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. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Fungal Genet Biol. 2012 Jan 5;49(2):180–188. doi: 10.1016/j.fgb.2011.12.006

The Neurospora crassa OS MAPK Pathway-Activated Transcription Factor ASL-1 Contributes to Circadian Rhythms In Pathway Responsive Clock-Controlled Genes

Teresa M Lamb 1, Katelyn E Finch 1,1, Deborah Bell-Pedersen 1
PMCID: PMC3278502  NIHMSID: NIHMS348143  PMID: 22240319

Abstract

The OS-pathway mitogen-activated protein kinase (MAPK) cascade of Neurospora crassa is responsible for adaptation to osmotic stress. Activation of the MAPK, OS-2, leads to the transcriptional induction of many genes involved in the osmotic stress response. We previously demonstrated that there is a circadian rhythm in the phosphorylation of OS-2 under constant non-stress inducing conditions. Additionally, several osmotic stress-induced genes are known to be regulated by the circadian clock. Therefore, we investigated if rhythms in activation of OS-2 lead to circadian rhythms in other known stress responsive targets. Here we identify three more osmotic stress induced genes as rhythmic: cat-1, gcy-1, and gcy-3. These genes encode a catalase and two predicted glycerol dehydrogenases thought to be involved in the production of glycerol. Rhythms in these genes depend upon the oscillator component FRQ. To investigate how the circadian signal is propagated to these stress induced genes, we examined the role of the OS-responsive transcription factor, ASL-1, in mediating circadian gene expression. We find that while the asl-1 transcript is induced by several stresses including an osmotic shock, asl-1 mRNA accumulation is not rhythmic. However, we show that ASL-1 is required for generating normal circadian rhythms of some OS-pathway responsive transcripts (bli-3, ccg-1, cat-1, gcy-1 and gcy-3) in the absence of an osmotic stress. These data are consistent with the possibility that post-transcriptional regulation of ASL-1 by the rhythmically activated OS-2 MAPK could play a role in generating rhythms in downstream targets.

Keywords: Neurospora crassa, Circadian output, MAPK pathway, ATF/CREB transcription factor, Osmotic stress, Oxidative stress

1. Introduction

The evolution of organisms in the cyclical environment of the earth has led to the development of internal biological clocks. Biological clocks provide a mechanism for organisms to anticipate cyclical daily stresses such as light, heat, and desiccation, and to mount appropriate physiological changes that will help the organism better adapt to their environment(Dodd et al., 2005; Ouyang et al., 1998; Woelfle et al., 2004; Yerushalmi and Green, 2009; Yerushalmi et al., 2011). We are beginning to understand that the circadian oscillator mechanism does this, at least in part, through the rhythmic activation of stress response pathways, even in the absence of obvious stress(de Paula et al., 2008). We previously showed that the Neurospora crassa clock transcription factor, the White Collar Complex (WCC), binds rhythmically to the promoter of the MAPK kinase kinase (Smith et al., 2010)of the OS-pathway (os-4), and drives daily rhythms in os-4 mRNA and protein(Lamb et al., 2011). This then leads to a rhythm in the phosphorylation of the terminal MAPK, OS-2(Vitalini et al., 2007). Daily rhythms in OS-2 phosphorylation occur in the absence of stress, and in constant conditions (constant temperature, constant darkness) under control of the endogenous circadian clock mechanism.

OS-2 is a homolog of both the Hog1 MAPK from yeast and the p38 MAPK from mammals(Zhang et al., 2002). This MAPK is critical for mounting responses that can help organisms adapt to increased extra-cellular osmotic pressure, oxidative stress, heat and many other stresses(Hohmann, 2002; Sheikh-Hamad and Gustin, 2004). In fungi, the OS-pathway provides the target by which many anti-fungal drugs work to promote toxicity(Ochiai et al., 2002; Vetcher et al., 2007; Zhang et al., 2002). In mammals, the p38 MAPK is a target of the pyridinyl imidazole class of compounds that have anti-inflammatory activity(Schindler et al., 2007). Furthermore, there are links between the p38 MAPKs and cell death, cell cycle progression, and cancer(Cuenda and Rousseau, 2007; Fu and Lee, 2003; Yaakov et al., 2009). Thus, understanding how the p38 MAPK pathway is connected to the circadian clock and downstream rhythmic gene expression may be important for many aspects of human health.

Several targets of the OS-2 MAPK have been discovered in Neurospora, and these are similar to the targets in yeast and mammals(Irmler et al., 2006; Noguchi et al., 2007; Watanabe et al., 2007; Yamashita et al., 2007). In S. cerevisiae, Hog1 phosphorylates ion channels, kinases, phosphatases, and transcription factors that bring about the adaptive response and modulate pathway activity(Hohmann, 2002; Proft et al., 2006; Proft and Struhl, 2002, 2004; Rep et al., 2000). In mammals, a similar complement of p38 MAPK targets is also observed. One common target found in yeast and mammals is the bZIP transcription factor Sko1 or ATF/CREB. In yeast, Sko1 is phosphorylated by Hog1 (Proft et al., 2001; Proft and Struhl, 2002; Rep et al., 2001)and in mammals ATF/CREB is phosphorylated by p38 MAPK in response to various stresses(Hazzalin et al., 1996; Sen et al., 2005; Zayzafoon et al., 2002). Phosphorylation of Sko1 by the MAPK(Hog1) is known to convert it from a repressor to an activator, rapidly increasing the expression of many stress responsive genes(Proft and Struhl, 2002; Rep et al., 2001). Neurospora possesses a homolog of Sko1 designated ASL-1 for an as cospore lethal phenotype(Colot et al., 2006), and was subsequently referred to as ATF-1 for homology to the mammalian transcription factor(Yamashita et al., 2008). ASL-1 is required for the induction of some genes in response to the fungicide fludioxonil, and in response to osmotic stress(Yamashita et al., 2008).

Following the demonstration that the OS-2 MAPK is rhythmically phosphorylated during the course of a day in the absence of stress, it was of interest to determine if OS-2 phosphorylation rhythms would lead to rhythms in genes known to be stress-induced targets of the OS pathway. Indeed, some of the targets of the OS pathway, including bli-3(blue light induced-3, unknown function), ccg-1(clock controlled gene-1, unknown function), ccg-9(trehalose synthase), ccg-13(cell wall protein), and ccg-14(cell wall protein) are both induced by an activated OS-2 pathway and clock-controlled (Bell-Pedersen et al., 1996; Loros et al., 1989; Shinohara et al., 2002B; Watanabe et al., 2007; Zhu et al., 2001). However, there are several other genes induced by the OS-pathway that have not been specifically studied for rhythmicity. Furthermore, the role of the ASL-1 transcription factor in generating rhythmicity of OS-pathway output genes has not been examined. In this study we investigate the connection between the OS-pathway and circadian gene expression, with a focus on the role of the ASL-1 transcription factor.

2. Materials and Methods

2.1. Strains and culture conditions

N. crassa strains utilized in this study were grown and maintained as previously described(Davis and de Serres, 1970). All strains are “clock strains” that contain the ras-1bd allele, a mutation that reduces the growth rate, and clarifies the rhythmic asexual conidial banding pattern on racetubes(Belden et al., 2007; Sargent et al., 1966), but that does not alter the rhythm in phosphorylated OS-2 (Vitalini et al., 2007). Time course experiments were carried out as described (Lamb et al., 2011). The genotype of the circadian clock deficient ΔFRQ strain(frq10) we analyzed was ras-1bd, Δfrq::hph(Aronson et al., 1994), which was obtained from the Fungal Genetic Stock Center (FGSC 7490). Stress treatments were performed on mycelial discs grown in 1x Vogels, 2% glucose, 0.5% arginine pH 6.0 liquid media in constant light at 25°C after 24 h of growth, except the 30 min light pulse was performed after growth in constant darkness for 24 h. The stress treatments were all for 30 min except the hydrogen peroxide treatment, which was for 15 min. The stress treatments consisted of 100% water, 4% NaCl, 1M sorbitol, 1μg/ml fludioxonil “Pestanal” (Sigma 46102), and 10 mM hydrogen peroxide. Tissue was harvested immediately following the stress treatments.

2.2. Deletion of the ASL-1 transcription factor

Deletion of the transcription factor ASL-1 causes an ascospore lethal phenotype(Colot et al., 2006), and thus a ras-1bd Δasl-1 strain could not be obtained via crossing of the available Δasl-1 strain (FGSC 21336). Generation of the Δasl-1 strain in the ras-1bd background required new strain construction. Primers used in the construction of the new knockout are identical to those used in the gene knockout project (see http://www.dartmouth.edu/~neurosporagenome/primers.html), however, rather than using yeast to recombine the 5’asl-1 flank, the hph gene, and 3’asl-1 flank, a hybrid PCR was performed to fuse them together. This hybrid PCR product was then used to transform a ras-1bd strain (DBP1258). Heterokaryons were selected for on plates containing 200μg/ml hygromycin. Homokaryons of the Δasl-1::hph genotype were identified by PCR screening of single colonies derived from the heterokaryons. Three homokaryons derived from three independent transformants called DBP889 , DBP1314, and DBP1315 were obtained, and we have observed no differences between these strains. Consistent with previous data (Colot et al., 2006; Yamashita et al., 2008), we find that the ras-1bd Δasl-1::hph strain is ascospore lethal, has reduced conidial germination rates, and it is not sensitive to osmotic stress similar to the Δasl-1::hph strain (data not shown).

2.3. Gene expression analysis

RNA was extracted and purified from ground tissue and 10 to 15 μg were run on denaturing formaldehyde gels(Bell-Pedersen et al., 1996). RNA from control and test strains were always run side by side on the same gel so that changes in gene expression relative to the control could be determined. Gels were blotted to NitroPure membranes (GE, WP4HY00010) and hybridized to gene specific probes: cat-1, dak-1, fbp-1, gyc-1, gcy-3, and pck-1 were detected with[α-32P]-dATP-labeled DNA probes, while asl-1,bli-3, ccg-1, and ccg-9 were detected with [α-32P]-UTP labeled anti-sense RNA probes. As is standard in the field, gene expression was normalized to the ribosomal RNA visualized on the membrane with ethidium bromide. For time course experiments, the average normalized expression level of a strain was set to one and then to permit sine wave curve fitting, one was subtracted from the expression level at each time point.

2.4 Statistical Analysis

Nonlinear regression to fit the rhythmic data to a sine wave (fitting period, phase, and amplitude) and a line (fitting slope and intercept), as well as Akaike’s information criteria tests (Burnham and Anderson, 2002)to compare the fit of each data set to the 2 equations, were carried out using the Prism software package (GraphPad Software, San Diego, CA). The p values reflect the probability that, for instance, the sine wave fits the data better than a straight line.

3. Results

3.1. The OS-pathway stress induced genes, cat-1, gcy-1 and gcy-3 are circadianly regulated

Several Neurospora genes are induced after a salt shock or fungicide treatment, with induction being dependent on activation of the OS pathway. These include the catalase gene cat-1, the glycerol synthesis genes gcy-1, gcy-3, and dak-1, and the gluconeogenesis genes fbp-1 and pck-1, amongst others(Noguchi et al., 2007;Watanabe et al., 2007; Yamashita et al., 2007). The OS-pathway is activated by the circadian clock in the absence of stress; therefore, we postulated that genes that are stress regulated by the OS pathway would also be circadianly regulated in the absence of stress. Thus, we examined if these mRNAs accumulated with a circadian rhythm in constant darkness and temperature, conditions in which the only time of day information comes from the organism’s internal circadian clock. We observed rhythms in mRNA accumulation for cat-1, gcy-1 and gcy-3 with periods of 22.7+/−1.1,19.8+/−0.8h, and 17.2 +/−0.5h, respectively (Figure 1). With measurements taken every 4 hours for roughly two days, these period values are imprecise (+/− 8 h); however, all are within range of the endogenous 22-h period of the organism, while the gcy-1 and gcy-3 periods are somewhat shorter. No clear rhythms were observed for dak-1, fbp-1 or pck-1 mRNA accumulation (Supplementary Figure 1). Subjective morning peaks in expression were observed for cat-1 and gcy-3, while gcy-1 showed peak expression in the subjective night. Consistent with the idea that these genes depend on the Neurospora circadian oscillator, rhythms in abundance of cat-1, gcy-1 and gcy-3 were abolished in cells that lacked the core oscillator component, FREQUENCY (FRQ).

Figure 1. cat-1, gcy-1 and gcy-3 are clock-dependent rhythmic targets of the OS-pathway.

Figure 1

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The left panel shows representative northern blots of RNA harvested from a circadian time course. The time (h) after the shift to constant darkness (DD) is indicated at the top. The bars represent the phases that the circadian clock is experiencing with “subjective night” in black and “subjective day” in white. The ras-1bd(WT) and clock mutant, ras-1bd &Delta;frq (Δfrq), strains were examined for cat-1, gcy-1 and gcy-3 mRNA levels. The associated ethidium bromide stained rRNA loading control for each blot is shown underneath. The right panel indicates the normalized (gene signal/rRNA signal) average expression for the WT strain (black line) (N=4,± SEM) and the Δfrq strain (gray line) (N=3, ± SEM). The best fit to a sine wave for the wild type strain is indicated by a red line for the morning phase genes (cat-1 and gcy-3) and by a blue line for the late night phase gene (gcy-1) (p<0.0001). The best fit for all of the genes examined in the Δfrq strain was a line(not shown).

3.2. asl-1 expression is regulated by stress, but not by the circadian clock

ASL-1 is involved in mediating part of the stress response downstream of the OS-pathway. Therefore, we examined if asl-1 expression is regulated by stress. We found that asl-1 mRNA levels increased after 4% NaCl, 1M sorbitol, 1μg/ml fludioxonil, 10 mM hydrogen peroxide treatments and after a light pulse, and decreased after water treatment (Figure 2A and B).

Figure 2. Expression of asl-1 is induced by multiple stress treatments, but is not clock-regulated at the level of transcript abundance.

Figure 2

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(A) Northern blots showing expression of asl-1 in a ras-1 bdstrain (WT) after treatment with the indicated stresses (N=2 with similar results in both experiments)and (B) in a circadian time course, with the hours in DD and the circadian phase indicated as in Figure 1. Symbols: U= untreated, N= 4% NaCl, W= 100% water, S= 1 M sorbitol, F= 1 μg/ml fludioxonil, X= 10 mM H2O2,, D= constant darkness, and LP = light pulse. In B, the ASL-1 deletion strain was used as a control (Δ) (N=3 with similar results obtained in all experiments). rRNA is shown as a loading control below each blot in both A and B.

To define the role ASL-1 plays in mediating circadian output from the OS-pathway, we first tested if asl-1 mRNA accumulates with a circadian rhythm. A time course experiment was conducted as described above, and asl-1 mRNA was analyzed by northern blot (Figure 2B). While there is some random variation in the levels of asl-1 mRNA throughout the time course, no reproducible circadian rhythm was detected.

Next, we examined if ASL-1 is involved in generating the rhythmic pattern of asexual spore development (conidiation)observed in constant darkness on racetubes. To accomplish this goal, we first needed to generate a ras-1bd Δ;asl-1 strain. The ras-1bd allele clarifies the developmental rhythm on racetubes, and therefore, serves as the standard laboratory clock strain (Belden et al., 2007; Sargent et al., 1966). To generate the ras-1bd Δasl-1 strain, the asl-1open reading frame was replaced with the hygromycin resistance (hph) gene (see Materials and Methods). Heterokaryotic Δasl-1::hph transformants were identified as hygromycin resistant colonies whose genomic DNA yielded the correct sized DNA fragments in PCRs with “insertion test” primer sets (Figure 3A and B). Homokaryons of the Δasl-1::hph genotype were isolated, and the absence of the WT asl-1 gene in this strain was verified by PCR (Figure 3C). Furthermore, northern blots were used to confirm the absence of asl-1 mRNA in the ras-1bd Δasl-1 homokaryon strain (Figure 3D). In racetube assays, we observed that the period of the conidiation rhythm in the ras-1bd Δasl-1 strain(22.3+/−0.3 h) was similar to the period in the control ras-1bdstrain ( 22.1 +/− 0.2 h) (data not shown), indicating that the core FRQ/WCC oscillator functions normally in cells that lack ASL-1.

Figure 3. Generation of a ras-1bd Δasl-1 strain.

Figure 3

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(A) Schematic representation of the desired asl-1 deletion locus is shown with the white bars representing the approximately 1 kb of homology flanking the asl-1 gene used to recombine the knockout construct in which the asl-1 ORF is replaced by the hph ORF conferring resistance to hygromycin. The black line represents the genomic locus outside the homology region. (B) PCR using the indicated primers in A to examine proper insertion of the knockout cassette. The expected 2376 bp fragment is observed using the 5’F and YG primers, and the expected 3378 bp fragment is observed using the HY and 3’R primers in a PCR using genomic DNA from strains containing the desired deletion of ASL-1 (Δ). No products are expected or observed from ras-1bd (+) genomic DNA. Molecular weight standards are shown on the left (kb). (C) PCR using primer sets to detect the presence of the asl-1 and the os-4 gene open reading frames using genomic DNA from ras-1bd (+) and a homokaryotic ras-1bd Δasl-1 strain (Δ). The control os-4 gene is present in both strains, but the asl-1 gene is found only in the ras-1bd strain, demonstrating that a homokaryon with all copies of asl-1 deleted has been obtained. Molecular weight standards are shown on the left (kb). (D) Northern assay of ras-1bd (+) and homokaryotic ras-1bd Δasl-1 (Δ) RNA probed with a radio labeled asl-1 riboprobe. rRNA is shown as a loading control.

Together, these data suggest that circadian rhythms in ASL-1-target genes are not the result of clock-controlled asl-1 promoter activation or mRNA stability, but are likely due to rhythms in the activation of ASL-1 through the rhythmically activated OS-2 MAPK. Furthermore, any effect that Δasl-1 has on rhythmic gene expression will be due to its role in circadian output, rather than effects on the circadian oscillator.

3.3. The role of asl-1 in stress induction of OS-pathway output genes

The effect of deleting asl-1 on the stress induction of several OS-pathway output genes has been examined previously in wild type germinating conidia(Yamashita et al., 2008). However, because we are analyzing the effects of Δasl-1 in the ras-1bd background, as well as in mycelial mats grown in submerged culture, we needed to determine the role that ASL-1 plays in stress responses in this context (Figure 4). This was particularly important given the recent findings of Lara-Rojas et al (Lara-Rojas et al., 2011b), which showed cell-type-specific roles for the Aspergillus homolog of asl-1.

Figure 4. ASL-1 is required for proper stress-induced gene expression of OS-pathway targets.

Figure 4

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(A)The expression of bli-3, cat-1, ccg-1, ccg-9, gcy-1 and gcy-3 was examined by northern blot after various stress treatments in ras-1bd(WT) and ras-1bd Δasl-1(Δasl-1 ) strains. Quantitation of the expression levels from two biological replicates is shown in Supplemental Figure 1. Symbols: U= untreated, N= 4% NaCl, W= 100% water, S= 1M sorbitol, F= 1μg/ml fludioxonil, X= 10mM H2O2. (B) The expression of bli-3 and cat-1 in constant darkness (D) and after a light pulse (LP) was examined by northern blot in ras-1bd (WT) and ras-1bd Δasl-1 (Δasl-1 ) strains. rRNA is shown in A and B as a loading control.

Various osmotic stressors (4% NaCl, 1M sorbitol, 1μg/ml fludioxonil) induced the expression of bli-3, cat-1, ccg-1,ccg-9, gcy-1 and gcy-3 inras-1bd mycelia. However, induction of these genes in the ras-1bd Δasl-1 strain was seriously impaired (Figure 4A and Figure S1). cat-1 expression was nearly undetectable in all conditions in the ras-1bd Δasl-1 strain. bli-3, ccg-1, and gcy-3 were notinduced by salt (N) or fludioxonil (F)treatments, but were still weakly induced by sorbitol (S) treatment in the ras-1bd Δasl-1 strain. Induction of gcy-1 by these treatments was impaired, but not abolished. ccg-9 was still induced by salt and fludioxonil treatments, but not by sorbitol. Furthermore, ccg-1,ccg-9, and gcy-3 expression levels were elevated in the ras-1bd Δasl-1 strain as compared to the ras-1bd strainin the untreated samples (U). Shifting tissue from normal growth medium to 100% water (W) constitutes a hypoosmotic stress that may affect the expression of OS-pathway responsive genes. Expression of bli-3, gcy-1 and gcy-3 was strongly reduced after hypoosmotic stress treatment (Figure 4A and Figure S1). There was only partial reduction of bli-3, gcy-1 and gcy-3 expression in the ras-1bd Δasl-1 strain in response to a hypoosmotic stress, indicating an impaired response in the absence of ASL-1. We also examined the response of these genes to oxidative stress. Following a 15 min treatment with 10 mM H2O2(X), bli-3, cat-1, ccg-1, gcy-1 and gcy-3 were induced, and this induction required asl-1. Finally, we examined the expression of bli-3 and cat-1 in constant darkness (D) and after a light pulse (LP) (Figure 4B). We found that both genes were induced by a light pulse in the ras-1bdstrain, but only bli-3 was induced in the ras-1bd Δasl-1 strain. Thus, ASL-1 plays a crucial role in activating cat-1 expression in all conditions, while for other genes, it is involved in activation after hyperosmotic(and drug induced) stress, repression after hypoosmotic stress, and repression in non-stressed conditions.

3.4. ASL-1 is required for proper circadian rhythms of ccg-1, bli-3, gcy-1 and gcy-3 and for cat-1 expression

To determine the role ASL-1 plays in generating circadian expression of genes known to be regulated by the OS-pathway following stress, a time course experiment was performed with ras-1bd and ras-1bd Δasl-1 strains under constant conditions, and expression of OS-pathway output genes was examined (Figure 5). Similar to the stress response experiments, ASL-1 was found to be required for cat-1 expression at all times of the day. Rhythms in ccg-1 were still detected in the ASL-1 deletion; however, the amplitude of ccg-1 expression rhythms was reduced 2-fold as compared to the control ras-1bd strain. A similar expression profile was observed for bli-3, with a nearly 2-fold reduction in amplitude in the ASL-1 deletion strain. Alternatively, the rhythm in gcy-1 expression depended on ASL-1. The rhythm in gcy-3 was severely impaired in the ras-1bd Δasl-1 strain, and the amplitude was reduced almost 2-fold. Lastly, ASL-1 was found to not be required for proper circadian regulation of ccg-9.

Figure 5. ASL-1 is involved in generating proper circadian rhythms of several downstream OS- pathway genes.

Figure 5

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Representative northern blots (left panels)showing accumulation of bli-3, cat-1, ccg-1, ccg-9, gcy-1 and gcy-3 mRNA in circadian time courses in the ras-1bd Δasl-1 strain, with the hours after dark shift (DD) and the circadian phase indicated as in Figure 1. rRNA is shown as a loading control. The right panel shows the average expression for each gene (N≥3, ± SEM). Results from the ras-1bd strain (WT) are plotted in grey (the northern blots are not shown), ras-1bd Δasl-1 (Δasl-1) in black, and the best fit (line or sine) for the ras-1bd Δasl-1 strain is plotted in red. The predicted sine wave for each of the genes in ras-1bd has a p≤value 0.004. In the ras-1bd Δasl-1 strain, the p value for the best fit to a sine wave for ccg-1, bli-3 and ccg-9 is p≤0.001, and for gcy-3 is p≤0.03.

4.1 Discussion

In this paper we have explored the connection between the circadian clock and the osmotic stress pathway in Neurospora. We have identified three genes that were previously known to be induced by osmotic stress downstream of the OS-MAPK pathway, as also being under control of the circadian clock. We have defined a role of the ATF/CREB related transcription factor, ASL-1 in hyperosmotic, hypoosmotic, and oxidative stress responses. Finally, we have examined the role ASL-1 plays in driving circadian gene expression. Our results suggest that ASL-1 is receiving time of day information from the OS-pathway to contribute to circadian gene expression (Figure 6).

Figure 6. Model for the circadian and stress regulation of OS-pathway outputs.

Figure 6

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When the circadian transcription factor White Collar Complex (WCC) is in the subjective morning phase of activity, or when cells are exposed to elevated osmotic conditions, the OS- MAPKpathway is activated, leading to high levels of phospho-OS-2. Phospho-OS-2 activates several transcription factors, including ASL-1, which in turn stimulate the expression of numerous output genes either directly or indirectly. ASL-1 is required for both stress induction and normal circadian rhythms of ccg-1, bli-3, cat-1, gcy-1 and gcy-3, although the levels of dependence on ASL-1 vary from gene to gene. Circadian control of the morning specific genes by ASL-1 may be direct, but control of the evening specific gene, gcy-1, is likely to be indirect (dashed line). The circadian expression of ccg-9 is independent of ASL-1. When the WCC is in the subjective evening phase of inactivity or when cells are un-stressed, the OS-pathway MAPK cascade is inactive, leading to low phospho-OS-2 levels. Here, ASL-1 functions as a repressor, keeping the expression of ccg-1 and ccg-9low. While repression of ccg-1 is probably direct, it is unknown whether ccg-9 is a direct target.

4.1 Identification of three new OS-pathway outputs as clock-controlled genes

The OS-pathway of Neurospora has been shown to receive time of day input from the circadian clock via the transcriptional control of the OS-4 MAPKKK(Lamb et al., 2011). This input then leads to the rhythmic phosphorylation of the OS-2 MAPK(Vitalini et al., 2007). One prediction from these findings is that genes that are regulated by the OS-pathway in response to osmotic stress would also be circadianly regulated. Several of the OS-pathway responsive genes were already known to be rhythmic: ccg-1, ccg-9, ccg-13, ccg-14, and bli-3(Bell-Pedersen et al., 1996; Loros et al., 1989; Shinohara et al., 2002a; Watanabe et al., 2007; Zhu et al., 2001). However, there are many other known OS-pathway outputs that were not known to be rhythmic. Here we tested several of these genes and found three more to be circadianly regulated in constant conditions (Figure 1). Furthermore, the rhythmicity of these genes was shown to depend on the canonical clock component, FRQ. Not all genes that are downstream of the OS-pathway have rhythms in their steady state transcript levels. No rhythms were detected in dak-1, fbp-1 or pck-1. It is possible that the rhythms in OS-2 phosphorylation do not lead to rhythms in these genes because the amplitude of phosphorylated OS-2 does not reach a critical threshold for activating these genes in a time-of-day-dependent manner. Alternatively, the semRNAs may have stability characteristics that maskcircadian regulation of transcription initiation or perhaps in the unstressed circadian conditions of our experiments, regulation of these genes by the OS-pathway is only a minor contributor compared to other factors.

Thus, while not an absolute predictor, other genes that are downstream of rhythmic stress signaling pathways (rhythmic phospho-OS-2), are likely to also be rhythmic. Rhythmicity of stress response genes is predicted to be important for the ability of the organism to rapidly respond to daily environmental stresses. Most of the genes examined in this study have peak expression in the late-night to early-morning, a time that anticipates the daytime associated stresses of light, UV light, heat, and desiccation. Elevated catalase (cat-1) is expected to detoxify the reactive oxygen species associated with UV light exposure and metabolism(Peraza and Hansberg, 2002). The glycerol dehydrogenases (gcy-1 and -3) are expected to perform both forward and reverse reactions, so upregulation in the morning (gcy-3) could promote the production of the osmoprotectant, glycerol, from dihydroxyacetone (DHA) or glyceraldehyde (GA), while upregulation in the night (gcy-1) could be mediating recycling of glycerol back to DHA or GA when it is no longer needed(Viswanath-Reddy et al., 1978). The trehalose synthase gene (ccg-9) expression peaks in the mid-day and is expected to increase the production of trehalose, a storage form of glucose that is also an osmoprotectant(Iturriaga et al., 2009; Shinohara et al., 2002b; Soto et al., 1999). Moreover, rhythmicity of the OS pathway is functionally relevant as it promotes a more rapid induction of glycerol after salt stress treatment in the subjective morning compared to treatment in the subjective evening (Lamb et al., 2011). Over evolutionary time, or in competition with other organisms, it is tempting to speculate that the clock regulation of this pathway gives organisms an advantage.

4.2. The role of ASL-1 in stress responses

Although previous studies have examined the role of ASL-1 in output from the OS pathway, those studies were done on wild type strains in germinating conidia. As our studies focused on myceliain the standard laboratory clock ras-1bdstrain, we reexamined the role ASL-1 plays in such conditions. Our results are largely in agreement with the published work(Yamashita et al., 2008), showing reduced induction of the OS output genes bli-3, cat-1, ccg-1, gcy-1 and gcy-3 in the ASL-1 deleted strain after NaCl, sorbitol, and fludioxonil treatments. One difference in our study is that we found ASL-1 to be unimportant for the NaCl induction of ccg-9 (discussed below, see section 4.3). Furthermore, we extend the understanding of stress responses by examining expression of OS output genes in hypoosmotic (100% H2O), oxidative stress (10 mM H2O2) and light pulse treatments. In the control strain, some of the output genes are repressed under hypoosmotic conditions, however in the Δasl-1 strain, repression is impaired for bli-3, gcy-1 and gcy-3. This suggests a function for ASL-1 in the hypoosmotic response, which could be related to the role the yeast homolog Sko1 plays in repressing osmotic shock genes in unstressed conditions(Proft et al., 2001; Rep et al., 2001). We also demonstrate that bli-3, cat-1, ccg-1, gcy-1 and gcy-3 are induced by oxidative stress and dependent on ASL-1 for induction. A role for ASL-1 in oxidative stress is thus conserved in fungi and mammals (Aggeli et al., 2006; Alonso-Monge et al., 2010; Kurata, 2000; Lara-Rojas et al., 2011a; Rep et al., 2001; Sakamoto et al., 2009). Finally we demonstrate that the light-induced expression of bli-3 is not dependent on ASL-1, while cat-1 expression remains completely dependent on ASL-1 even after a light pulse. Taken together these data suggest that ASL-1 plays several roles in stress responses. It is absolutely required for cat-1 expression, it is important for repression of some genes in unstressed and in hypoosmotic conditions, it is required for hyperosmotic induction of several genes, and it is involved in the oxidative stress response of several genes.

4.3 The role of ASL-1 in circadian gene expression

Although asl-1 steady state mRNA levels do not show a circadian rhythm (Figure 2), and Δasl-1 strains have normal oscillator function, we propose that the ASL-1 transcription factor plays a role in circadian clock output by driving rhythmic expression of stress response genes. Consistent with the demonstration that yeast HOG1 directly phosphorylates the ASL-1 homolog SKO-1 (Proft et al, 2001; Proft and Struhl, 2002), we hypothesize that rhythms in the levels of phosphorylated OS-2 MAPK (Vitalini et al., 2007)lead to rhythms in phosphorylation and activation of ASL-1, which drives rhythmic expression of some, but not all, OS-pathway output genes. While direct or circadian association of Neurospora ASL-1 with the OS-2 MAPK has yet to be demonstrated, our hypothesis is supported by the role ASL-1 plays in circadian gene expression and the known activities of ASL-1 homologs. ASL-1 is required for rhythmic gcy-1expression and is involved in the rhythms in ccg-1, bli-3, and gcy-3 expression. Additionally, ASL-1 is necessary for cat-1 expression under all conditions tested. Previous studies have shown the direct binding of purified ASL-1 protein to the promoters of the ccg-1 and cat-1 genes in vitro(Yamashita et al., 2008), suggesting direct regulation of these two genes. Because both ccg-1 and cat-1 genes peak in expression in the morning and are likely to be bound directly by ASL-1, the mechanism by which ASL-1 generates rhythms in the evening specific gene, gcy-1, is predicted to be indirect.

Now a nearly complete molecular pathway for circadian gene expression of ccg-1 and cat-1 can be constructed (Figure 6). The pathway begins with the circadian transcription factor (WCC) rhythmically binding to and driving expression of the MAPKK kinase (OS-4)(Lamb et al., 2011). Rhythms in the levels of the OS-4 protein lead to rhythms in the phosphorylation state of the MAPK(OS-2) in constant non-stress conditions(Lamb et al., 2011; Vitalini et al., 2007). Rhythms in OS-2 phosphorylation are expected to lead to rhythms in activity of the ASL-1 transcription factor, which directly binds the cat-1 and ccg-1 promoters (Yamashita et al., 2008)to drive expression and rhythms. During the daytime when the WCC activates os-4 expression or after a hyperosmotic stress treatment, phospho-OS-2 MAPK levels increase. This activated MAPK promotes the activity of the ASL-1 transcription factor to drive bli-3, ccg-1, cat-1 and gcy-3 expression during the day or under osmotic stress conditions. It is not currently known whether bli-3 or gcy-3 are direct ASL-1 targets for activation. Osmotic stress also drives the expression of ccg-9, but ASL-1 is not required for that induction nor is it required for ccg-9 rhythms. gcy-1 is also induced by an osmotic stress, and that induction requires ASL-1, however expression in the evening of a circadian cycle is likely to be due to indirect circadian control by ASL-1. During the evening when the levels of active WCC are very low, or in unstressed conditions, there are very low levels of phosphorylated OS-2. This allows ASL-1 to function as a repressoron the ccg-1 promoter directly and on the ccg-9 promoter either directly or indirectly . While this model is consistent with current information, many aspects require clarification. Future studies will test if ASL-1 rhythmically associates with and is phosphorylated by OS-2. ChIP-seq analysis will define the direct targets of ASL-1 after osmotic and/or oxidative shock, which we predict will also identify additional ASL-1 circadian targets.

The only gene we tested that ASL-1 did not adversely affect following salt treatment or for circadian rhythmicity was ccg-9. ASL-1 did, however, play a clear role in repressing this gene. This suggests that multiple factors are controlling ccg-9. Rhythmicity of ccg-9 is not likely to be the result of direct regulation by the WCC, as ccg-9 was not identified as a direct target of the WCC in ChIP-seq studies (Smith et al, 2010). What drives the rhythm of ccg-9 and the low amplitude rhythms observed in ccg-1, bli-3, and gcy-3 in the absence of ASL-1? There are two additional transcription factors that are predicted to mediate output from the OS-pathway based on homology to yeasts, NCU02671(MSN2 homolog) and NCU02558 (SMP1/RLM1 homolog)(Hohmann, 2002). These two transcription factors may also be involved in clock output from the OS pathway, and future studies will address this question.

4.4 Conservation of MAPK pathway rhythmicity and transcription factor targets

There is evidence that the OS-pathway is not the only rhythmic MAPK pathway in Neurospora (Bennett and Bell-Pedersen, in preparation). Furthermore, MAPK pathways in other organisms may also be controlled by the clock(Coogan and Piggins, 2003; Dziema et al., 2003; Hasegawa and Cahill, 2004; Kako et al., 1996; Koyanagi et al., 2011; Obrietan et al., 1998). While a role for MAPK and ATF/CREB in mediating light input to the central oscillator has been extensively studied in mammals and birds(Butcher et al., 2002; Coogan and Piggins, 2004; Dziema et al., 2003; Ko et al., 2009; Koyanagi et al., 2011; Obrietan et al., 1998; Pizzio et al., 2003; Shimizu and Fukada, 2007)our findings with the fungal homolog, ASL-1, indicate a new role for this transcription factor in mediating circadian output. It is possible that a role for p38 MAPK and ATF/CREB in mammalian circadian output has been masked by its established role in mediating light input to the central pacemaker.

Supplementary Material

01

Supplemental Figure 1. dak-1, fbp-1 and pck-3 are not expressed with a circadian rhythm. The left panel shows representative northern blots of RNA harvested from a circadian time course. The time (h) after the shift to constant darkness (DD) is indicated at the top. The bars represent the phases that the circadian clock is experiencing with “subjective night” in black and “subjective day” in white. The ras-1bd strain was examined for dak-1, fbp-1 and pck-1 mRNA levels. The associated ethidium bromide stained rRNA loading control for each blot is shown underneath. The right panel indicates the normalized (gene signal/rRNA signal) average expression for the WT strain (black line) (N=3, ± SEM for dak-1 and N=2, ±SEM for gcy-1 and gcy-3). The best fit of the data for each of these genes is a line shown in grey.

02

Supplemental Figure 2. Quantitation of mRNA levels from treated tissue. Plotted are the average normalized signal (mRNA signal/rRNA signal) ± SD (N=2) for the genes indicated in ras-1bd (WT) (black bars) and ras-1bd Δasl-1 (Δasl-1 ) (grey bars) strains. The average value of the ras-1bd (WT) untreated sample was set to 1 for each gene. Symbols: U= untreated, N= 4% NaCl, W= 100% water, S= 1M sorbitol, F= 1μg/ml fludioxonil, X= 10mM H2O2. Plots are on a log base 2 scale.

  • Circadian clock control of the OS MAPK pathway leads to rhythms in expression of target genes.

  • The ASL-1 transcription factor is necessary for normal stress responses of the target genes.

  • ASL-1 is required for normal rhythmic expression of the target genes.

  • Rhythmic posttranslational modification of ASL-1 is likely responsible for target gene rhythms.

Acknowledgments

We thank Dr. Richard Gomer for help with the statistical analysis and Lindsay Bennett and Stephen Caster for supplying Neurospora tissue. This work was supported by NIH GM58529.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Supplemental Figure 1. dak-1, fbp-1 and pck-3 are not expressed with a circadian rhythm. The left panel shows representative northern blots of RNA harvested from a circadian time course. The time (h) after the shift to constant darkness (DD) is indicated at the top. The bars represent the phases that the circadian clock is experiencing with “subjective night” in black and “subjective day” in white. The ras-1bd strain was examined for dak-1, fbp-1 and pck-1 mRNA levels. The associated ethidium bromide stained rRNA loading control for each blot is shown underneath. The right panel indicates the normalized (gene signal/rRNA signal) average expression for the WT strain (black line) (N=3, ± SEM for dak-1 and N=2, ±SEM for gcy-1 and gcy-3). The best fit of the data for each of these genes is a line shown in grey.

NIHMS348143-supplement-01.tif (1.5MB, tif)
02

Supplemental Figure 2. Quantitation of mRNA levels from treated tissue. Plotted are the average normalized signal (mRNA signal/rRNA signal) ± SD (N=2) for the genes indicated in ras-1bd (WT) (black bars) and ras-1bd Δasl-1 (Δasl-1 ) (grey bars) strains. The average value of the ras-1bd (WT) untreated sample was set to 1 for each gene. Symbols: U= untreated, N= 4% NaCl, W= 100% water, S= 1M sorbitol, F= 1μg/ml fludioxonil, X= 10mM H2O2. Plots are on a log base 2 scale.

NIHMS348143-supplement-02.tif (1.5MB, tif)

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