determined by Student’s 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/↗) (Liu et al., 2021; Parsons et al., 2020). CircaCompare was used to compare the differences between two rhythmic datasets, and CircaSingle was used to re-analyze the rhythmic parameters of the RNA-seq results by eJTK Cycle. Amplitude refers to half of the difference between the peak and trough of a given response variable, phase refers to the time at which the response variable peaks, and mesor refers to the rhythm-adjusted mean level of a response variable around which a wave function oscillates. Statistical tests for the presence of rhythmicity and statistically significant differences between two groups of rhythmic parameters are indicated by p-values. A p-value <0.05 indicates the presence of rhythmicity, but a p-value >0.05 indicates the loss of rhythmicity. The results of the CircaSingle statistical tests are added in Supplementary file 1. The results of the CircaCompare statistical tests are summarized in Supplementary file 2.

All 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 Hurley et al., 2014 were previously deposited to the NCBI SRA under accession number SRP046458 (Hurley et al., 2014). Materials are available from the corresponding authors upon reasonable request.