Complexity of the Neurospora crassa circadian clock system: multiple loops and oscillators.

Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. We review evidence from Neurospora crassa that suggests that the circadian clock is organized as a network of genes and proteins that form coupled evening- and morning-specific oscillatory loops that can function autonomously, respond differently to environmental inputs, and regulate phase-specific outputs. There is also evidence for coupled morning and evening oscillator loops in plants, insects, and mammals, suggesting conservation of clock organization. From a systems perspective, fungi provide a powerful model organism for investigating oscillator complexity, communication between oscillators, and addressing reasons why the system has evolved to be so complex.

Effects of altered Clock gene expression on the pacemaker properties of SCN2.2 cells and oscillatory properties of NIH/3T3 cells.

While peripheral tissues and serum-shocked fibroblasts express rhythmic oscillations in clock gene expression, only the suprachiasmatic nucleus (SCN) is capable of endogenous, self-sustained rhythmicity and of functioning as a pacemaker by imposing rhythmic properties upon other cells. To differentially examine the molecular elements necessary for the distinctive rhythm-generating and pacemaking properties of the SCN, the effects of antisense inhibition of Clock expression on the rhythms in 2-deoxyglucose uptake and Per gene expression were compared in immortalized SCN cells and a fibroblast cell line. Similar to changes in molecular and physiological rhythmicity observed in the SCN of Clock mutant mice, the rhythmic pattern of Per2 expression was disrupted and the period of metabolic rhythmicity was increased in SCN2.2 cells subjected to antisense inhibition of Clock. NIH/3T3 fibroblasts cocultured with antisense-treated SCN2.2 cells showed metabolic rhythms with comparable increases in period and decreases in rhythm amplitude. Per2 expression in these cocultured fibroblasts exhibited a similar reduction in peak levels, but was marked by non-24 h or irregular peak-to-peak intervals. In serum-shocked NIH/3T3 fibroblasts, oscillations in Per2, Bmal1, and Cry1 expression persisted with some change in rhythm amplitude during antisense inhibition of CLOCK, demonstrating that feedback interactions between Clock and other core components of the clock mechanism may be regulated differently in SCN2.2 cells and fibroblasts. The present results suggest that CLOCK is differentially involved in the generation of endogenous molecular and metabolic rhythmicity within SCN2.2 cells and in the regulation of their specific outputs that control rhythmic processes in NIH/3T3 cells.

Overexpression of White Collar-1 (WC-1) activates circadian clock-associated genes, but is not sufficient to induce most light-regulated gene expression in Neurospora crassa.

Many processes in fungi are regulated by light, but the molecular mechanisms are not well understood. The White Collar-1 (WC-1) protein is required for all known blue-light responses in Neurospora crassa. In response to light, WC-1 levels increase, and the protein is transiently phosphorylated. To test the hypothesis that the increase in WC-1 levels after light treatment is sufficient to activate light-regulated gene expression, we used microarrays to identify genes that respond to light treatment. We then overexpressed WC-1 in dark-grown tissue and used the microarrays to identify genes regulated by an increase in WC-1 levels. We found that 3% of the genes were responsive to light, whereas 7% of the genes were responsive to WC-1 overexpression in the dark. However, only four out of 22 light-induced genes were also induced by WC-1 overexpression, demonstrating that changes in the levels of WC-1 are not sufficient to activate all light-responsive genes. The WC proteins are also required for circadian rhythms in dark-grown cultures and for light entrainment of the circadian clock, and WC-1 protein levels show a circadian rhythm in the dark. We found that representative samples of the mRNAs induced by over-expression of WC-1 show circadian fluctuations in their levels. These data suggest that WC-1 can mediate both light and circadian responses, with an increase in WC-1 levels affecting circadian clock-responsive gene regulation and other features of WC-1, possibly its phosphorylation, affecting light-responsive gene regulation.

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.

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

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

The Neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg-2) gene.

The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock-controlled genes, including the well-characterized eas(ccg-2) gene. Dissection of the eas(ccg-2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light-regulated nuclear protein factors that bind specifically to the ACE in a time-of-day-dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg-2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg-2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg-2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild-type cells, indicating that other proteins directly control eas(ccg-2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg-2) at specific times of the day.

vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation.

con-10 and con-6 are two of the conidiation (con) genes of Neurospora crassa that were identified based on their preferential expression during macroconidiophore development. They are also regulated by several other environmental stimuli independent of development, including a transient induction by light. We identified an allele of vivid (vvd) in a mutant screen designed to obtain strains with altered expression of con-10. vvd mutants display enhanced carotenoid pigmentation in response to light. In addition, con-10 and con-6 show a heightened response to photoinduction. We tested the function of the light-responsive circadian clock in the vvd mutant and found no major defect in the circadian rhythm of conidiation or light regulation of a key clock component, frequency (frq). We conclude that vvd is primarily involved in a process of light-dependent gene repression, called light adaptation. Although a number of gene products are known to control light induction in fungi, vvd is the first gene shown to have a role in adaptation to constant light.

Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes.

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 approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 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.

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

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

Circadian clock-controlled genes isolated from Neurospora crassa are late night- to early morning-specific.

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs preferentially accumulate during the late night to early morning, although they vary with respect to steady-state mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper metallothionein, providing the suggestion that not all clock-regulated genes in N. crassa are specifically involved in the development of conidia. This was supported by finding that half of the new ccgs, including cmt(ccg-12), are not transcriptionally induced by developmental or light signals. These data suggest a major role for the clock in the regulation of biological processes distinct from development.

Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene.

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.

The genetic and molecular dissection of a prototypic circadian system.

A great deal is known about this archetypal circadian system, and it is likely that Neurospora will represent the first circadian system in which it will be possible to provide a complete description of the flow of information from the photoreceptor, through the components of oscillator, out to a terminal aspect of regulation. In Neurospora the strongest case has been made for there being a state variable of clock identified (Hall, 1995), it has now been shown that light resetting of the clock is mediated by the rapid light induction of the gene encoding this state variable, and a number of defined clock-regulated output genes have been identified, in two of which the clock-specific parts of the promoters have been localized. In addition to the importance of these factoids themselves, our efforts towards understanding of this system has allowed the development of tools and paradigms (e.g. Loros et al., 1989; Loros and Dunlap, 1991; Aronson et al., 1994a) that will help to pave the way for proving the identity of clock components in more complex systems, for understanding how clocks are regulated by entraining factors, and for showing how time information eventually is used to regulate the behaviors of clock cells, and of whole organisms.

The genetic basis of the circadian clock: identification of frq and FRQ as clock components in Neurospora.

Genetic approaches to the identification of clock components have succeeded in two model systems, Neurospora and Drosophila. In each organism, genes identified through screens for clock-affecting mutations (frq in Neurospora, per in Drosophila) have subsequently been shown to have characteristics of central clock components: (1) mutations in each gene can affect period length and temperature compensation, two canonical characteristics of circadian systems; (2) each gene regulates the timing of its own transcription in a circadian manner; and (3) in the case of frq, constitutively elevated expression will set the phase of the clock on release into normal conditions. Despite clear genetic and molecular similarities, however, the two genes are neither molecular nor temporal homologues. The timing of peak expression is distinct in the two genes, frq expression peaking after dawn and per expression peaking near midnight. Also, although expression of per from a constitutive promoter can rescue rhythmicity in a fly lacking the gene, constitutive expression of frq will not rescue rhythmicity in Neurospora frq-null strains, and in fact causes arrhythmicity when expressed in a wild-type strain. These data suggest that frq is and/or encodes a state variable of the circadian oscillator. Recent molecular genetic analyses of frq have shed light on the origin of temperature compensation and strongly suggest that this property is built into the oscillatory feedback loop rather than appended to it. It seems plausible that clocks are adjusted and reset through adjustments in central clock components such as frq, and, by extension, per.

Circadian rhythms.

Circadian rhythms are a ubiquitous adaptation of eukaryotic organisms to the most reliable and predictable of environmental changes, the daily cycles of light and temperature. Prominent daily rhythms in behavior, physiology, hormone levels and biochemistry (including gene expression) are not merely responses to these environmental cycles, however, but embody the organism's ability to keep and tell time. At the core of circadian systems is a mysterious mechanism, located in the brain (actually the suprachiasmatic nucleus of the hypothalamus) of mammals, but present even in unicellular organisms, that functions as a clock. This clock drives circadian rhythms. It is independent of, but remains responsive to, environmental cycles (especially light). The interest in temporal regulation--its organization, mechanism and consequences--unites investigators in diverse disciplines studying otherwise disparate systems. This diversity is reflected in the brief reviews that summarize the presentations at a meeting on circadian rhythms held in New York City on October 31, 1992. The meeting was sponsored by the Fondation pour l'Etude du Système Nerveux (FESN) and followed a larger meeting held 18 months earlier in Geneva, whose proceedings have been published (M. Zatz (Ed.), Report of the Ninth FESN Study Group on 'Circadian Rhythms', Discussions in Neuroscience, Vol. VIII, Nos. 2 + 3, Elsevier, Amsterdam, 1992). Some speakers described progress made in the interim, while others addressed aspects of the field not previously covered.

The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer.

The Neurospora crassa clock-controlled gene (ccg-2) is transcriptionally activated by the circadian clock in a time-of-day-specific manner. Transcript and sequence analyses of ccg-2 reveal that the predicted Ccg-2 polypeptide bears significant similarity to a class of low-molecular-weight, cysteine-rich, hydrophobic proteins (hydrophobins), first identified in Schizophyllum, and including the product of the developmentally regulated Aspergillus gene, rodletless, required for spore surface rodlets. Allelism between ccg-2 and easily wettable (eas) (one of the first developmental genetic loci identified in Neurospora) was predicted on the basis of this similarity, their close genetic linkage, and previous findings demonstrating that eas mutants lack rodlets. In this study allelism is confirmed experimentally by showing that (1) transformation of an eas mutant strain with ccg-2 DNA results in phenotypic complementation, including restoration of surface rodlets, (2) inactivation of the ccg-2 gene, by RIP, results in an eas phenotype including loss of rodlet fascicles, and (3) the original eas strain has dramatically reduced levels of ccg-2 mRNA. Thus, the clock-controlled ccg-2 gene encodes an integral component of fungal asexual spores important for spore dispersal. The dramatic reduction of ccg-2 expression in the eas mutant has no apparent effect on the normal operation of the circadian clock, confirming that there is no feedback of this clock output on the oscillator itself. These data, in conjunction with the previous observation that ccg-2 is light induced, serve to focus attention on the dual interacting role of light and the circadian clock in the regulation of fungal spore development.

I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.

Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread.

A transcription terminator in the thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a viable thyA::Kmr deletion.

A transcription terminator has been identified within the coding sequence of the Escherichia coli thyA gene. Fusion of a relevant segment of the thyA structural gene to galK sequences showed that the terminator functions in vivo. Primer extension and Northern hybridization (RNA blot) analysis of thyA RNA suggested that the terminator acts as the transcription stop signal for an upstream gene and for thyA-specific transcripts. Results from antitermination studies utilizing a lambda PL-thyA fusion also offer evidence that the terminator is capable of attenuating thyA expression by reducing the amount of full-length thyA transcripts. This gene arrangement suggested that previous unsuccessful attempts to create a chromosomal thyA deletion in E. coli were attributable to the presence of the overlapping transcript. Introducing a deletion into the nonoverlapping portion of the cloned thyA gene and inserting a gene encoding kanamycin resistance produced a (delta thyA::Kmr) that was easily transferred to the chromosome of a recD host by marker replacement. This delta thyA::Kmr allele provides a useful and readily transducible chromosomal marker.

Intron mobility in phage T4 is dependent upon a distinctive class of endonucleases and independent of DNA sequences encoding the intron core: mechanistic and evolutionary implications.

Although mobility of the phylogenetically widespread group I introns appears to be mechanistically similar, the phage T4 intron-encoded endonucleases that promote mobility of the td and sunY introns are different from their eukaryotic counterparts. Most notably, they cleave at a distance from the intron insertion sites. The td enzyme was shown to cleave 23-26 nt 5' and the sunY endonuclease 13-15 nt 3' to the intron insertion site to generate 3-nt or 2-nt 3'-OH extensions, respectively. The absolute coconversion of exon markers between the distant cleavage and insertion sites is consistent with the double-strand-break repair model for intron mobility. As a further critical test of the model we have demonstrated that the mobility event is independent of DNA sequences that encode the catalytic intron core structure. Thus, in derivatives in which the lacZ or kanR coding sequences replace the intron, these marker genes are efficiently inserted into intron-minus alleles when the cognate endonuclease is provided in trans. The process is therefore endonuclease-dependent, rather than dependent on the intron per se. These findings, which imply that the endonucleases rather than the introns themselves were the primordial mobile elements, are incorporated into a model for the evolution of mobile introns.

A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4.

The product of the td intron open reading frame (ORF) of phage T4 is required for high-frequency transfer of the intervening sequence from intron-plus (In+) to intron-minus (In-) alleles. In vivo studies have demonstrated that the td ORF product targets cleavage of td In- DNA, and that cleavage is correlated with intron inheritance [Quirk et al., Cell 56 (1989) 455-465]. In the present study we show by in vitro synthesis of the td intron ORF product, that the protein possesses endonuclease activity and efficiently cleaves double-stranded DNA at or near the site of intron integration. In addition, we demonstrate that intron insertion is accompanied by co-conversion of the flanking exon sequences. Co-conversion of markers within 50 nt surrounding the site of intron insertion occurred at a high frequency (80-100%), and decreased at greater distance from the intervening sequence. Co-conversion may provide a mechanism for maintaining exon-intron RNA contacts required for accurate splicing of the relocated intron. Cleavage of target DNA by an intron endonuclease and co-conversion of flanking exon sequences are both features associated with mobile introns of eukaryotes, indicating a common mechanism for intron transfer in the eukaryotic and prokaryotic kingdoms.

Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames.

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.