Regulation of Gene Expression in Neurospora crassa with a Copper Responsive Promoter

N. crassa tcu-1 gene expression is controlled by copper availability

We examined whether the high-affinity copper transporter (tcu-1, NCU00830) is regulated by copper availability. WT N. crassa was grown in liquid media with copper sulfate (50−250 µM), or a copper chelator (BCS, 50−250 µM) and harvested after 8 hr. The levels of tcu-1 and control actin mRNAs were examined. Supporting Information, Figure S1 shows that although actin expression was unaffected by the abundance of copper, the endogenous tcu-1 message was highly responsive to copper availability, with a 40-fold expression range under these conditions. Excess copper turned down tcu-1 expression whereas increasing concentrations of the copper chelator, BCS, induced tcu-1 expression, as predicted (Korripally et al. 2010).

The kinetics of tcu-1 activation depend on the copper concentration, and the kinetics of turn off are very rapid

We investigated tcu-1 expression at several time points after the addition of 50 µM and 250 µM BCS in a WT strain. Figure 1A shows that tcu-1 was induced 4 hr after the addition of 50 µM BCS and 2 hr after the addition of 250 µM BCS. Induction of tcu-1 was quantitated in two independent experiments, and the average expression level was plotted in Figure S2. These data shows that the concentration of copper chelator affected the kinetics of induction. Maximal levels of induction occurred between 8 and 24 hr, and expression remained high at 24 hr after addition of either concentration of BCS.

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Figure 1

The kinetics of tcu-1 gene expression responses to Cu/BCS in a WT strain. (A) Levels of endogenous tcu-1 mRNA at the indicated times after the addition of 50 or 250 µM BCS or 250 µM CuSO₄ (Cu) were determined by Northern analysis. Ethidium bromide staining of the rRNA shows even loading in all panels. Quantitation of two independent experiments is shown in Figure S2. (B) Levels of tcu-1 mRNA were examined in untreated (U) or BCS treated (either 50 or 250 µM) cultures that were subsequently treated with 200 or 100 µM CuSO_4, respectively, for the indicated times by Northern analysis. (C) Induction and repression of tcu-1 are long-lasting. Cultures grown for the indicated times were either untreated (lanes 1−3), treated with 200 µM BCS for the indicated times (lanes 4−14), or treated with 200 µM BCS and then subsequently treated with 250 µM CuSO₄ for the indicated times (lanes 9−14). Levels of tcu-1 mRNA were determined by Northern analysis. Two independent experiments were analyzed with similar results.

To study the effect of gene turn down/off, it is useful to have rapid and concentration insensitive kinetics. To test whether the tcu-1 promoter fulfills this criterion, we activated tcu-1 expression with either 50 µM BCS or 250 µM BCS for 4 hr, added CuSO₄ at 200 µM or 100 µM respectively, and then analyzed the effect on tcu-1 message over time in a WT strain. Figure 1B shows that turn off of tcu-1 was robust in both experiments, with nearly all expression extinguished by 0.25 hr, and expression remained reliably low for at least 6 hr.

To examine how long tcu-1 expression is either activated by BCS or repressed by Cu, we treated cultures with 200 µM BCS for various times with or without the subsequent addition of 250 µM CuSO₄ (Figure 1C). tcu-1 expression is quite low in the untreated samples independent of the culture time. Treatment with BCS for 10 hr maximally induced tcu-1, with longer treatments yielding lower induction. However, even after 48 hr in BCS, tcu-1 is still greatly induced over the untreated samples. All samples treated with BCS first, and then treated with Cu for different amounts of time, maintained repression of tcu-1. Thus, induction of tcu-1 by BCS or repression of tcu-1 by CuSO₄ can last for at least 2 days.

Copper control of tcu-1 gene expression is highly specific

Ideally, a promoter used to drive controlled gene expression would respond primarily to the specific signal (in this case copper availability) and not to other external factors. To determine whether the tcu-1 promoter fulfills this criterion, we tested the effects of media composition and various stresses on the native expression of tcu-1 in a WT strain. We compared the expression of both tcu-1 and the quinic acid responsive gene, qa-2, under these conditions. Figure 2 shows that in 2% glucose growth conditions, BCS strongly induced tcu-1. Osmotic, oxidative, heat, and cell wall stresses as well as growth in Westergaard’s medium, which stimulates sexual development, and air exposure, which stimulates conidiation, only weakly affected the expression of tcu-1. As predicted, qa-2 was not induced in 2% glucose growth conditions, even when the inducer quinic acid was provided (Giles et al. 1985). In 0.01% glucose conditions tcu-1 was still induced by BCS. There was weak induction of tcu-1 by quinic acid, most likely resulting from the acidic environment inhibiting copper import, and thus depleting copper (Blackwell et al. 1995). As expected, qa-2 was strongly induced by quinic acid in 0.01% glucose conditions (Giles et al. 1985). Loading of the RNA samples was uniform as shown in the bottom panel, with the exceptions that the detergent treated samples were somewhat degraded, and the Westergaards and air treated samples were low. These data indicated that tcu-1 was specifically controlled by copper availability, and was generally insensitive to other tested perturbations. In addition previous studies have shown that tcu-1 gene expression is independent of glucose, light, or circadian clock control (Lewis et al. 2002; Smith et al. 2010; Xie et al. 2004). Thus, the tcu-1 promoter can be used for copper-controlled exogenous gene expression in both low- and high-glucose conditions and under many environmental stress conditions.

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Figure 2

Effects of stress and nutrition on copper and quinic acid−controlled gene expression in a WT strain. The levels of tcu-1 and qa-2 mRNA were examined by Northern analysis in control untreated (Ø), 250 µM CuSO₄ (C), 250 µM BCS (B), 10 mM quinic acid (Q), 1 µg/mL fludioxonil (F), 10 mM H₂O₂ (oxidative stress, X), 0.4M NaCl (salt, S), 0.05% SDS (detergent, D), and 30 min 42°C heat shock (HS) in 0.01% and 2% glucose conditions, as well as in 24-hr DD, a 30 min-light pulse (LP), Westergaard’s medium (W), and air exposure (A). The exposure times of the membranes with film are indicated for each blot. Ethidium bromide staining of the rRNA is shown in the bottom panel.

A PCR-based strategy to integrate P_tcu-1 in front of any target gene

Figure 3 diagrams the strategy for integrating P_tcu-1 in front of any gene. This strategy is similar to the split marker integration scheme introduced (Catlett et al. 2003) and further explored by the N. crassa gene knockout project (Colot et al. 2006). The design of the required primers is shown in Table 1. Two PCR-generated fragments are cotransformed into N. crassa that can integrate the copper promoter into the desired genomic locus (Figure 3A). Transformants are selected by virtue of their resistance to the drug glufosinate upon recombination of the bar gene (Avalos et al. 1989).

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Figure 3

Scheme for integrating P_tcu-1 to a target genomic locus. (A) An overview of the scheme shows integration of two DNA fragments containing homology to the 5′ UTR of the target gene, the bar gene, the copper transporter promoter, and finally homology to the ORF of the target gene. Three recombination events (as denoted by parallel lines) are required to properly integrate the two fragments. (B) Production of the two integration fragments is described in the results. (C) The final desired genomic locus can be confirmed by PCR using primers P9 and P4 to validate correct 5′ integration, and P5 and P10 to validate correct 3′ integration. YFG, your favorite gene.

Construction of each PCR fragment for integration depends on a two-step process, as shown in Figure 3B. PCR1 with primers P1 and P2 amplifies 500 bp of the 5′ flank of the target gene from genomic DNA. PCR2 with primers P3 and P4 amplifies the 3′ end of the bar gene from the plasmid pCR blunt bar P_tcu-1. PCR3 with primers P1 and P4 on PCR1 and PCR2 templates generates the 5′ integration fragment. PCR4 with primers P5 and P6 amplifies the 5′ end of the bar gene fused to the tcu-1 promoter from the plasmid pCRblunt bar P_tcu-1. PCR5 with primers P7 and P8 on genomic DNA amplifies 500 bp of the target gene ORF. PCR 6 with primers P5 and P8 on PCR4 and PCR5 templates generates the 3′ integration fragment. Proper integration at the 5′ and 3′ ends of the genomic locus can be verified by PCR with use of the primer sets P9 and P4 and P5 and P10, respectively (Figure 3C).

Copper control of a P_tcu-1wc-1 strain recapitulates deletion and overexpression phenotypes

As proof of principle, we chose to integrate P_tcu-1 into the wc-1 genomic locus for two reasons. First, WC-1 protein levels can be easily monitored by Western blot (Gorl et al. 2001). Second, the phenotypes of WC-1 knockout (Δwc-1) and overexpression strains (P_qa-2::wc-1) have been previously published (Cheng et al. 2001; Lee et al. 2003), thus providing predictable outcomes for P_tcu-1 control of WC-1.

The primers used to create bar::P_tcu-1::wc-1 integrating fragments are listed (Table S1). A defined 1.5-kb region of the wc-1 promoter drives clock-controlled expression (Froehlich et al. 2002, 2003); therefore primers (WC-1 P1 and WC-1 P2) were designed to delete the clock control region. In this way, the desired genomic locus should only be under P_tcu-1 control. PCR products 1 through 6 were generated and the expected fragment sizes were confirmed (Figure S3A). PCR products 3 and 6 were cotransformed into N. crassa, and glufosinate-resistant colonies were examined for proper integration. Of nine glufosinate-resistant heterokaryotic clones, three (1, 7, and 9) displayed the proper 5′ genomic structure as indicated by the production of the expected 1.5 kb-DNA fragment using WC-1 P9F and P4R primers in a PCR on genomic DNA (Figure S3B). Clones 1 and 7 also displayed the proper 3′ genomic structure as indicated by the production of the expected 3.1-kb DNA fragment using P5F and WC-1 P10 R primers in a PCR on genomic DNA. Clone 9 did not show the proper 3′ genomic structure.

To determine whether expression of WC-1 in heterokaryons containing the integrated bar::P_tcu-1::wc-1 construct was dependent on copper availability, protein extracts from strains grown in 200 µM CuSO₄ or 200 µM BCS (plus 50 µM CuSO₄) were examined by Western blot (Figure S3C). Expression of WC-1 in a control strain (Ø) and in clone #9 was independent of copper availability. Clones 1 and 7 showed clear differential regulation of WC-1 in high copper vs. BCS. We crossed clone 7 as described in the Materials and Methods to obtain a homokaryotic P_tcu-1wc-1 strain.

To determine the efficiency of P_tcu-1-driven WC-1 control, the effects of Cu and BCS on WC-1 protein levels was assayed in the P_tcu-1wc-1 strain. Our ultimate goal was to test how P_tcu-1 control of WC-1 affects rhythms in conidial development in DD (a condition in which the clock is functional). Therefore, the levels of WC-1 protein were first examined in two time points in the dark (DD18, representing subjective afternoon and DD6, representing subjective late night). Figure 4 shows an inverse correlation between copper availability and WC-1 protein in the P_tcu-1wc-1 strain, which is independent of the time in DD. WC-1 protein levels in a P_wc-1wc-1 strain were unaffected by copper availability, and were not strongly influenced by the time in DD. Although it has been reported that WC-1 protein levels peak in abundance around DD6 and display a trough around DD18 (Lee et al. 2000), the amplitude of this rhythm can be quite low (Cheng et al. 2001), so our failure to observe a clear time of day difference in just two time points is not surprising. Finally, the WC-1 expression in the P_tcu-1wc-1 strain at 10 µM BCS most closely matched expression of the native P_wc-1wc-1 strain.

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Figure 4

Effects of copper and BCS on P_tcu-1-driven WC-1 protein production. Levels of WC-1 protein were analyzed by western blot (WB) on extracts from P_wc-1wc-1 and P_tcu-1wc-1 strains treated with copper and/or BCS as indicated for 6 hr. Extracts were generated from tissue grown for a total of 24 hr with 18 hr (DD18) or 6 hr (DD6) of that time in the dark at 25. The amido black staining (Amido) of the membrane in the lower panel demonstrates equal protein loading. Protein size markers (S) are shown, with the molecular weight (kDa) as indicated. Relative WC-1 expression (±SD) from two biological replicates is plotted below.

Rhythms in conidial development are typically assayed on medium with 0.1% glucose as a carbon source (Liu et al. 1997). We were worried that the WC-1 protein might not be fully induced under these conditions given the weaker response of tcu-1 expression to BCS in 0.01% glucose medium (Figure 2). Therefore, we examined the effects of copper concentration on WC-1 protein levels in the P_tcu-1wc-1 strain at 0.1% and 2% glucose in two time points in DD (DD6 and DD18) and LL (Figure S4). WC-1 was well controlled by copper availability in both glucose concentrations, and independent of the light conditions.

Finally, to assess growth and rhythmicity, P_wc-1wc-1 and P_tcu-1wc-1 strains were inoculated on race tubes containing 200, 100, and 50 µM final CuSO₄, or 50 µM CuSO₄ with 10, 25, 50, 100, and 200 µM BCS (Figure 5A). Growth rates of both strains were only very mildly affected by changes in the availability of CuSO₄ (Figure S5), suggesting that copper toxicity or starvation is not achieved under these conditions. The P_wc-1wc-1 strain grew ~50% of the time as linear hyphae compared with conidiating aerial hyphae, and developed bands of conidia with a period of ~22.3 hr independent of copper availability (Figure 5, B and C). In high copper, the P_tcu-1wc-1 strain was largely arrhythmic with conidiation predominating (only ~10% of the growth was linear hyphae), and had a long period, when a period could be measured (~24.5 hr), as expected for strains with reduced or no expression of WC-1 (Lee et al. 2003). As copper was reduced and BCS increased, normal rhythmicity was restored at 50 µM CuSO₄ with or without 10 µM BCS. Above this level of BCS, rhythmicity became unreliable, and the oscillations accelerated (shorter period), as expected for strains overexpressing WC-1 (Cheng et al. 2001).

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Figure 5

Effects of P_tcu-1-controlled wc-1 on circadian rhythms. (A) P_wc-1wc-1 and P_tcu-1wc-1 strains (denoted here, P_wc-1 and P_tcu-1) were inoculated on race tubes containing the indicated concentrations of CuSO₄ (Cu in µM) and BCS (BCS in µM), grown in LL for 24 hr at 30°, and then shifted to DD at 25°. The solid black line on the left marks the growth front at the time of the shift. Every 24 hr the growth front was marked under safe red lights (smaller tick marks). Two independent tubes are shown for each strain and condition. (B) The period of the conidiation rhythm vs. the Cu/BCS levels is plotted for P_wc-1wc-1 and P_tcu-1wc-1 strains. Each data point is the average period calculated from at least three race tubes (±SEM). (C) Percent of growth that is linear hyphae (and not aerial hyphae/conidia) is plotted vs. the Cu/BCS levels for P_wc-1wc-1 and P_tcu-1wc-1 strains.

Together, these data demonstrate that the effects of copper availability on rhythmicity of the P_tcu-1wc-1 strain correlated with the protein expression levels of WC-1. Increased WC-1 expression accelerated, and decreased WC-1 expression decelerated, oscillations. When copper-controlled WC-1 expression matched native gene expression, circadian oscillations mimicked native rhythmicity and period on race tubes.

Copper repression of an essential gene, hpt-1, phenocopies inviabiliy

To test whether P_tcu-1 is suitable to knock down expression of an essential gene and recapitulate loss of viability, we inserted P_tcu-1 into the 5′ region of the essential gene hpt-1, which encodes the sole histidine phospho-transferase in N. crassa (Banno et al. 2007; Borkovich et al. 2004). The primers used for this construction are listed in Table S2. Figure S6 shows the expression of HPT-1-FLAG in the control P_hpt-1hpt-1 strain, as well as in several P_tcu-1hpt-1 primary transformants after growth in 250 µM CuSO₄ and 200 µM BCS. Four of six glufosinate-resistant transformants had the expected copper regulation of HPT-1-FLAG. A homokaryotic P_tcu-1hpt-1 strain was isolated from transformant #6 as described in the Material and Methods.

To examine how rapidly CuSO₄ reduces HPT-1 protein in the P_tcu-1hpt-1 strain, BCS-induced cultures were treated with 250 µM CuSO₄ for varying lengths of time, and the protein was analyzed by Western blot (Figure 6A). Compared with the P_hpt-1hpt-1 control strain, HPT-1 protein was well induced by BCS in the P_tcu-1hpt-1 strain. However, after treatment with Cu for 6 hr, the level of HPT-1 protein was already below that of the control strain, and longer Cu treatments reduced HPT-1 to below the level of detection. Treatment of the control strain with Cu for 48 hr had no effect on the level of HPT-1 protein, as expected. Thus, copper repression of HPT-1 is efficient and long lived.

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Figure 6

Effects of copper on HPT-1 protein levels and viability of in a P_tcu-1hpt-1 strain. (A) Strains with hpt-1::FLAG expression driven by the native (hpt-1) or copper promoters (tcu-1) were treated with 100 µM BCS for 10 hr, subsequently treated with 250 µM CuSO₄ for the indicated times, and protein extracts were analyzed by Western blot for HPT-1::FLAG. The amido black staining (Amido) of the membrane in the lower panel demonstrates equal protein loading. This experiment was performed twice (N = 2), and the average HPT-1::FLAG/Amido signal (±SD) is plotted below. Expression from the native promoter at time 0 was set to 1 for each experiment. (B) The average growth rate (cm/d ± SD) of P_tcu-1 hpt-1 (tcu-1, light bars) and P_hpt-1hpt-1 (hpt-1, dark bars) strains is plotted vs. the copper/BCS content as indicated below. A significant difference (****T-test, P < 0.0004) in the growth rate of the P_tcu-1hpt-1 strain at low (250 µM BCS) vs. high copper (500 µM CuSO₄), but not in the strain driven by the native promoter (P_hpt-1hpt-1), is shown.

To determine whether copper repression of P_tcu-1hpt-1 could phenocopy an hpt-1 deletion, which is known to be lethal (Banno et al. 2007), race tubes containing CuSO₄ and/or BCS were inoculated with P_hpt-1hpt-1 and P_tcu-1hpt-1 strains and allowed to grow at 30° for 3 d. The growth rate of the P_hpt-1hpt-1 strain was largely unaffected by the copper/BCS content of the race tubes, while the growth rate of the P_tcu-1hpt-1 strain was strongly inhibited by increasing copper content as predicted (Figure 6B). The growth rate of the P_tcu-1hpt-1 strain was low but constant over the course of several days, suggesting that HPT-1 protein was depleted but not completely abolished.