Transcription factor CBF-1 helps control daily gene activity by influencing the clock protein complex at the frq gene

To identify new components that regulate the transcription of clock gene frq in N. crassa, we generated viable knockout mutants of transcription factors and performed race tube assays to screen for mutants with defects in circadian conidiation rhythms. We found that the deletion of cbf-1 gene (NCU08999) resulted in 2-hour longer period and much lower amplitude of circadian conidiation rhythm than those of the wild-type strain (Fig 1A). To confirm the period of the cbf-1 mutant at the molecular level, we introduced a plasmid that carries a luciferase reporter construct (frq-luc) into the cbf-1^KO strain at the his-3 locus. As shown in Fig 1B, the bioluminescence rhythm of the cbf-1^KO, frq-luc strains was of very low amplitude and long period comparing to the wt, frq-luc strains. Sequence alignment revealed that the helix-loop-helix (HLH) region of CBF-1 protein is highly conserved from yeast to mammals (Fig 1C).

To determine how CBF-1 influences the circadian clock, we examined the FRQ expression profile at different time points in constant darkness (DD). FRQ protein levels were lower in the cbf-1^KO strain than in the wild-type strain in DD. Moreover, the peak of FRQ protein was delayed in the cbf-1^KO strain relative to the wild-type strain (Fig 1D), which is consistent with its long period phenotype. The levels of frq mRNA in the cbf-1^KO strain were increased in DD (Fig 1E), suggesting that CBF-1 suppresses frq transcription. We then evaluated FRQ stability after the addition of the protein synthesis inhibitor cycloheximide (CHX). FRQ stability was similar in the cbf-1^KO strain and the wild-type strain (S1 Fig). The mechanism that results in low FRQ protein levels but high frq mRNA levels in the cbf-1^KO strain is unknown, but these molecular data indicate that the circadian clock is dampened in the cbf-1^KO strain due to impaired regulation of frq expression.

To further confirm the function of CBF-1 in the circadian clock, a construct in which the expression of Myc-tagged CBF-1 is driven by quinic acid (QA)-inducible qa-2 promoter was introduced to the cbf-1^KO strain. We quantified Myc-CBF-1 expression in growth medium containing different QA concentrations (0 to 10⁻² M). The amount of Myc-CBF-1 with 10⁻⁷ M QA and without QA (S2A Fig) partially restore the conidiation rhythm of cbf-1 mutant (Fig 2A), indicating that the low levels of Myc-CBF-1 partially rescue the circadian conidiation defects of cbf-1^KO strain. The conidiation rhythm of the cbf-1^KO, qa-Myc-CBF-1 strain was completed rescued when the QA concentration was between 10⁻⁶ and 10⁻⁴ M in the medium. QA at 10⁻³ M, however, resulted in a slow growth rate and arrhythmic conidiation rhythm in the cbf-1^KO, qa-Myc-CBF-1 strain (Fig 2A), suggesting that the proper level of CBF-1 is critical for circadian clock function.

QA-inducible gene expression was known to be suppressed by catabolites [38]. To determine whether the leaky expression of Myc-CBF-1 is subjected to catabolite repression, we evaluated the conidiation rhythm of the cbf-1^KO, qa-Myc-CBF-1 strain in different concentrations of glucose in the race tube medium without QA. The race tube phenotype of the cbf-1^KO, qa-Myc-CBF-1 strain was not affected by glucose concentration (S3 Fig), suggesting that the leaky expression of Myc-CBF-1 in the absence of QA is probably unrelated to a glucose effect.

To verify whether the levels of CBF-1 protein are important for conidiation rhythm, the pqa-Myc-CBF-1 plasmid was introduced into the wild-type stain. Addition of QA (0 to 10⁻² M) resulted in increased levels of Myc-CBF-1 in a dose-dependent manner (S2B Fig). As expected, the race tube assay showed that QA (10⁻³ M) induced expression of high levels of Myc-CBF-1 in the wt, qa-Myc-CBF-1 strain and that the overexpression of Myc-CBF-1 resulted in growth and circadian conidiation defects (Fig 2B). To further confirm these results, we introduced a luciferase reporter construct (frq-luc) into the wt, qa-Myc-CBF-1 strain. Overexpression of CBF-1 in the wild-type strain resulted in very low amplitude circadian bioluminescence rhythm (Fig 2C). Thus, CBF-1 is critical for the clock function and high levels of CBF-1 protein interfere with growth and circadian clock function in N. crassa.

We then examined the rhythmic expression of FRQ protein and frq mRNA in the wild-type and wt, qa-Myc-CBF-1 strains. High levels of Myc-CBF-1 were induced in the presence of 10⁻³ M QA (S2C Fig) at different time points in DD. Overexpression of Myc-CBF-1 caused a marked decrease in FRQ expression, and FRQ cycling amplitude was also affected (Fig 2D). Northern blot showed that the level of frq mRNA was also reduced in the wt, qa-Myc-CBF-1 strain compared to that in wild-type strain in the first 24 hours (Fig 2E), indicating that CBF-1 suppresses frq transcription. After 24 hours in DD, however, the level of frq mRNA was comparable in mutant and wild-type strains, suggesting that overexpressed CBF-1 also affected FRQ expression at a post-transcriptional level.

The low levels of FRQ protein in the wt, qa-Myc-CBF-1 strain promoted us to examine the expression of WC-1 and WC-2 in this strain. The levels of WC-1 and WC-2 proteins in the wt, qa-Myc-CBF-1 strain were lower than those in the wild-type strain (S4A Fig). However, the levels of wc-1 and wc-2 mRNA were similar (S4B Fig). These results suggest that overexpressed CBF-1 negatively regulates the levels of WC-1 and WC-2 proteins in a post-transcriptional manner.

To further investigate whether CBF-1 directly regulates frq transcription by binding to the frq promoter, we performed electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays. For the EMSA assay, purified GST-CBF-1 or GST-CBF-1^{Δ(187–214)} fusion proteins or GST only (S5A Fig) were incubated with a radioactively labeled C-box oligonucleotide probe. A migrating complex was observed when the GST-CBF-1 fusion protein was present, but not when GST or the GST-CBF-1^{Δ(187–214)} fusion proteins were used (Fig 3A). The interaction was specific for the C-box sequence as an unlabeled C-box oligonucleotide disrupted the complex, but an oligonucleotide with a different sequence did not (Fig 3A). These results suggest that CBF-1 binds directly to the C-box of frq promoter sequence.

To test whether CBF-1 binds to the frq promoter in vivo, we generated a CBF-1-specific antibody, which recognized a specific band at predicted molecular weight in the wild-type strain but not in the cbf-1^KO strain (Fig 3B). A ChIP assay using this antibody showed that the enrichment of CBF-1 at the C-box of frq promoter was specific and rhythmic, peaking at DD14 when frq transcription and WCC binding are high (Fig 3C). The levels of CBF-1 protein, however, were constant (Fig 3D).

Because both CBF-1 and WCC are transcription factors and rhythmically bind to the C-box, we evaluated the relationship between the two factors in frq promoter binding. We examined the binding of CBF-1 to the frq promoter in wc-1^KO, wc-2^KO, and frq⁹ mutant strains. In the latter, a frame-shift mutation of frq results in truncated FRQ protein and defective negative feedback loop [39]. The binding of CBF-1 to the C-box was constantly low in the wc-1^KO and wc-2^KO strains but was constantly high in the frq⁹ mutant strain with high levels WCC activity (Fig 3E and 3F). Moreover, the CBF-1 protein levels were not altered in these mutants (Fig 3G). These data suggested that the rhythmic CBF-1 association with the frq promoter is regulated by WCC activity.

To determine how CBF-1 regulates frq transcription, we performed ChIP assays using WC-1 and WC-2 antibodies [27]. Our results showed that WCC rhythmically bound to the C-box of the frq promoter in DD with a peak at DD14 in wild-type strain (Fig 4A and 4B). However, the robust rhythmic binding of WCC to the C-box was dramatically decreased with a low amplitude and delayed peak in the cbf-1^KO strain (Fig 4A and 4B). Previous studies showed that hypophosphorylated WC-1 and WC-2 efficiently bound to the C-box activating frq transcription [16, 17] and that hyperphosphorylated WC-1 and WC-2 had lower affinity for the C-box of frq promoter in DD [40]. Western blots showed that WC-1 and WC-2 were hyperphosphorylated in the cbf-1^KO strain compared with the wild-type strain throughout DD (Fig 4C). However, we observed no significant differences in levels of WC-1 or WC-2 in the cbf-1^KO strain compared to those in the wild-type strain (S5B Fig). These results suggest that the loss of CBF-1 decreases WCC activity by promoting phosphorylation of WC-1 and WC-2. A previous study showed that hyperphosphorylation of WC-1 and WC-2, which was mediated by FRQ, resulted in less binding to the C-box of frq [41]. Thus, the low WCC activity in the cbf-1^KO strain may be mediated by FRQ.

The decreased binding of WC-1 and WC-2 at C-box of frq promoter and the high levels of frq mRNA in the cbf-1^KO strain prompted us to examine whether there is WC-independent frq transcription in the cbf-1^KO mutant. Thus, we generated the cbf-1^KOwc-2^KO double mutant and compared FRQ levels with those in the wc-2^KO single mutant. As expected, constant levels of FRQ expression were observed in cbf-1^KOwc-2^KO double mutant but not in wc-2^KO single mutant (Fig 4D). Further, constant high levels of frq mRNA were also observed in the cbf-1^KOwc-2^KO double mutant (Fig 4E), indicating that CBF-1 is required for suppression of WC-independent transcription of frq. These results indicate that the constant expression of FRQ in the cbf-1^KO strain mediates the hyperphosphorylation of WC proteins, resulting in decreased binding of WCC to the C-box of the frq promoter. WC-independent frq expression leads to the increased frq mRNA level in the cbf-1 mutant. However, low luc expression levels in the cbf-1^KO, frq-luc strains suggest the lack of WC-independent transcription. The entire ORF and 3’UTR of frq are replaced by luc in frq-luc transgene and the frq-luc transgene is targeted to the his-3 locus. So, the lack of WC-independent transcription of the frq-luc reporter may be caused by the chromatin state of the frq-luc locus being different from the chromatin state of the frq locus [27].

Our previous study showed that the transcriptional co-repressor RCM-1 suppresses WC-independent frq transcription by binding to the frq locus and that hyperphosphorylation of RCM-1 impairs its suppressor activity [28]. The level of RCM-1 enrichment at the C-box of frq promoter was dramatically reduced and RCM-1 became hyperphosphorylated in the cbf-1^KO strain compared to the wild-type strain (Fig 4F and 4G), but the levels of RCM-1 were similar in the two strains (Fig 4H). Therefore, the WC-independent frq transcription in the cbf-1^KO strain may be caused at least partially by hyperphosphorylation of RCM-1.

Our results suggest that the decreased binding of WCC to the C-box in the cbf-1^KO strains is due to FRQ-mediated hyperphosphorylation of WCC. To further test whether CBF-1 directly regulates WCC binding of the C-box independent of FRQ, we generated the cbf-1^KOfrq⁹ double mutant (S6A Fig). A ChIP assay showed that WC-2 was significantly enriched at the C-box of the cbf-1^KOfrq⁹ double mutant compared to the frq⁹ single mutant (Fig 5A) even though the WC levels were similar in the two strains (S6B Fig). The results indicate that CBF-1 can suppress WC-2 binding at the C-box independently of FRQ. The levels of frq mRNA were higher in the cbf-1^KOfrq⁹ double mutant than in the frq⁹ single mutant (Fig 5B). WC-1 and WC-2 were both hypophosphorylated in the cbf-1^KOfrq⁹ double mutant and in the frq⁹ single mutant (Fig 5C), suggesting that the increased binding of WC-2 to the frq C-box was due to the absence of CBF-1 protein but was not affected by WC phosphorylation status. Taken together, these results suggest that CBF-1 suppresses WCC binding to the C-box of frq promoter through a FRQ-independent mechanism.

The HLH DNA binding domains are conserved in all CBF-1 homologues from yeast to human (Fig 1C). To test the role of CBF-1 binding to the frq promoter, we generated FLAG-tagged CBF-1 constructs that contain a E195A point mutation or deletion of the 187–214 amino acid region in the HLH domain [42]. Unlike the wild-type FLAG-CBF-1 protein, the mutant FLAG-CBF-1 proteins failed to rescue the long period conidiation phenotype of the cbf-1^KO strain (Fig 6A and S7A Fig), suggesting that the DNA binding activity of CBF-1 is required for its circadian clock function. A ChIP assay showed that CBF-1 enrichment at the C-box in cbf-1^KO, pcbf-1-FLAG-CBF-1^E195A, and cbf-1^KO, pcbf-1-FLAG-CBF-1^{Δ(187–214)} strains was abolished (Fig 6B). These results demonstrate that CBF-1 regulates the circadian clock via its HLH DNA binding domain. Similarly, neither the defect of WCC binding to the C-box nor the hyperphosphorylation of WC-1 and WC-2 was rescued by mutant FLAG-CBF-1 proteins (Fig 6C–6F). The levels of WC proteins were not affected in the cbf-1^KO mutants (S7B and S7C Fig). These results suggest that CBF-1 binding at the C-box of frq promoter is required for its role in the circadian clock.

To determine whether the decreased levels of frq mRNA in the wt, qa-Myc-CBF-1 strain is caused by impaired WCC recruitment to the C-box of frq promoter, we performed ChIP assays with WC-1 and WC-2 antibodies. ChIP data showed that robust rhythmic binding of WCC to the C-box was markedly decreased by the overexpression of Myc-CBF-1 in the wt, qa-Myc-CBF-1 strain compared to the wild-type strain (Fig 7A and 7B). In contrast, ChIP assays with CBF-1 antibody showed that the recruitment of CBF-1 to the C-box of frq promoter was increased in the wt, qa-Myc-CBF-1 strain (Fig 7C). These results suggest that overexpression of CBF-1 interferes with WC recruitment to the C-box.

To confirm this conclusion, we created a wt, qa-Myc-CBF-1^E195A strain and a wt, qa-Myc-CBF-1^Δ187–214 strain. As expected, the circadian conidiation phenotype was not affected by overexpression of these mutant Myc-CBF-1 proteins that cannot bind to frq C-box (Fig 7D). Furthermore, ChIP assays with WC-1 and WC-2 antibodies showed that binding of WCC to the C-box was similar in the wt, qa-Myc-CBF-1^E195A and wt, qa-Myc-CBF-1^{Δ(187–214)} strains to that in the wild-type strain (Fig 7E and 7F). In addition, the levels of WCC and FRQ proteins were not affected in the wt, qa-Myc-CBF-1^E195A or wt, qa-Myc-CBF-1^Δ187–214 strains (S8 Fig). Together, these results are consistent with a model in which CBF-1 binding to the C-box region impairs WCC binding.

As WCC and CBF-1 appear to be mutual regulators in frq promoter binding, we tested whether they interact. Co-immunoprecipitation assays were performed using pre-immune serum as the negative control. We found that CBF-1 co-immunoprecipitated with WC-1 and WC-2, suggesting that these proteins interact to regulate frq transcription (Fig 7G and 7H). Taken together, our results suggest that the proper level of CBF-1 is critical for modulating the binding of WCC at the C-box of the frq promoter to allow rhythmic WC-dependent frq transcription.

The 87–3 (bd, a) strain was used as the wild-type strain in this study [50]. The ku70^RIP (bd, a) strain, generated previously [16], was used as the host strain for creating the cbf-1 knockout mutant. The cbf-1^KO strain was created by deleting the entire cbf-1 ORF through homologous recombination using a protocol described previously [51]. The wc-1^KO, wc-2^KO, rcm-1^RIP and frq⁹ strains, generated previously [28, 52–55], were also used in this study. The newly created cbf-1^KOwc-2^KO and cbf-1^KOfrq⁹ double mutants were obtained by crossing. The 301–6 (bd, his-3, A) and cbf-1^KO (bd, his-3, A) strains were the host strains for his-3 targeting construct transformation.

The wt, pqa-Myc-CBF-1, wt, pqa-Myc-CBF-1^E195A, and wt, pqa-Myc-CBF-1^{Δ(187–214)} strains were created by transferring pqa-Myc-CBF-1, pqa-Myc-CBF-1^E195A, and pqa-Myc-CBF-1^{Δ(187–214)} constructs into the his-3 locus of 301–6 host strain. Using the same method, the cbf-1^KO, pqa-Myc-CBF-1, cbf-1^KO, pqa-Myc-CBF-1^E195A, cbf-1^KO, pqa-Myc-CBF-1^{Δ(187–214)}, cbf-1^KO, pcbf-1-FLAG-CBF-1, cbf-1^KO, pcbf-1-FLAG-CBF-1^E195A, and cbf-1^KO, pcbf-1-FLAG-CBF-1^{Δ(187–214)} strains were generated. For each transformation, the transformants were first analyzed by western blot for the expression of tagged CBF-1 proteins, and the positive transformants were examined by race tube assays. Escherichia coli BL21 cells and pGEX-4T-1 plasmid were used for expression of GST-CBF-1 and GST-CBF-1^{Δ(187–214)} fusion proteins.

The medium for race tube assays contained 1x Vogel’s salts, 0.1% glucose, 0.17% arginine, 50 ng/mL biotin, and 1.5% agar. In the race tube medium containing QA, 0.1% glucose was replaced with the desired concentration of QA (0-10^-2 M). Strains were grown in constant light at 25°C for 24 hours before being transferred to DD at 25°C. Densitometric analyses of race tubes and calculations of period length were performed as described [56]. Growth conditions were as described previously [57]. Liquid cultures were grown in minimal medium (1x Vogel’s, 2% glucose). When QA was used, liquid cultures were grown in low-glucose medium (1x Vogel’s, 0.1% glucose, 0.17% arginine) with different concentration of QA (0-10^-2 M).

The luciferase reporter assays were performed as described previously [58, 59]. The 301–6 (bd, A), frq-luc strain was used as control strain in this study. The cbf-1^KO strains were crossed with the 301–6 (bd, A), frq-luc strain to obtain the cbf-1^KO, frq-luc strain. The luciferase reporter construct was co-transformed with a pBT6 plasmid into CBF-1 overexpression strain to obtain wt, qa-Myc-CBF-1, frq-luc strain. LumiCycle (ACTIMETRICS) and the autoclaved fructose-glucose-sucrose (FGS)-Vogel’s medium (1x FGS, 1x Vogel’s medium, 50 μg/L biotin, and 1.8% agar) containing 50 μM firefly D-luciferin were used for the luciferase assay. Conidia suspensions in water were 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 one day in DD. The data were normalized with LumiCycle Analysis Software by subtracting the baseline luciferase signal which increases as cell grows. Under our experimental conditions, luciferase signals are highly variable during the first day in the LumiCycle and become stabilized afterwards, which is likely due to an artifact caused by the light-dark transfer of the cultures. Thus, the results presented were recorded after one day in DD.

The GST-CBF-1 (amino acids M1-N136), GST-WC-1 (amino acids G291-E708), GST-WC-2 (amino acids M8-C423), GST-RCM-1 (amino acids D480-P883) [28], and GST-FRQ (amino acids S249-K315 and E359-G766) [60] fusion proteins were expressed in E. coli BL21 cells, and the recombinant proteins were purified and used as the antigens to generate rabbit polyclonal antiserum as described previously [61].

Protein extraction, quantification, western blot analyses, and co-immunoprecipitation assays were performed as previously described [62]. Equal amounts of total protein (40 μg) were loaded in each protein lane. After electrophoresis, proteins were transferred onto PVDF membrane, and western blot analysis was performed. To analyze the phosphorylation profiles of WC-1, WC-2, and RCM-1, phosphatase inhibitors were added to protein extraction buffer and 7.5% SDS-PAGE gels containing a ratio of 149:1 acrylamide/bis-acrylamide were used. Otherwise, 7.5% SDS-PAGE gels contained a ratio of 37.5:1 acrylamide/bis-acrylamide were employed.

Total RNA was extracted using Trizol, and then further purified with 2.5 M LiCl as described previously [63]. For northern blot analysis, equal amounts of total RNA (20 μg) were loaded onto agarose gels. After electrophoresis, the RNA was transferred onto Amersham Hybond-N+ membrane. The membrane was probed with ³²P-UTP-labeled RNA probes specific for frq, wc-1, or wc-2. RNA probes were transcribed in vitro from PCR products by T7 RNA polymerase. The northern primer sequences used for the template amplification were frq-N term F (5’-GGGTAGTCGTGTACTTTGTCAGGCATAGATCTC-3’), frq-N-term T7 + R (5’-TAATACGACTCACTATAGGGGGCAGGGTTACGATTGGATT-3’), wc1 F (5’-GTTATACCTGGTTTGAAAGC-3’), wc1 T7 + R (5’-TAATACGACTCACTATAGGGACAACTGTTGCATAGATCTC-3’), wc2 F (5’-CTGCAGATGACTTCCGACCC-3’), and wc2 T7 + R (5’-TAATACGACTCACTATAGGGATCTCGGTCTAGGGGAATC-3’). The T7 promoter regions are underlined.

EMSA assays were performed in a manner similar to that described previously [64]. To make the probe, oligonucleotides were dissolved in double distilled H₂O (ddH₂O) to a final concentration of 10 μM. We then mixed 10 μL each of complementary oligonucleotides with 30 μL ddH₂O and heated at 95°C for 10 minutes. After overnight at room temperature, oligonucleotide labeling was performed at 37°C for 30 minutes in 50 μL reaction with 5 μL of double-stranded oligonucleotide, 5 μL of T4 polynucleotide kinase buffer (NEB), 2.5 μL of T4 polynucleotide kinase (NEB), 7.5 μCi γ³²P-ATP and 30 μL ddH₂O. After the kinase reaction, the sample was purified using Bio-Gel P-30 chromatography columns (Bio-Rad). The oligonucleotides annealed for use as probe were ACF57 (5’-CGTCCTGATGCCGCTGCAAGACCGATGACGCTGCAAAATTGAGATCTA-3’); and ACF58 (5’-TAGATCTCAATTTTGCAGCGTCATCGGTCTTGCAGCGGCATCAGGACG-3’).

Binding reactions using BL21 cells expressing fusion protein contained 1x binding buffer [20 mM HEPES, pH 7.9, 1 mM EDTA, 2 mM MgCl₂, 10% (v/v) glycerol, 20 μM ZnCl₂], 0.1 μg poly (dI-dC), 1 μL probe, and 3 μg of GST, GST-CBF-1, or GST-CBF-1^{Δ(187–214)} fusion protein (which was added last to the binding reactions) in a total volume of 20 μL. Binding reactions were incubated for 30 minutes on ice prior to electrophoresis at 4°C on nondenaturing 4% polyacrylamide gels containing 0.5x TBE and 2.5% (v/v) glycerol. Gels were dried at 80°C for 15 minutes and were exposed it to X-ray film for 2 to 10 hours.

ChIP assays were performed as described previously [62]. Briefly, N. crassa tissues were fixed with 1% formaldehyde for 15 minutes at 25°C with shaking. Glycine was added at a final concentration of 125 mM, and samples were incubated for another 5 minutes. The crosslinked tissues are ground and resuspended at 0.5 g in 6 mL lysis buffer containing protease inhibitors (1 mM PMSF, 1 μg/mL leupeptin and 1 μg/mL pepstatin A). Chromatin was sheared by sonication to approximately 200–500 base pair fragments. A 1mL aliquot of protein solution (2 mg/mL) was used for each immunoprecipitation reaction, and 10 μL was kept as the input DNA. The chromatin immunoprecipitations were carried out with 2.5 μL WC-2, 3.5 μL WC-1, 2.5 μL RCM-1, or 3 μL CBF-1 antibodies. The corresponding knock-out strains were used as the negative controls. Immunoprecipitated DNA was quantified using real-time PCR. The primer sets used are frq C-box F (5’-GTCAAGCTCGTACCCACATC-3’) and frq C-box R (5’-CCGAAAGTATCTTGAGCCTCC-3’) were described in a previous study [29]. Occupancies were normalized by the ratio of ChIP to Input. The relative values of protein occupancy were calculated using the 2^-ΔΔCT method by comparing the cycle number for ChIP sample with that for the Input control [65].

Quantification of western blot and northern blot data were performed using Quantity One software. All experiments were performed at least three independent times. For blots, representative images are shown. Error bars are standard deviations of triplicate data. Statistical significance was determined by Student’s t test for ChIP analyses.

All relevant data are within the paper and its Supporting Information files.