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Intracellular calcium signaling has been implicated in control of a variety of circadian processes in animals and plants but its role in microbial clocks has remained largely cryptic. To examine the role of intracellular Ca2+ in the Neurospora clock we screened knockouts of calcium transporter genes and identified a gene encoding a calcium exporter, nca-2, uniquely as having significant period effects. Loss of NCA-2 results in an increase in cytosolic calcium level, and this leads to hyper-phosphorylation of core clock components, FRQ and WC-1, and a short period as measured by both the core oscillator and overt clock. Genetic analyses showed that mutations in certain frq phospho-sites, and in Ca2+-calmodulin-dependent kinase (camk-2), are epistatic to nca-2 in controlling the pace of the oscillator. These data are consistent with a model in which elevated intracellular Ca+2 leads to increased activity of CAMK-2 leading to enhanced FRQ phosphorylation, accelerated closure of the circadian feedback loop, and a shortened circadian period length. At a mechanistic level some CAMKs undergo more auto-phosphorylations in Δnca-2, consistent with high calcium in the Δnca-2 mutant influencing the enzymatic activity of CAMKs. NCA-2 interacts with multiple proteins including CSP-6, a protein known to be required for circadian output. Most importantly, expression of nca-2 is circadian clock-controlled at both the transcriptional and translational levels, and this in combination with the period effects seen in strains lacking NCA-2, firmly places calcium signaling within the larger circadian system where it acts as both an input to and output from the core clock.ImportanceCircadian rhythms are based on cell-autonomous, auto-regulatory, feedback loops formed by interlocked positive and negative arms, and they regulate myriad molecular and cellular processes in most eukaryotes including fungi. Intracellular calcium signaling is also a process that impacts a broad range of biological events in most eukaryotes. Clues have suggested that calcium signaling can influence circadian oscillators through multiple pathways; however, mechanistic details have been lacking in microorganisms. Building on prior work describing calcium transporters in the fungus Neurospora, one such transporter, NCA-2, was identified as a regulator of circadian period length. Increased intracellular calcium levels caused by loss of NCA-2 results in over-activation of calcium-responsive protein kinases, in turn leading to a shortened circadian period length. Importantly, expression of NCA-2 is itself controlled by the molecular clock. In this way calcium signaling can be seen as providing both input to and output from the circadian system.

Circadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. InNeurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and Casein Kinase I (CKI), which inhibits the activity of the clock’s positive arm, the White Collar Complex (WCC). Here, we show that theperiod-2gene, whose mutation is characterized by recessive inheritance of a long 26-hour period phenotype, encodes an RNA-binding protein that stabilizes theck-1atranscript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period ofperiod-6mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically-derivedNeurosporaclock mutants each arise from disruption ofck-1aregulation is consistent with circadian period being exquisitely sensitive to levels ofcasein kinase I.

The circadian clock broadly governs immune cell function, leading to time-of-day differences in inflammatory responses and subsequently, pathogen clearance. However, the effect of inflammatory signals on circadian machinery is poorly understood. We found that in bone marrow-derived macrophages, some host-derived pro-inflammatory cytokines, e.g., IFN-γ or TNF-α, and pathogen-associated molecular patterns, e.g., LPS or Pam3Csk4, suppress the amplitude in oscillations of circadian negative feedback arm clock components such as PER2, and when examined, specific combinations of these immune-related signals suppressed the amplitude of these oscillations to a greater degree in both bone marrow-derived and peritoneal macrophages. At the transcript level, multiple components of the circadian clock were affected in different ways by pro-inflammatory stimulus, including Per2 and Nr1d1. This suppressive effect on PER2 did not arise from nor correlate with cell death or clock resetting. Suppression of the clock by IFN-γ was dependent on its cognate receptor; however, pharmacological inhibition of the canonical JAK/STAT and MEK pathways did not hinder suppression, suggesting a mechanism involving a non-canonical pathway. In contrast, anti-inflammatory signals such as IL-4 and dexamethasone enhanced the expression of PER2 protein and Per2 mRNA. Our results suggest that the circadian system in macrophages can differentially respond to pro- and anti-inflammatory signals in their microenvironments.

Work on the filamentous fungus Neurospora crassa has contributed to or pioneered many aspects of research on circadian clock mechanism, a process that is functionally conserved across eukaryotes. Biochemical assays of the fungal circadian clock typically involve growth in liquid medium where Neurospora forms a spherical ball of submerged mycelium. Here, we revive a method for dispersed growth of Neurospora in batch culture using polyacrylic acid as an additive to the medium. We demonstrate that dispersed growth cultures utilize more carbon than mycelial balls, but nonetheless retain a functional circadian clock. This culturing method is suited for use in circadian experiments where uniform exposure to nutrients and/or increased biomass is required.

Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins.

Circadian rhythms govern immune cell function, giving rise to time-of-day variation in the recognition and clearance of bacterial or viral pathogens; to date, however, no such regulation of the host-fungal interaction has been described. In this report, we use murine models to explore circadian control of either fungal-macrophage interactions in vitro or pathogen clearance from the lung in vivo. First, we show that expression of the important fungal pattern recognition receptor Dectin-1 ( clec7a), from either bone marrow-derived or peritoneum-derived macrophages, is not under circadian regulation at either the level of transcript or cell surface protein expression. Consistent with this finding, the phagocytic activity of macrophages in culture against spores of the pathogen Aspergillus fumigatus also did not vary over time. To account for the multiple cell types and processes that may be coordinated in a circadian fashion in vivo, we examined the clearance of A. fumigatus from the lungs of immunocompetent mice. Interestingly, animals inoculated at night demonstrated a 2-fold enhancement in clearance compared with animals inoculated in the morning. Taken together, our data suggest that while molecular recognition of fungi by immune cells may not be circadian, other processes in vivo may still allow for time-of-day differences in fungal clearance from the lung.

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δcsp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δwhi-2 mutant phenotypes resemble Δcsp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δcsp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δcsp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output., Author summary Though molecules and components in the core circadian oscillator are well studied in Neurospora, the mechanisms through which output pathways are coupled with core components are less well understood. In this study we investigated a HAD phosphatase, CSP-6; loss-of-function Δcsp-6 strains are overtly arrhythmic but have a functional core circadian oscillation. CSP-6 in association with WHI-2 dephosphorylates the core clock component WC-1 to regulate light-responses and development. To dissect the functions of CSP-6 in core clock and output, we screened known WC-1 targets and found that loss of CSP-6 causes misregulation of transcriptional/translational activation of ADV-1, a key regulator of output. Thus, loss of CSP-6-mediated dephosphorylation of WC-1 leads to loss of ADV-1 activation and is responsible for the complete loss of overt developmental rhythmicity in Δcsp-6.

The capacity for biological timekeeping arose at least three times through evolution, in prokaryotic cyanobacteria, in cells that evolved into higher plants, and within the group of organisms that eventually became the fungi and the animals. Neurospora is a tractable model system for understanding the molecular bases of circadian rhythms in the last of these groups, and is perhaps the most intensively studied circadian cell type. Rhythmic processes described in fungi include growth rate, stress responses, developmental capacity, and sporulation, as well as much of metabolism; fungi use clocks to anticipate daily environmental changes. A negative feedback loop comprises the core of the circadian system in fungi and animals. In Neurospora , the best studied fungal model, it is driven by two transcription factors, WC-1 and WC-2, that form the White Collar Complex (WCC). WCC elicits expression of the frq gene. FRQ complexes with other proteins, physically interacts with the WCC, and reduces its activity; the kinetics of these processes is strongly influenced by progressive phosphorylation of FRQ. When FRQ becomes sufficiently phosphorylated that it loses the ability to influence WCC activity, the circadian cycle starts again. Environmental cycles of light and temperature influence frq and FRQ expression and thereby reset the internal circadian clocks. The molecular basis of circadian output is also becoming understood. Taken together, molecular explanations are emerging for all the canonical circadian properties, providing a molecular and regulatory framework that may be extended to many members of the fungal and animal kingdoms, including humans.

In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology.

Summary Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed intrinsically disordered proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein FREQUENCY (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to FREQUENCY-Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via a pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.

Filamentous fungi are important model systems for understanding eukaryotic cellular processes, including the study of protein expression. A salient feature of fungi is the ability of the protein-processing machinery to perform all of the extensive posttranslational modifications needed in the complex world of eukaryotic organisms, making them great hosts for production of eukaryotic proteins. In the model organism Neurospora crassa, several regulatable promoters have been used for heterologous gene expression but all suffer from leaky expression absent stimuli or an inability to induce protein expression at levels greater than those seen in vivo. To increase and better control in vivo protein expression in Neurospora, we have harnessed the light-induced vvd promoter. vvd promoter-driven mRNA expression is dependent upon light, shows a graded response, and is rapidly shut off when returned to the dark. The vvd promoter is a highly tunable and regulatable system, which could be a useful instrument for those interested in efficient and controllable gene expression.

A periodic luciferase reporter system from cell cycle–regulated promoters in synchronous U2OS cells measures periodic, cell cycle–regulated gene expression in live cells. This assay is used to identify Forkhead transcription factors that control periodic gene expression, and it identifies FOXK1 as an activator of key cell cycle genes., We developed a system to monitor periodic luciferase activity from cell cycle–regulated promoters in synchronous cells. Reporters were driven by a minimal human E2F1 promoter with peak expression in G1/S or a basal promoter with six Forkhead DNA-binding sites with peak expression at G2/M. After cell cycle synchronization, luciferase activity was measured in live cells at 10-min intervals across three to four synchronous cell cycles, allowing unprecedented resolution of cell cycle–regulated gene expression. We used this assay to screen Forkhead transcription factors for control of periodic gene expression. We confirmed a role for FOXM1 and identified two novel cell cycle regulators, FOXJ3 and FOXK1. Knockdown of FOXJ3 and FOXK1 eliminated cell cycle–dependent oscillations and resulted in decreased cell proliferation rates. Analysis of genes regulated by FOXJ3 and FOXK1 showed that FOXJ3 may regulate a network of zinc finger proteins and that FOXK1 binds to the promoter and regulates DHFR, TYMS, GSDMD, and the E2F binding partner TFDP1. Chromatin immunoprecipitation followed by high-throughput sequencing analysis identified 4329 genomic loci bound by FOXK1, 83% of which contained a FOXK1-binding motif. We verified that a subset of these loci are activated by wild-type FOXK1 but not by a FOXK1 (H355A) DNA-binding mutant.

Photoadaptation, the ability to attenuate a light response on prolonged light exposure while remaining sensitive to escalating changes in light intensity, is essential for organisms to decipher time information appropriately, yet the underlying molecular mechanisms are poorly understood. In Neurospora crassa , VIVID (VVD), a small LOV domain containing blue-light photoreceptor protein, affects photoadaptation for most if not all light-responsive genes. We report that there is a physical interaction between VVD and the white collar complex (WCC), the primary blue-light photoreceptor and the transcription factor complex that initiates light-regulated transcriptional responses in Neurospora . Using two previously characterized VVD mutants, we show that the level of interaction is correlated with the level of WCC repression in constant light and that even light-insensitive VVD is sufficient partly to regulate photoadaptation in vivo. We provide evidence that a functional GFP-VVD fusion protein accumulates in the nucleus on light induction but that nuclear localization of VVD does not require light. Constitutively expressed VVD alone is sufficient to change the dynamics of photoadaptation. Thus, our results demonstrate a direct molecular connection between two of the most essential light signaling components in Neurospora , VVD and WCC, illuminating a previously uncharacterized process for light-sensitive eukaryotic cells.

In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1 -dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq . Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.

Light is a key environmental signal for most life on earth. Over 5% of Neurospora crassa genes are expressed in response to light stimulation in a temporally regulated cascade that includes several transcription factors. Fungal genomes, including Neurospora's, may encode several different proteins capable of binding chromophores with the ability to harvest light energy as well as proteins that can interact with primary photoreceptors or further propogate the light signal. The best understood photo- receptors are the evolutionarily conserved White Collar proteins, and the related Vivid protein, but fungi may also encode phytochromes, cryptochromes and opsins.

Temperature compensation is a poorly understood, defining property of circadian rhythms. Here, we critically interpret and expand the interpretation of some of our recently published work along with preliminary new data regarding this question. We describe, in greater detail, the cloning of two subunits of casein kinase 2 (CK2) that affect compensation. We briefly review and comment on key data including dose dependence of CK2 on compensation, the differences between CK2 and CK1, and the nature of phenocopying frequency (frq) alleles. We present preliminary data on in vitro phosphorylation of full-length FRQ and a new hypothesis regarding PEST-1 (proline (P), glutamic acid (E), serine (S), and threonine (T) rich) phosphorylation. Finally, we describe an extended model of temperature compensation.

Circadian systems are comprised of multiple proteins functioning together to produce feedback loops driving robust, approximately 24 hour rhythms. In all circadian systems, proteins in these loops are regulated through myriad physically and temporally distinct post-translational modifications (PTMs). To better understand how PTMs impact a circadian oscillator we implemented a proteomics-based approach by combining purification of endogenous FREQUENCY (FRQ) and its interacting partners with quantitative mass spectrometry (MS). We identify and quantify time-of-day specific protein-protein interactions in the clock and show how these provide a platform for temporal and physical separation between the dual roles of FRQ. Additionally, by unambiguously identifying over 75 phosphorylated residues, following their quantitative change over a circadian cycle, and examining the phenotypes of strains that have lost these sites, we demonstrate how spatially and temporally regulated phosphorylation has opposing effects directly on overt circadian rhythms and FRQ stability.

Circadian rhythms are considered to play an essential part in the adaptation of organisms to their environments. The occurrence of circadian oscillations appears to be based on the presence of transcriptional-translational negative feedback loops. In Neurospora crassa, the protein FREQUENCY (FRQ) is part of such a negative feedback loop apparently by a direct interaction with its transcription factor WHITE COLLAR-1 (WC-1). Based on the observation that nuclear FRQ levels are significantly lower than nuclear WC-1 levels, it was suggested that FRQ would act more like a catalyst in inhibiting WC-1 rather than binding to WC-1 and making an inactive FRQ:WC-1 complex. Intrigued by this hypothesis, we constructed a model for the Neurospora circadian clock, which includes expression of the frq and the wc-1 genes and their possible interactions. The model suggests that even small amounts of nuclear FRQ-protein are capable of inhibiting frq transcription in a rhythmic manner by binding to WC-1 and promoting its degradation. Our model predicts the importance of a FRQ dependent degradation of WC-1 in closing the negative feedback loop. The model shows good agreement with experimental levels in nuclear and cytosolic FRQ and WC-1, their phase relationships, and several clock mutant phenotypes.

The large (l) and small (s) isoforms of FREQUENCY (FRQ) are elements of interconnected feedback loops of the Neurospora circadian clock. The expression ratio of l-FRQ vs. s-FRQ is regulated by thermosensitive splicing of an intron containing the initiation codon for l-FRQ. We show that this splicing is dependent on light and temperature and displays a circadian rhythm. Strains expressing only l-FRQ or s-FRQ support short and long temperature-compensated circadian rhythms, respectively. The thermosensitive expression ratio of FRQ isoforms influences period length in wt. Our data indicate that differential expression of FRQ isoforms is not required for temperature compensation but rather provides a means to fine-tune period length in response to ambient temperature.

Circadian output comprises the business end of circadian systems in terms of adaptive significance. Work on Neurospora pioneered the molecular analysis of circadian output mechanisms, and insights from this model system continue to illuminate the pathways through which clocks control metabolism and overt rhythms. In Neurospora, virtually every strain examined in the context of rhythms bears the band allele that helps to clarify the overt rhythm in asexual development. Recent cloning of band showed it to be an allele of ras-1 and to affect a wide variety of signaling pathways yielding enhanced light responses and asexual development. These can be largely phenocopied by treatments that increase levels of intracellular reactive oxygen species. Although output is often unidirectional, analysis of the prd-4 gene provided an alternative paradigm in which output feeds back to affect input. prd-4 is an allele of checkpoint kinase-2 that bypasses the requirement for DNA damage to activate this kinase; FRQ is normally a substrate of activated Chk2, so in Chk2(PRD-4), FRQ is precociously phosphorylated and the clock cycles more quickly. Finally, recent adaptation of luciferase to fully function in Neurospora now allows the core FRQ/WCC feedback loop to be followed in real time under conditions where it no longer controls the overt rhythm in development. This ability can be used to describe the hierarchical relationships among FRQ-Less Oscillators (FLOs) and to see which are connected to the circadian system. The nitrate reductase oscillator appears to be connected, but the oscillator controlling the long-period rhythm elicited upon choline starvation appears completely disconnected from the circadian system; it can be seen to run with a very long noncompensated 60-120-hour period length under conditions where the circadian FRQ/WCC oscillator continues to cycle with a fully compensated circadian 22-hour period.

In the Neurospora circadian system, the White Collar complex (WCC) of WC-1 and WC-2 drives transcription of the circadian pacemaker gene frequency (frq), whose gene product, FRQ, as a part of the FRQ-FRH complex (FFC), inhibits its own expression. The WCC is also the principal Neurospora photoreceptor; WCC-mediated light induction of frq resets the clock, and all acute light induction is triggered by WCC binding to promoters of light-induced genes. However, not all acutely light-induced genes are also clock regulated, and conversely, not all clock-regulated direct targets of WCC are light induced; the structural determinants governing the shift from WCC's dark circadian role to its light activation role are poorly described. We report that the DBD region (named for being defective in binding DNA), a basic region in WC-1 proximal to the DNA-binding zinc finger (ZnF) whose function was previously ascribed to nuclear localization, instead plays multiple essential roles assisting in DNA binding and mediating interactions with the FFC. DNA binding for light induction by the WCC requires only WC-2, whereas DNA binding for circadian functions requires WC-2 as well as the ZnF and DBD motif of WC-1. The data suggest a means by which alterations in the tertiary and quaternary structures of the WCC can lead to its distinct functions in the dark and in the light.

INTRODUCTION Circadian oscillators allow individual organisms to coordinate metabolism with day/night cycles and to anticipate such changes. Such oscillators in fungi and animals share a common regulatory architecture centered on transcription and translation-based negative feedback loops. Within such oscillators, extensive coordinated and progressive phosphorylation of negative element proteins leads to their proteasome-mediated degradation. Current clock models posit that this turnover event is the final essential step in the loop and that the time taken to achieve phosphorylation and turnover determines the speed of the circadian clock. The clock in Neurospora exemplifies such oscillators: FREQUENCY (FRQ) is a negative element, and its half-life is well correlated with circadian period length. Surprisingly, however, using real-time reporters in cells with compromised proteasomal turnover, we unveiled an unexpected uncoupling between negative element half-life and circadian period determination. RATIONALE We followed FRQ dynamics as well as transcriptional activity of the frq promoter in vivo using luciferase-based reporters. FRQ turnover was tracked through Western blotting, and kinase inhibitors helped to test the correlation between phosphorylation and period length. Strains bearing frq alleles causing abnormal period lengths were used, as were strains with diminished FRQ turnover, including knockouts of both the F-box protein FWD-1 (a ubiquitin ligase that mediates FRQ proteasomal degradation) and individual components of the COP9 signalosome. RESULTS Without FWD-1, FRQ turnover is severely compromised and circadian regulation of development is lost; however, in such Δfwd-1 cells, the amount of FRQ still oscillated, the result of cyclic transcription of frq and reinitiation of FRQ synthesis. The circadian nature of these rhythms was confirmed by examining well-established frq mutants having altered periods. Analyses of additional strains bearing knockouts of individual COP9 signalosome components further confirmed circadian oscillations in FRQ amounts, despite compromised FRQ turnover. Broadly accepted oscillator models posit that negative element stability determines clock period length; thus, Δfwd-1 strains with long FRQ half-lives are predicted to have extremely long periods. This, however, is not seen: Period is mainly determined by the characteristics of the frq allele irrespective of the half-life of this negative element. Partial inhibition of overall phosphorylation provided additional evidence that clock protein phosphorylation events, not the resulting stability changes, provide key information in determining period length. DISCUSSION The long-standing and assumed causal loop uniting clock protein phosphorylation, stability, and period determination should be revisited. Data indicate that qualities of FRQ—in particular, its phosphorylation status rather than its quantity—are crucial for determining when the circadian feedback loop is completed and can be restarted. Previously described strong correlations between clock protein phosphorylation and half-life and between half-life and period length are, in fact, just correlations that do not always imply cause and effect. Although degradation is the final outcome of FRQ posttranslational modifications, phosphorylation and its effects of secondary, tertiary, and quaternary protein structure may actually be the key elements determining clock speed. Although it may be premature to broadly generalize these findings to all circadian oscillators, diverse data from several animal circadian systems are not inconsistent with this revised model.

The expression of FREQUENCY, a central component of the circadian clock in Neurospora crassa, shows daily cycles that are exquisitely sensitive to the environment. Two forms of FRQ that differ in length by 99 amino acids, LFRQ and SFRQ, are synthesized from alternative initiation codons and the change in their ratio as a function of temperature contributes to robust rhythmicity across a range of temperatures. We have found frq expression to be surprisingly complex, despite our earlier prediction of a simple transcription unit based on limited cDNA sequencing. Two distinct environmentally regulated major promoters drive primary transcripts whose environmentally influenced alternative splicing gives rise to six different major mRNA species as well as minor forms. Temperature-sensitive alternative splicing determines AUG choice and, as a consequence, the ratio of LFRQ to SFRQ. Four of the six upstream ORFs are spliced out of the vast majority of frq mRNA species. Alternative splice site choice in the 5′ UTR and relative use of two major promoters are also influenced by temperature, and the two promoters are differentially regulated by light. Evolutionary comparisons with the Sordariaceae reveal conservation of 5′ UTR sequences, as well as significant conservation of the alternative splicing events, supporting their relevance to proper regulation of clock function.

A light-entrainable circadian clock controls development and physiology in Neurospora crassa. Existing simple models for resetting based on light pulses (so-called nonparametric entrainment) predict that constant light should quickly send the clock to an arrhythmic state; however, such a clock would be of little use to an organism in changing photoperiods in the wild, and we confirm that true, albeit dampened, rhythmicity can be observed in extended light. This rhythmicity requires the PAS/LOV protein VIVID (VVD) that acts, in the light, to facilitate expression of an oscillator that is related to, but distinguishable from, the classic FREQUENCY/WHITE-COLLAR complex (FRQ/WCC)-based oscillator that runs in darkness. VVD prevents light resetting of the clock at dawn but, by influencing frq RNA turnover, promotes resetting at dusk, thereby allowing the clock to run through the dawn transition and take its phase cues from dusk. Consistent with this, loss of VVD yields a clock whose performance follows the simple predictions of earlier models, and overexpression of VVD restores rhythmicity in the light and sensitivity of phase to the duration of the photoperiod.

The eukaryotic filamentous fungus Neurospora crassa has proven to be a durable and dependable model system for the analysis of the cellular and molecular bases of circadian rhythms. Pioneering genetic analyses identified clock genes, and beginning with the cloning of frequency ( frq), work over the past 2 decades has revealed the molecular basis of a core circadian clock feedback loop that has illuminated our understanding of circadian oscillators in microbes, plants, and animals. In this transcription/translation-based feedback loop, a heterodimer of the White Collar-1 (WC-1) and WC-2 proteins acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene. FRQ dimerizes and feeds back to block the activity of its activators (making a negative feedback loop), as well as feeding forward to promote the synthesis of its activator, WC-1. Phosphorylation of FRQ by several kinases leads to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle. Light resetting of the clock can be understood through the rapid light induction of frq expression and temperature resetting through the influence of elevated temperaturesin driving higher levels of FRQ. Several FRQ- and WC-independent, noncircadian FRQ-less oscillators (FLOs) have been described, each of which appears to regulate aspects of Neurospora growth or development. Overall, the FRQ/white collar complex feedback loop appears to coordinate the circadian system through its activity to regulate downstream-target clock-controlled genes, either directly or via regulation of driven FLOs.

Circadian rhythms in the level of intracellular Mg appear to be widely conserved phylogenetically, and have the potential to impact nearly all aspects of metabolism. Moreover, the clock regulates the ion channels that generate the rhythm, demonstrating that the whole cell operates as a circadian system.

In the fungus Neurospora crassa , the blue light photoreceptor(s) and signaling pathway(s) have not been identified. We examined light signaling by exploiting the light sensitivity of the Neurospora biological clock, specifically the rapid induction by light of the clock component frequency ( frq ). Light induction of frq is transcriptionally controlled and requires two cis-acting elements (LREs) in the frq promoter. Both LREs are bound by a White Collar–1 (WC-1)/White Collar–2 (WC-2)–containing complex (WCC), and light causes decreased mobility of the WCC bound to the LREs. The use of in vitro–translated WC-1 and WC-2 confirmed that WC-1, with flavin adenine dinucleotide as a cofactor, is the blue light photoreceptor that mediates light input to the circadian system through direct binding (with WC-2) to the frq promoter.

The circadian clock of Neurospora crassa regulates the rhythmic expression of a number of genes encoding diverse functions which, as an ensemble, are adaptive to life in a rhythmic environment of alternating levels of light and dark, warmth and coolness, and dryness and humidity. Previous differential screens have identified a number of such genes based solely on their cycling expression, including clock-controlled gene 9 ( ccg-9 ). Sequence analysis now shows the predicted CCG-9 polypeptide to be homologous to a novel form of trehalose synthase; as such it would catalyze the synthesis of the disaccharide trehalose, which plays an important role in protecting many cells from environmental stresses. Consistent with this, heat, glucose starvation, and osmotic stress induce ccg-9 transcript accumulation. Surprisingly, however, a parallel role in development is suggested by the finding that inactivation of ccg-9 results in altered conidiophore morphology and abolishes the normal circadian rhythm of asexual macroconidial development. Examination of a clock component, FRQ, in the ccg-9 -null strain revealed normal cycling, phosphorylation, and light induction, indicating that loss of the conidiation rhythm is not due to changes in either the circadian oscillator or light input into the clock but pointing instead to a defect in circadian output. These data imply an interplay between a role of trehalose in stress protection and an apparent requirement for trehalose in clock regulation of conidiation under constant environmental conditions. This requirement can be bypassed by a daily light signal which drives a light-entrained rhythm in conidiation in the ccg-9 -null strain; this bypass suggests that the trehalose requirement is related to clock control of development and not to the developmental process itself. Circadian control of trehalose synthase suggests a link between clock control of stress responses and that of development.

vvd , a gene regulating light responses in Neurospora , encodes a novel member of the PAS/LOV protein superfamily. VVD defines a circadian clock–associated autoregulatory feedback loop that influences light resetting, modulates circadian gating of input by connecting output and input, and regulates light adaptation. Rapidly light induced, vvd is an early repressor of light-regulated processes. Further, vvd is clock controlled; the clock gates light induction of vvd and the clock gene frq so identical signals yield greater induction in the morning. Mutation of vvd severely dampens gating, especially of frq , consistent with VVD modulating gating and phasing light-resetting responses. vvd null strains display distinct alterations in the phase–response curve to light. Thus VVD, although not part of the clock, contributes significantly to regulation within the Neurospora circadian system.

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalianperiod gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in culturedmCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) ofmCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophilais conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner.

Common regulatory patterns can now be discerned among eukaryotic circadian systems, extending from fungi through to mammals. Complexes of two distinct PAS domain-containing transcription factors play positive roles in clock-associated feedback loops by turning on classic clock proteins such as FRQ, PER and TIM. These in turn appear to act as negative elements, interfering with their own activation and thus giving rise to an oscillatory negative feedback loop. Post-transcriptional control governs the amount and type of FRQ and makes the clock responsive to temperature.

To understand how light might entrain a mammalian circadian clock, we examined the effects of light on mPer1 , a sequence homolog of Drosophila per , that exhibits robust rhythmic expression in the SCN. mPer1 is rapidly induced by short duration exposure to light at levels sufficient to reset the clock, and dose–response curves reveal that mPer1 induction shows both reciprocity and a strong correlation with phase shifting of the overt rhythm. Thus, in both the phasing of dark expression and the response to light mPer1 is most similar to the Neurospora clock gene frq . Within the SCN there appears to be localization of the induction phenomenon, consistent with the localization of both light-sensitive and light-insensitive oscillators in this circadian center.

The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydrophobin, is transcriptionally regulated by the circadian clock. In addition, eas (ccg-2) is positively regulated by light and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analyses of the eas (ccg-2) promoter were carried out to localize the cis-acting elements mediating clock, light, and developmental control. The primary sequence determinants of a positive activating clock element (ACE) were found to reside in a 45-bp region, just upstream from the TATA box. Using a novel unregulated promoter/reporter system developed for this study, we show that a 68-bp sequence encompassing the ACE is sufficient to confer clock regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using the ACE reveal factors present in N. crassa protein extracts that recognize and bind specifically to DNA containing this element. Separate regions of the eas (ccg-2) promoter involved in light induction and developmental control are identified and shown not to be required for clock-regulated expression of eas (ccg-2). The distinct nature of the ACE validates its use as a tool for the identification of upstream regulatory factors involved in clock control of gene expression.

Summary To understand how light entrains circadian clocks, weexamined the effects of light on a gene known to encode a state variable of a circadian oscillator, the frequency ( frq ) gene. frq is rapidly induced by short pulses of visible light; clock resetting is correlated with frq induction and is blocked by drugs that block the synthesis of protein or translatable RNA. The speed and magnitude of frq induction suggest that this may be the initial clock-specific event in light resetting. Light induction overcomes frq negative autoregulation so that frq expression can remain high in constant light. These data explain how a simple unidirectional signal (light and the induction of frq ) may be turned into a bidirectional clock response (time of day-specific advances and delays). This light entrainment model is easily generalized and may be the common mechanism by which the intracellular feedback cycles that comprise circadian clocks are brought into synchrony with external cycles in the real world.

The pacemaker of the Neurospora circadian clock is composed of a transcriptional-translational feedback loop that has been intensively studied during the last two decades. Invaluable information has been derived from measuring the expression of the central clock component frequency ( frq ) under liquid culture conditions. Direct analyses of frq mRNA and protein levels on solid media – where overt circadian rhythms are normally visualized – have not been trivial due to technical issues. Nevertheless, a frq promoter-luciferase reporter has recently allowed the study of frq transcription under these conditions. It is known that FRQ undergoes extensive posttranslational modifications, and changes in its levels provide important information regarding the clockworks. Here we describe a FRQ-luciferase translational fusion reporter that directly tracks FRQ levels, granting access to a better understanding and analysis of FRQ dynamics in vivo . More generally the method, which allows the investigator to follow continuous gene expression in real time in a spatially and temporally unrestricted manner, should be widely applicable to analyses of environmentally and developmentally regulated gene expression in ascomycete filamentous fungi as well as in basidiomycetes.

The mammalian circadian clock influences the timing of many biological processes such as the sleep/wake cycle, metabolism, and cell division. Environmental cues such as light exposure can influence the timing of this system through the posttranslational modification of key components of the core molecular oscillator. We have previously shown that DNA damage can reset the circadian clock in a time-of-day—dependent manner in the filamentous fungus Neurospora crassa through the modulation of negative regulator FREQUENCY levels by PRD-4 (homologue of mammalian Chk2). We show that DNA damage, generated with either the radiomimetic drug methyl methane sulfonate or UV irradiation, in mouse embryonic fibroblasts isolated from PER2::LUC transgenic mice or in the NIH3T3 cell line, elicits similar responses. In addition to induction of phase advances, DNA damage caused a decrease in luciferase signal in PER2::LUC mouse embryonic fibroblast cells that is indicative of PER2 degradation. Finally, we show that the activity of the BMAL1 promoter is enhanced during DNA damage. These findings provide further evidence that the DNA damage-mediated response of the clock is conserved from lower eukaryotes to mammals.

The pace has quickened in circadian biology research. In particular, an abundance of results focused on post-translational modifications (PTMs) is sharpening our view of circadian molecular clockworks. PTMs affect nearly all aspects of clock biology; in some cases they are essential for clock function and in others, they provide layers of regulatory fine-tuning. Our goal is to review recent advances in clock PTMs, help make sense of emerging themes, and spotlight intriguing (and perhaps controversial) new findings. We focus on PTMs affecting the core functions of eukaryotic clocks, in particular the functionally related oscillators in Neurospora crassa, Drosophila melanogaster, and mammalian cells.

Although an extensive number of biological processes are under the daily control of the circadian biological clock, little is known about how the clock maintains its regulatory networks within a cell. An important aspect of this temporal control is the daily control of gene expression. Previously we identified two morning-specific genes that are regulated by the clock through daily control of gene expression (J. Loros, S. Denome, and J.C. Dunlap, Science 243:385-388, 1989). We have now introduced a method for transcriptional analysis in Neurospora crassa and used this nuclear run-on procedure to show that regulation of mRNA abundance for these two morning-specific genes occurs at the level of transcription. This transcriptional regulation by the circadian clock provides a basis for isolating circadian rhythm mutants.

In Neurosporacrassa the FRQ/WC feedback loop has been shown to be central to the function of the circadian clock. Similar to other eukaryotic systems it is based on a transcription-translation PAS heterodimer type feedback. FRQ levels cycle with a period identical to that of the Neurospora circadian cycle and its expression is rapidly induced by light. A complex of White Collar 1 (WC-1) and White Collar 2 (WC-2) (the WCC) is required for the transcriptional activation of frq. The oscillation in frq message is transcriptionally regulated via a single necessary and sufficient cis-acting element in the frq promoter, the Clock-Box (CB) bound by WCC. Light-induction of frq transcription is mediated by WCC binding to two cis-acting elements (LREs) in the frq promoter. WC-1, with flavin adenine dinucleotide (FAD) as a cofactor, is the blue-light photoreceptor. The original description of a frq-null strain, frq9, (Loros et al 1986) included a description of oscillations in asexual conidial banding that occasionally appeared following 3 to 7 days of arrhythmic development now referred to as FLO for FRQ-less oscillator. Unlike the intact clock, FLO period is sensitive to media composition. We have identified a circadianly regulated gene whose mutation interferes with FLO even under temperature entrainment conditions. This same mutation affects the circadian clock in a frq+ background causing a shorter period length as well as temperature response defects. This gene may be an entry point to study the connection between the biological clock and other basic cellular mechanisms.

In Neurospora , metabolic oscillators coexist with the circadian transcriptional/translational feedback loop governed by the FRQ (Frequency) and WC (White Collar) proteins. One of these, a choline deficiency oscillator (CDO) observed in chol-1 mutants grown under choline starvation, drives an uncompensated long-period developmental cycle (≈60–120 h). To assess possible contributions of this metabolic oscillator to the circadian system, molecular and physiological rhythms were followed in liquid culture under choline starvation, but these only confirmed that an oscillator with a normal circadian period length can run under choline starvation. This finding suggested that long-period developmental cycles elicited by nutritional stress could be masking output from the circadian system, although a caveat was that the CDO sometimes requires several days to become consolidated. To circumvent this and observe both oscillators simultaneously, we used an assay using a codon-optimized luciferase to follow the circadian oscillator. Under conditions where the long-period, uncompensated, CDO-driven developmental rhythm was expressed for weeks in growth tubes, the luciferase rhythm in the same cultures continued in a typical compensated manner with a circadian period length dependent on the allelic state of frq . Periodograms revealed no influence of the CDO on the circadian oscillator. Instead, the CDO appears as a cryptic metabolic oscillator that can, under appropriate conditions, assume control of growth and development, thereby masking output from the circadian system. frq -driven luciferase as a reporter of the circadian oscillator may in this way provide a means for assessing prospective role(s) of metabolic and/or ancillary oscillators within cellular circadian systems.

In the Neurospora circadian system, the transcription factors White Collar-1 (WC-1) and White Collar-2 (WC-2) activate expression of frq, whose gene product inhibits its own expression. The WC proteins are thought to form an obligate complex; however, chromatin immunoprecipitation (ChIP) indicates that WC-2 binds to the frq promoter in a rhythmic fashion, whereas WC-1 is bound continuously. Small oscillations in histone acetylation are detected over the circadian cycle with a marked reduction upon light-induced activation. Nuclease accessibility experiments indicate chromatin rearrangement at the frq promoter; therefore, 19 genes with homology to ATP-dependent chromatin-remodeling enzymes were deleted and the strains examined for clock phenotypes. One gene, designated clockswitch (csw-1), is required for clock function; its product localizes to the frq promoter, is required for proper frq expression, and has an impact on chromatin structure. The data suggest that CSW-1 regulates accessibility of promoter DNA, thus generating the sharp transition from the transcriptionally active to the repressed state.

The eukaryotic filamentous fungus Neurospora crassa is a tractable model system that has provided numerous insights into the molecular basis of circadian rhythms. In the core circadian clock feedback loop, WC-1 and WC-2 interact via PAS domains to heterodimerize, and this complex acts both as the circadian photoreceptor and, in the dark, as a transcription factor that promotes the expression of the frq gene. In the negative step of the loop, dimers of FRQ feed back to block the activity of the WC-1/WC-2 complex (WCC) and, in a positive step, to promote the synthesis of WC-1. Several kinases phosphorylate FRQ, leading to its ubiquitination and turnover, releasing the WC-1/WC-2 dimer to reactivate frq expression and restart the circadian cycle. Light and temperature entrainment of the clock arise from rapid light induction of frq expression and from the effect of elevated temperatures in driving higher levels of FRQ. Noncircadian candidate slave oscillators, termed FRQ-less oscillators (FLOs), have been described, each of which appears to regulate aspects of Neurospora growth or development. Overall, the core FRQ/WCC feedback loop coordinates the circadian system by regulating downstream clock-controlled genes either directly or via regulation of driven FLOs. This article provides a brief synopsis of the system and describes current assays for the Neurospora clock. Methods for genetic and molecular manipulation of the core clock are summarized, and accompanying chapters address more specifically aspects of photobiology and output.

The true biology of chronobiology lies in the spectrum of processes that are controlled by the circadian clock. Although this biology plays out at the level of the whole organism, it derives, finally, from clock-driven changes in physiology, and frequently in gene expression, that occur at the level of individual cells. For this reason, analysis of gene expression rhythms measured in cell culture or in organisms that elaborate only a few cell types provides insights not possible in multicellular organisms. In this context we have used mammalian fibroblasts in culture as well as the eukaryotic filamentous fungus Neurospora crassa to study circadian output, in particular the output rhythms in gene expression that underlie so much of circadian biology. Each cell type has its own advantages: Data from mammalian cells are obviously immediately pertinent to animal cell rhythms, but the system allows little genetics and only limited amounts of material can be collected. Alternatively, Neurospora allows genetic and molecular analyses and is useful for developing concepts and models of output that can be examined in other contexts. This methods article focuses on these two systems for analysis, providing an overview of how control is presently viewed followed by current methods for its analysis.

The biological clock of Neurospora crassa includes interconnected transcriptional and translational feedback loops that cause both the transcript and protein encoded by the frequency gene ( frq ) to undergo the robust daily oscillations in abundance, which are essential for clock function. To understand better the mechanism generating rhythmic frq transcript, reporter constructs were used to show that the oscillation in frq message is transcriptionally regulated, and a single cis-acting element in the frq promoter, the Clock Box (C box), is both necessary and sufficient for this rhythmic transcription. Nuclear protein extracts used in binding assays revealed that a White Collar (WC)-1- and WC-2-containing complex (WCC) binds to the C box in a time-of-day-specific manner. Overexpression of an ectopic copy of FRQ or addition of in vitro -generated FRQ resulted in reduced WCC binding to the C box. These data suggest that oscillations in frq transcript result from WCC binding to the frq promoter and activating transcription with subsequent changes in FRQ levels having an inverse effect on WCC binding. In this way rhythmic expression and turnover of FRQ drives the rhythm in its own transcription.

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.

Eukaryotic circadian clocks comprise feedback loops where PAS domain-containing transcriptional activators drive gene expression of negative elements. In NEUROSPORA:, clock models posit a White Collar complex (WCC) containing WC-1 and WC-2 that activates expression of the central clock gene frequency (frq); FRQ protein is hypothesized to feed back to block the activity of the WCC. We have characterized the WC-2 protein and its role in this complex: WC-2 is an abundant constitutive nuclear protein, in contrast to rhythmically expressed FRQ and WC-1. WC-2 interacts with WC-1 and FRQ but, significantly, WC-1 and FRQ do not interact in the absence of WC-2. By quantifying the relative numbers of WC-2, FRQ and WC-1 proteins and complexes in cell extracts, both the numbers and types of complexes at different circadian times were estimated, yielding results consistent with the model. Constitutive and abundant WC-2 appears to provide a scaffold allowing for the interaction of two limiting and rhythmically out-of-phase proteins, FRQ and WC-1, and this temporal and physical relationship may be responsible for rhythmic expression of frq.

In Neurospora crassa , white collar 1 (WC-1), a transcriptional activator and positive clock element, is rhythmically expressed from a nonrhythmic steady-state pool of wc-1 transcript, consistent with posttranscriptional regulation of rhythmicity. Mutations in frq influence both the level and periodicity of WC-1 expression, and driven FRQ expression not only depresses its own endogenous levels, but positively regulates WC-1 synthesis with a lag of about 8 hours, a delay similar to that seen in the wild-type clock. FRQ thus plays dual roles in the Neurospora clock and thereby, with WC-1, forms a second feedback loop that would promote robustness and stability in this circadian system. The existence also of interlocked loops in Drosophila melanogaster and mouse clocks suggests that such interlocked loops may be a conserved aspect of circadian timing systems.

A recent explosion in the identification of new clock components in cyanobacteria, fungi, insects, mammals as well as potential candidates in plants has uncovered common themes among the structure, function and regulation of these components. Positive and negative interactions that are organized in negative feedback loops have been found crucial for clock function. Both transcriptional and posttranscriptional mechanisms appear to be important for circadian rhythm generation in all of these organisms.