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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.

Strategies for promoting high-efficiency homologous gene replacement have been developed and adopted for many filamentous fungal species. The next generation of analysis requires the ability to manipulate gene expression and to tag genes expressed from their endogenous loci. Here we present a suite of molecular tools that provide versatile solutions for fungal high-throughput functional genomics studies based on locus-specific modification of any target gene. Additionally, case studies illustrate caveats to presumed overexpression constructs. A tunable expression system and different tagging strategies can provide valuable phenotypic information for uncharacterized genes and facilitate the analysis of essential loci, an emerging problem in systematic deletion studies of haploid organisms.

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.

White collar-1 (WC-1) and white collar-2 (WC-2) are essential for light-mediated responses in Neurospora crassa, but the molecular mechanisms underlying gene induction and the roles of other real and putative photoreceptors remain poorly characterized. Unsupervised hierarchical clustering of genome-wide microarrays reveals 5.6% of detectable transcripts, including several novel mediators, that are either early or late light responsive. Evidence is shown for photoreception in the absence of the dominant, and here confirmed, white collar complex (WCC) that regulates both types of light responses. VVD primarily modulates late responses, whereas light-responsive submerged protoperithecia-1 (SUB-1), a GATA family transcription factor, is essential for most late light gene expression. After a 15-min light stimulus, the WCC directly binds the sub-1 promoter. Bioinformatics analysis detects many early light response elements (ELREs), as well as identifying a late light response element (LLRE) required for wild-type activity of late light response promoters. The data provide a global picture of transcriptional response to light, as well as illuminating the cis- and trans-acting elements comprising the regulatory signalling cascade that governs the photobiological response.

In Neurospora crassa, a transcription factor, WCC, activates the transcription of frq. FRQ forms homodimers as well as complexes with an RNA helicase, FRH, and the WCC, and translocates into the nucleus to inactivate the WCC, closing the time-delayed negative feedback loop. The detailed mechanism for closing this loop, however, remains incompletely understood. In particular within the nucleus, the low amount of FRQ compared with that of WC-1 creates a conundrum: How can the nuclear FRQ inactivate the larger amount of WCC? One possibility is that FRQ might function as a catalytic component in phosphorylation-dependent inhibition. However, in silico experiments reveal that stoichiometric noncatalytic binding and inhibition can generate a robust oscillator, even when nuclear FRQ levels are substantially lower than nuclear WCC, so long as there is FRQ-dependent clearance of WC-1 from the nucleus. Based on this model, we can predict and now demonstrate that WC-1 stability cycles, that WC-1 is stable in the absence of FRQ, and that physical binding between FRQ and WCC is essential for closure of the negative feedback loop. Moreover, and consistent with a noncatalytic clearance-based model for inhibition, appreciable amounts of the nuclear FRQ:WCC complex accumulate at some times of day, comprising as much as 10% of the nuclear WC-1.

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.

We report the complete reconstruction of the firefly luciferase gene, fully codon optimized for expression in Neurospora crassa . This reporter enhances light output by approximately 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase , shows the highest levels of luciferase activity near the growth front and newly formed conidial bands. Further, we have established a sorbose medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15-h period rhythms at high temperatures, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.

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.

The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Here, we present an analysis of the sterile mutant pro41 that is unable to produce mature fruiting bodies. The mutant carries a deletion of 4 kb and is complemented by the pro41 open reading frame that is contained within the region deleted in the mutant. In silico analyses predict PRO41 to be an endoplasmic reticulum (ER) membrane protein, and a PRO41-EGFP fusion protein colocalizes with ER-targeted DsRED. Furthermore, Western blot analysis shows that the PRO41-EGFP fusion protein is present in the membrane fraction. A fusion of the predicted N-terminal signal sequence of PRO41 with EGFP is secreted out of the cell, indicating that the signal sequence is functional. pro41 transcript levels are upregulated during sexual development. This increase in transcript levels was not observed in the sterile mutant pro1 that lacks a transcription factor gene. Moreover, microarray analysis of gene expression in the mutants pro1, pro41 and the pro1/41 double mutant showed that pro41 is partly epistatic to pro1. Taken together, these data show that PRO41 is a novel ER membrane protein essential for fruiting body formation in filamentous fungi.

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.

The regulation of metabolism by circadian systems is believed to be a key reason for the extensive representation of circadian rhythms within the tree of life. Despite this, surprisingly little work has focused on the link between metabolism and the clock in Neurospora, a key model system in circadian research. The analysis that has been performed has focused on the unidirectional control from the clock to metabolism and largely ignored the feedback from metabolism on the clock. Recent efforts to understand these links have broken new ground, revealing bidirectional control from the clock to metabolism and vise-versa, showing just how strongly interconnected these two cellular systems can be in fungi. This review describes both well understood and emerging links between the clock and metabolic output of fungi as well as the role that metabolism plays in influencing the rhythm set by the clock.

Most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. In the filamentous fungus Neurospora crassa VIVID (VVD) is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. Detailed in vitro analysis of the photochemistry of the blue light sensing, FAD binding, LOV domain of VVD has revealed residues around the site of photo-adduct formation that influence the stability of the adduct state (light state), that is, altering the photocycle length. We have examined the biological significance of VVD photocycle length to photoadaptation and report that a double substitution mutant (vvdI74VI85V), previously shown to have a very fast light to dark state reversion in vitro, shows significantly reduced interaction with the White Collar Complex (WCC) resulting in a substantial photoadaptation defect. This reduced interaction impacts photoreceptor transcription factor WHITE COLLAR-1 (WC-1) protein stability when N. crassa is exposed to light: The fast-reverting mutant VVD is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation as assayed both by attenuation of gene expression and the ability to respond to increasing light intensity. Additionally, transcription of the clock gene frequency (frq) is sensitive to changing light intensity in a wild-type strain but not in the fast photo-reversion mutant indicating that the establishment of this dynamic VVD-WCC pool is essential in general photobiology and circadian biology. Thus, VVD photocycle length appears sculpted to establish a VVD-WCC reservoir of sufficient size to sustain photoadaptation while maintaining sensitivity to changing light intensity. The great diversity in photocycle kinetics among photoreceptors may be viewed as reflecting adaptive responses to specific and salient tasks required by organisms to respond to different photic environments., Author Summary Sensing light from the environment using a variety of photoreceptors is of great adaptive significance for most eukaryotes. A key feature of photoreceptors is the photocycle length, the time taken to decay from the initial signaling light state back to the receptive dark state; however, the significance of photocycle length, or adduct decay length, has not been tested in a biological setting. The photocycle length is determined by the chemical environment of the active site where a photon absorbing chromophore forms an adduct with a conserved amino acid. There is clear evidence of evolutionary selection for a particular photocycle length even between photoreceptors containing the same prototypic light-sensing domains suggesting functional relevance. Using defined in vitro mutations that change the photocycle length of the VIVID (VVD) protein over 4 orders of magnitude we were able to ascribe a pivotal role of the native photochemistry of the protein in its function as a photoreceptor in the light and circadian biology of Neurospora crassa. This study links in vitro photochemical studies with in vivo function and provides evidence that the true evolutionary and functional significance of native photochemistry of photoreceptors can be enhanced by studying photocycle mutants in their native systems.

The circadian clock exists to synchronize inner physiology with the external world, allowing life to anticipate and adapt to the continual changes that occur in an organism's environment. The clock architecture is highly conserved, present in almost all major branches of life. Within eukaryotes, the filamentous fungus Neurospora crassa has consistently been used as an excellent model organism to uncover the basic circadian physiology and molecular biology. The Neurospora model has elucidated our fundamental understanding of the clock as nested positive and negative feedback loop, regulated by transcriptional and posttranscriptional processes. This review will examine the basics of circadian rhythms in the model filamentous fungus N. crassa as well as highlight the output of the clock in Neurospora and the reasons that N. crassa has continued to be a strong model for the study of circadian rhythms. It will also synopsize classical and emerging methods in the study of the circadian clock.

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 circadian system in Neurospora remains a premier model system for understanding circadian rhythms, and evidence has now begun to accumulate suggesting broad conservation of rhythmicity amongst the filamentous fungi. A well-described transcription-translation-based negative feedback loop involving the FREQUENCY, WHITE COLLAR-1 and WHITE COLLAR-2 proteins is integral to the Neurospora system. Recent advances include descriptions of the surprisingly complex frequency transcription unit, an enhanced appreciation of the roles of kinases and their regulation in the generation of the circadian rhythm and their links to the cell cycle, and strong evidence for an additional WHITE COLLAR-associated feedback loop. Documentation of sequence homologs of integral circadian and photoresponsive proteins amongst the 42 available sequenced fungal genomes suggests unexpected roles for circadian timing among both pathogens and saprophytes.

The clock gene period-4 ( prd-4 ) in Neurospora was identified by a single allele displaying shortened circadian period and altered temperature compensation. Positional cloning followed by functional tests show that PRD-4 is an ortholog of mammalian checkpoint kinase 2 ( Chk2 ). Expression of prd-4 is regulated by the circadian clock and, reciprocally, PRD-4 physically interacts with the clock component FRQ, promoting its phosphorylation. DNA-damaging agents can reset the clock in a manner that depends on time of day, and this resetting is dependent on PRD-4. Thus, prd-4 , the Neurospora Chk2 , identifies a molecular link that feeds back conditionally from circadian output to input and the cell cycle.

Sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. To analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene Smta-1 encoding a high-mobility group domain (HMG) protein. The DeltaSmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. To identify genes expressed under control of Smta-1, we performed a cross-species microarray analysis using Neurospora crassa cDNA microarrays hybridized with S. macrospora targets. We identified 107 genes that are more than twofold up- or down-regulated in the mutant. Functional classification revealed that 81 genes have homologues with known or putative functions. Comparison of array data from DeltaSmta-1 with those from three phenotypically similar mutants revealed that only a limited set of ten genes is deregulated in all mutants. Remarkably, the ppg2 gene encoding a putative lipopeptide pheromone is 500-fold down-regulated in the DeltaSmta-1 mutant while in all other sterile mutants this gene is up-regulated.

Investigation of the red bread mold that contaminated French bakeries nearly two centuries ago has led to a wealth of discoveries that have impacted our understanding of genetic, biochemical, and molecular mechanisms in microbes, from Mendelian genetics and the gene-enzyme relationship to circadian rhythm and plant cell wall degradation. Early Neurospora research focused on elucidating mechanisms of genetic recombination and gene action and later progressed to addressing complex biological questions of eukaryotic microbes. Here we review the evolution of the filamentous fungus Neurospora as a model microbe over the past century. We discuss the origins of Neurospora as a model microbe, the immediate scientific impacts from work in this filamentous fungus, and how the introduction of other model organisms (i.e., Escherichia coli and Saccharomyces cerevisiae) redirected the focus of Neurospora research. Neurospora has and continues to inform our understanding of a myriad of basic scientific concepts and now has the opportunity to forge into the applied biosciences and biotechnology.

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.

Phytochromes (Phys) comprise a superfamily of red-/far-red-light-sensing proteins. Whereas higher-plant Phys that control numerous growth and developmental processes have been well described, the biochemical characteristics and functions of the microbial forms are largely unknown. Here, we describe analyses of the expression, regulation, and activities of two Phys in the filamentous fungus Neurospora crassa . In addition to containing the signature N-terminal domain predicted to covalently associate with a bilin chromophore, PHY-1 and PHY-2 contain C-terminal histidine kinase and response regulator motifs, implying that they function as hybrid two-component sensor kinases activated by light. A bacterially expressed N-terminal fragment of PHY-2 covalently bound either biliverdin or phycocyanobilin in vitro, with the resulting holoprotein displaying red-/far-red-light photochromic absorption spectra and a photocycle in vitro. cDNA analysis of phy-1 and phy-2 revealed two splice isoforms for each gene. The levels of the phy transcripts are not regulated by light, but the abundance of the phy-1 mRNAs is under the control of the circadian clock. Phosphorylated and unphosphorylated forms of PHY-1 were detected; both species were found exclusively in the cytoplasm, with their relative abundances unaffected by light. Strains containing deletions of phy-1 and phy-2 , either singly or in tandem, were not compromised in any known photoresponses in Neurospora , leaving their function(s) unclear.

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 filamentous fungus Sordaria macrospora forms complex three-dimensional fruiting bodies that protect the developing ascospores and ensure their proper discharge. Several regulatory genes essential for fruiting body development were previously isolated by complementation of the sterile mutants pro1, pro11 and pro22. To establish the genetic relationships between these genes and to identify downstream targets, we have conducted cross-species microarray hybridizations using cDNA arrays derived from the closely related fungus Neurospora crassa and RNA probes prepared from wild-type S. macrospora and the three developmental mutants. Of the 1,420 genes which gave a signal with the probes from all the strains used, 172 (12%) were regulated differently in at least one of the three mutants compared to the wild type, and 17 (1.2%) were regulated differently in all three mutant strains. Microarray data were verified by Northern analysis or quantitative real time PCR. Among the genes that are up- or down-regulated in the mutant strains are genes encoding the pheromone precursors, enzymes involved in melanin biosynthesis and a lectin-like protein. Analysis of gene expression in double mutants revealed a complex network of interaction between the pro gene products.

Summary The eukaryotic filamentous fungus Neurospora crassa has proven to be a dependable model system for the analysis of light-regulated gene expression and circadian rhythmicity. The molecular basis of the core circadian clock is a transcription/translationbased feedback loop in which a heterodimer of the white-collar 1 (WC-1) and white-collar 2 (WC-2) proteins act as a transcription factor to promote expression of the negative elements in the clock, frq mRNA and FRQ protein. Additionally, WC-1 is a flavoprotein that acts as the primary responder to environmental light and, in conjunction with WC-2, is the major photoreceptor for the clock as well as other light-regulated processes. Protein kinase C acts as a lightdependent regulator of the WC-1 protein.

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.

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.

The transcription factors WHITE COLLAR-1 (WC-1) and WHITE COLLAR-2 (WC-2) interact to form a heterodimeric complex (WCC) that is essential for most of the light-mediated processes in Neurospora crassa. WCC also plays a distinct non-light-related role as the transcriptional activator in the FREQUENCY (FRQ)/WCC feedback loop that is central to the N. crassa circadian system. Although an activator role was expected for WC-1, unanticipated phenotypes resulting from some wc-1 alleles prompted a closer examination of an allelic series for WC-1 that has uncovered roles for this central regulator in constant darkness and in response to light. We analyzed the phenotypes of five different wc-1 mutants for expression of FRQ and WC-1 in constant darkness and following light induction. While confirming the absolute requirement of WC-1 for light responses, the data suggest multiple levels of control for light-regulated genes.

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.

Many aspects of physiology and behavior are temporally organized into daily 24 hr rhythms, driven by an endogenous circadian clock. Studies in eukaryotes have identified a network of interacting genes forming interlocked autoregulatory feedback loops which underlie overt circadian organization in single cells [1, 2]. While in mammals the master oscillator resides in the suprachiasmatic nuclei of the hypothalamus [2], semiautonomous circadian oscillators also exist in peripheral tissues [3–5] and in immortalized fibroblasts, where rhythmicity is induced following a serum shock [6, 7]. We used this model system in combination with high-density cDNA microarrays to examine the magnitude and quality of clock control of gene expression in mammalian cells. Supported by application of novel bioinformatics tools, we find ∼2% of genes, including expected canonical clock genes, to show consistent rhythmic circadian expression across five independent experiments. Rhythmicity in most of these genes is novel, and they fall into diverse functional groups, highlighted by a predominance of transcription factors, ubiquitin-associated factors, proteasome components, and Ras/MAPK signaling pathway components. When grouped according to phase, 68% of the genes were found to peak during estimated subjective day, 32% during estimated subjective night, with a tendency to peak at a phase corresponding to anticipation of dawn or dusk.

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.

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation–based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter–luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.

In the negative feedback loop comprising the Neurospora circadian oscillator, the White Collar Complex (WCC) formed from White Collar-1 (WC-1) and White Collar-2 (WC-2) drives transcription of the circadian pacemaker gene frequency (frq). Although FRQ-dependent repression of WCC has been extensively studied, the mechanism by which the WCC initiates a circadian cycle remains elusive. Structure/function analysis of WC-1 eliminated domains previously thought to transactivate frq expression but instead identified amino acids 100–200 as essential for frq circadian expression. A proteomics-based search for coactivators with WCC uncovered the SWI/SNF (SWItch/Sucrose NonFermentable) complex: SWI/SNF interacts with WCC in vivo and in vitro, binds to the Clock box in the frq promoter, and is required both for circadian remodeling of nucleosomes at frq and for rhythmic frq expression; interestingly, SWI/SNF is not required for light-induced frq expression. These data suggest a model in which WC-1 recruits SWI/SNF to remodel and loop chromatin at frq, thereby activating frq expression to initiate the circadian cycle., Author Summary Circadian clocks govern behavior in a wide variety of organisms. These clocks are assembled in such a way that proteins encoded by a few dedicated “clock genes” form a complex that acts to reduce their own expression. That is, the genes and proteins participate in a negative feedback loop, and so long as the feedback has delays built in, this system will oscillate. The feedback loops that underlie circadian rhythms in fungi and animals are quite similar in many ways, and while much is known about the proteins themselves, both those that activate the dedicated clock genes and the clock proteins that repress their own expression, relatively little is known about how the initial expression of the clock genes is activated. In Neurospora, a fungal model for these clocks, the proteins that activate expression of the clock gene “frequency” bind to DNA far away from where the coding part of the gene begins, and a mystery has been how this action-at-a-distance works. The answer revealed here is that the activating proteins recruit other proteins to unwrap the DNA and bring the distal site close to the place where the coding part of the gene begins.

Visible light is an important source of energy and information for much of life on this planet. Though fungi are neither photosynthetic nor capable of observing adjacent objects, it is estimated that the majority of fungal species display some form of light response, ranging from developmental decision-making to metabolic reprogramming to pathogenesis. As such, advances in our understanding of fungal photobiology will likely reach the broad fields impacted by these organisms, including agriculture, industry and medicine. In this review, we will first describe the mechanisms by which fungi sense light and then discuss the selective advantages likely imparted by their ability to do so.

Light sensing and circadian rhythmicity are two related processes that promote the adaptation of many fungal species to their environment. This chapter begins with a description of fungal photoreceptors and their distributions across fungal lineages. We then discuss in some detail the molecular mechanisms of the photoresponse in two well-studied model fungi, Neurospora crassa and Aspergillus nidulans, placing an emphasis on the important similarities and differences between the two species. This will lead to a description of circadian rhythmicity in fungi in general, with a particular emphasis on Neurospora crassa. We highlight the core mechanism in this organism as well as discuss the inputs and outputs that can affect the clock. We also note at the end some new developments in molecular genetics in Neurospora.

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating ∼20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For ∼50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.

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.

Circadian rhythms control many physiological activities. The environmental entrainment of rhythms involves the immediate responses of clock components. Levels of the clock protein FRQ were measured in Neurospora at various temperatures; at higher temperatures, the amount of FRQ oscillated around higher levels. Absolute FRQ amounts thus identified different times at different temperatures, so temperature shifts corresponded to shifts in clock time without immediate synthesis or turnover of components. Moderate temperature changes could dominate light-to-dark shifts in the influence of circadian timing. Temperature regulation of clock components could explain temperature resetting of rhythms and how single transitions can initiate rhythmicity from characteristic circadian phases.

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.

Circadian rhythmicity is universally associated with the ability to perceive light, and the oscillators (“clocks”) giving rise to these rhythms, which are feedback loops based on transcription and translation, are reset by light. Although such loops must contain elements of positive and negative regulation, the clock genes analyzed to date— frq in Neurospora and per and tim in Drosophila —are associated only with negative feedback and their biochemical functions are largely inferred. The white collar–1 and white collar–2 genes, both global regulators of photoresponses in Neurospora , encode DNA binding proteins that contain PAS domains and are believed to act as transcriptional activators. Data shown here suggest that wc-1 is a clock-associated gene and wc-2 is a clock component; both play essential roles in the assembly or operation of the Neurospora circadian oscillator. Thus DNA binding and transcriptional activation can now be associated with a clock gene that may provide a positive element in the feedback loop. In addition, similarities between the PAS-domain regions of molecules involved in light perception and circadian rhythmicity in several organisms suggest an evolutionary link between ancient photoreceptor proteins and more modern proteins required for circadian oscillation.

Background Perturbations in the function of core circadian clock components such as the Period (Per) family of genes are associated with alcohol use disorder, and disruptions in circadian cycles may contribute to alcohol abuse and relapse. This study tested ethanol consumption, reinforcement, and metabolism in mice containing functional mutations in Per1 and/or Per2 genes on an ethanol-preferring background, C57BL/6J mice. Methods Mice were tested in: (A) free-access intake with ascending concentrations of ethanol (2–16%, v/v), (B) conditioned place preference using ethanol (2 g/kg for males; 2.5 g/kg for females) vs. saline injections, (C) recovery of the righting reflex following a 4 g/kg bolus of ethanol, and (D) blood ethanol levels 1 h after a 2 g/kg bolus of ethanol. Results All Per mutant (mPer) mice showed increased ethanol intake and condition place preference compared to controls. There were also genotypic differences in blood ethanol concentration: in males, only mPer1 mice showed a significantly higher blood ethanol concentration than WT mice, but in females, all mPer mice showed higher blood ethanol levels than WT mice. Conclusions Mutation of either Per1 or Per2, as well as mutations of both genes, increases ethanol intake and reinforcement in an ethanol-preferring mouse model. In addition, this increase in ethanol seeking behavior seems to result both from a change in ethanol metabolism and a change in reward responding to ethanol, but not from any change in sensitivity to ethanol's sedating effects.

Light is a pervasive environmental factor that regulates development, stress resistance, and even virulence in numerous fungal species. Though much research has focused on signaling pathways in Aspergillus fumigatus, an understanding of how this pathogen responds to light is lacking. In this report, we demonstrate that the fungus does indeed respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus light response is a reduction in conidial germination rates, increased hyphal pigmentation, enhanced resistance to acute ultraviolet and oxidative stresses, and an increased susceptibility to cell wall perturbation. By performing gene deletion analyses, we have found that the predicted blue light receptor LreA and red light receptor FphA play unique and overlapping roles in regulating the described photoresponsive behaviors of A. fumigatus. However, our data also indicate that the photobiology of this fungus is complex and likely involves input from additional photosensory pathways beyond those analyzed here. Finally, whole-genome microarray analysis has revealed that A. fumigatus broadly regulates a variety of metabolic genes in response to light, including those involved in respiration, amino acid metabolism, and metal homeostasis. Together, these data demonstrate the importance of the photic environment on the physiology of A. fumigatus and provide a basis for future studies into this unexplored area of its biology., IMPORTANCE Considerable effort has been taken to understand how the mold pathogen Aspergillus fumigatus senses its environment to facilitate growth within the immunocompromised host. Interestingly, it was shown that the deletion of a blue light photoreceptor in two divergent fungal pathogens, Cryptococcus neoformans and Fusarium oxysporum, leads to an attenuation of virulence in their respective animal infection models. This suggests that light signaling pathways are conservatively involved in the regulation of fungal pathogenesis. However, an understanding of whether and how A. fumigatus responds to light is lacking. Here we demonstrate that this organism coordinates broad aspects of its physiology with the photic environment, including pathways known to be involved in virulence, such as carbohydrate metabolism and oxidative stress resistance. Moreover, the photoresponse of A. fumigatus differs in notable ways from the well-studied model Aspergillus nidulans. Therefore, this work should represent a general advancement in both photobiology and A. fumigatus research communities.

Endogenous circadian clocks, present in both prokaryotes and eukaryotes, provide the machinery by which organisms keep in synchrony with the external world. The ability of the clock to measure accurate time allows changes in the environment (particularly light and temperature) to be anticipated so that biological activities can occur at the appropriate times of day. The observed circadian rhythms are, as a general feature, established through the daily control of specific target genes by the clock. Demonstrations of circadian rhythms are widespread, and in the fungi the clock has been shown to control daily rhythms in spore development and liberation. Within the fungi,Neurospora crassa provides the preeminent model system for investigations into the underlying processes of circadian rhythms. Through genetic and molecular approaches, significant progress has been made in describing theN crassa circadian system. As discussed in this review, the analysis of theN. crassa clock has provided important details on (i) how the clock is assembled and (ii) how environmental signals are perceived by the clock, and (iii) a description of rhythmic output genes that are regulated by the clock.

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.

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 fungus Neurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genes ccg-1 and ccg-2. In this paper we report studies on the regulation of ccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes in Neurospora, we report that ccg-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 of ccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction of ccg-1 requires the products of the Neurospora wc-1 and wc-2 genes, but surprisingly the requirement for wc-2 is suppressed in conditional mutants of cot-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 of ccg-1.