The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas).

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are "blind" to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. By contrast, ccg-2(eas) mRNA is photoinduced in clock-null strains such as frequency (bd;frq). The results in the clock mutants show that an intact circadian oscillator is not required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a dual regulation that involves separable regulation by light and circadian rhythm.     

A genetic selection for circadian output pathway mutations in Neurospora crassa

In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq-oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.     

Two circadian timing circuits in Neurospora crassa cells share components and regulate distinct rhythmic processes

In Neurospora crassa, FRQ, WC-1, and WC-2 proteins comprise the core circadian FRQ-based oscillator that is directly responsive to light and drives daily rhythms in spore development and gene expression. However, physiological and biochemical studies have demonstrated the existence of additional oscillators in the cell that function in the absence of FRQ (collectively termed FRQ-less oscillators [FLOs]). Whether or not these represent temperature-compensated, entrainable circadian oscillators is not known. The authors previously identified an evening-peaking gene, W06H2 (now called clock-controlled gene 16 [ccg-16]), which is expressed with a robust daily rhythm in cells that lack FRQ protein, suggesting that ccg-16 is regulated by a FLO. In this study, the authors provide evidence that the FLO driving ccg-16 rhythmicity is a circadian oscillator. They find that ccg-16 rhythms are generated by a temperature-responsive, temperature-compensated circadian FLO that, similar to the FRQ-based oscillator, requires functional WC-1 and WC-2 proteins for activity. They also find that FRQ is not essential for rhythmic WC-1 protein levels, raising the possibility that this WCFLO is involved in the generation of WC-1 rhythms. The results are consistent with the presence of 2 circadian oscillators within Neurospora cells, which the authors speculate may interact with each other through the shared WC proteins.     

Distinct signaling pathways from the circadian clock participate in regulation of rhythmic conidiospore development in Neurospora crassa

Several different environmental signals can induce asexual spore development (conidiation) and expression of developmentally regulated genes in Neurospora crassa. However, under constant conditions, where no environmental cues for conidiation are present, the endogenous circadian clock in N. crassa promotes daily rhythms in expression of known developmental genes and of conidiation. We anticipated that the same pathway of gene regulation would be followed during clock-controlled conidiation and environmental induction of conidiation and that the circadian clock would need only to control the initial developmental switch. Previous experiments showed that high-level developmental induction of the clock-controlled genes eas (ccg-2) and ccg-1 requires the developmental regulatory proteins FL and ACON-2, respectively, and normal developmental induction of fl mRNA expression requires ACON-2. We demonstrate that the circadian clock regulates rhythmic fl gene expression and that fl rhythmicity requires ACON-2. However, we find that clock regulation of eas (ccg-2) is normal in an fl mutant strain and ccg-1 expression is rhythmic in an acon-2 mutant strain. Together, these data point to the endogenous clock and the environment following separate pathways to regulate conidiation-specific gene expression.     

Circadian and light-induced expression of luciferase in Neurospora crassa

We have constructed a plasmid vector for expressing firefly luciferase in Neurospora crassa under control of the light- and clock-regulated ccg-2 (eas) promoter. The sequence of the luciferase gene in the vector has been modified to reflect the N. crassa codon bias. Both light-induced activity and circadian activity are demonstrated. Expression of luciferase in strains carrying mutant frequency alleles shows appropriate period length alterations. These data demonstrate that luciferase is a sensitive reporter of gene expression in N. crassa. Our results also show that the modified luciferase is expressed in Aspergillus nidulans.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

A PEST-like element in FREQUENCY determines the length of the circadian period in Neurospora crassa.

FREQUENCY (FRQ) is a crucial element of the circadian clock in Neurospora crassa. In the course of a circadian day FRQ is successively phosphorylated and degraded. Here we report that two PEST-like elements in FRQ, PEST-1 and PEST-2, are phosphorylated in vitro by recombinant CK-1a and CK-1b, two newly identified Neurospora homologs of casein kinase 1 epsilon. CK-1a is localized in the cytosol and the nuclei of Neurospora and it is in a complex with FRQ in vivo. Deletion of PEST-1 results in hypophosphorylation of FRQ and causes significantly increased protein stability. A strain harboring the mutant frq Delta PEST-1 gene shows no rhythmic conidiation. Despite the lack of overt rhythmicity, frq Delta PEST-1 RNA and FRQ Delta PEST-1 protein are rhythmically expressed and oscillate in constant darkness with a circadian period of 28 h. Thus, by deletion of PEST-1 the circadian period is lengthened and overt rhythmicity is dissociated from molecular oscillations of clock components.     

Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

OS-2 mitogen activated protein kinase regulates the clock-controlled gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.     

A connection between MAPK pathways and circadian clocks

Circadian clocks and mitogen-activated protein kinase (MAPK) signaling pathways are fundamental features of eukaryotic cells. Both pathways provide mechanisms for cells to respond to environmental stimuli, and links between them are known. We recently reported that the circadian clock in Neurospora crassa regulates daily rhythms in accumulation of phosphorylated, and thus active, OS-2 MAPK, a relative of mammalian p38 MAPK, when cells are grown in constant conditions. In the absence of acute stress, rhythmically activated MAPK then signals to downstream effector molecules to regulate rhythmic expression of target genes of the pathway. Clock regulation of MAPK signaling pathways provides a mechanism to coordinately control major groups of genes such that they peak at the appropriate times of day to provide a growth and survival advantage to the organism by anticipating stresses. MAPK pathways are well known for their role in cell proliferation and tumor suppression. New evidence reveals that some mammalian clock components also function as tumor suppressors and rhythms in phospho-MAPK have been observed in higher eukaryotes. Thus, the role of the clock in regulation of the activity of MAPK pathways provides important clues into the function of the circadian clock as a tumor suppressor.     

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.     

Understanding circadian rhythmicity in Neurospora crassa: from behavior to genes and back again

Circadian clocks have been described in organisms ranging in complexity from unicells to mammals, in which they function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology, including endocrine function, sleep/wake cycles, psychiatric illness, and drug tolerances and effectiveness. Because the clock in all organisms is assembled within the cell and clock mechanisms are evolutionarily conserved, simple eukaryotes provide appropriate experimental systems for dissecting the clock. Significant progress has been made in deciphering the circadian system in Neurospora crassa using both genetic and molecular approaches, and Neurospora has contributed greatly to our understanding of (1) the feedback cycle that comprises a circadian oscillator, (2) the mechanisms by which the clock is kept in synchrony with the environment, and (3) the genes that reside in rhythmic output pathways. Importantly, the lessons learned in Neurospora are relevant to our understanding of clocks in higher eukaryotes.