The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.     

Circadian clock-specific roles for the light response protein WHITE COLLAR-2

To understand the role of white collar-2 in the Neurospora circadian clock, we examined alleles of wc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2 (ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator of frq, is critical for establishing period length and temperature compensation in this circadian system.     

Molecular mechanism of suppression of circadian rhythms by a critical stimulus

Circadian singularity behavior (also called suppression of circadian rhythms) is a phenomenon characterized by the abolishment of circadian rhythmicities by a critical stimulus. Here we demonstrate that both temperature step up and light pulse, stimuli that activate the expression of the Neurospora circadian clock gene frequency (frq), can trigger singularity behavior in this organism. The arrhythmicity is transient and is followed by the resumption of rhythm in randomly distributed phases. In addition, we show that induction of FRQ expression alone can trigger singularity behavior, indicating that FRQ is a state variable of the Neurospora circadian oscillator. Furthermore, mutations of frq lead to changes in the amplitude of FRQ oscillation, which determines the sensitivity of the clock to phase-resetting cues. Our results further suggest that the singularity behavior is due to the loss of rhythm in all cells. Together, these data suggest that the singularity behavior is due to a circadian negative feedback loop driven to a steady state after the critical treatment. After the initial arrhythmicity, cell populations are then desynchronized.     

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.     

Circadian rhythms in Neurospora crassa: Dynamics of the clock component frequency visualized using a fluorescent reporter

The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism’s circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ–mCherryNC (FRQ–mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100×). Fluorescence of FRQ–mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150–200 μm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein’s nuclear trafficking may be more complex than previously realized.     

A new mutation affecting FRQ-less rhythms in the circadian system of Neurospora crassa

We are using the fungus Neurospora crassa as a model organism to study the circadian system of eukaryotes. Although the FRQ/WCC feedback loop is said to be central to the circadian system in Neurospora, rhythms can still be seen under many conditions in FRQ-less (frq knockout) strains. To try to identify components of the FRQ-less oscillator (FLO), we carried out a mutagenesis screen in a FRQ-less strain and selected colonies with altered conidiation (spore-formation) rhythms. A mutation we named UV90 affects rhythmicity in both FRQ-less and FRQ-sufficient strains. The UV90 mutation affects FRQ-less rhythms in two conditions: the free-running long-period rhythm in choline-depleted chol-1 strains becomes arrhythmic, and the heat-entrained rhythm in the frq(10) knockout is severely altered. In a FRQ-sufficient background, the UV90 mutation causes damping of the free-running conidiation rhythm, reduction of the amplitude of the FRQ protein rhythm, and increased phase-resetting responses to both light and heat pulses, consistent with a decreased amplitude of the circadian oscillator. The UV90 mutation also has small but significant effects on the period of the conidiation rhythm and on growth rate. The wild-type UV90 gene product appears to be required for a functional FLO and for sustained, high-amplitude rhythms in FRQ-sufficient conditions. The UV90 gene product may therefore be a good candidate for a component of the FRQ-less oscillator. These results support a model of the Neurospora circadian system in which the FRQ/WCC feedback loop mutually interacts with a single FLO in an integrated circadian system.     

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.     

Genetic interactions between clock mutations in Neurospora crassa: can they help us to understand complexity?

Recent work on circadian clocks in Neurospora has primarily focused on the frequency (frq) and white-collar (wc) loci. However, a number of other genes are known that affect either the period or temperature compensation of the rhythm. These include the period (no relationship to the period gene of Drosophila) genes and a number of genes that affect cellular metabolism. How these other loci fit into the circadian system is not known, and metabolic effects on the clock are typically not considered in single-oscillator models. Recent evidence has pointed to multiple oscillators in Neurospora, at least one of which is predicted to incorporate metabolic processes. Here, the Neurospora clock-affecting mutations will be reviewed and their genetic interactions discussed in the context of a more complex clock model involving two coupled oscillators: a FRQ/WC-based oscillator and a 'frq-less' oscillator that may involve metabolic components.