From cyanobacteria to mammals, organisms have evolved timing mechanisms to adapt to environmental changes in order to optimize survival and improve fitness. To anticipate these regular daily ...cycles, many organisms manifest ∼24h cell-autonomous oscillations that are sustained by transcription–translation-based or post-transcriptional negative-feedback loops that control a wide range of biological processes. With an eye to identifying emerging common themes among cyanobacterial, fungal, and animal clocks, some major recent developments in the understanding of the mechanisms that regulate these oscillators and their output are discussed. These include roles for antisense transcription, intrinsically disordered proteins, codon bias in clock genes, and a more focused discussion of post-transcriptional and translational regulation as a part of both the oscillator and output.
An apparent lack of structure is a common feature of some of the negative-arm proteins in fungi and animals, and may be essential to their proper function in the circadian clock.
The mechanism of circadian period determination does not lie in the half-life of the negative-arm proteins, leaving the potential for alternative methods, such as post-transcriptional regulation, to fill that role.
Transcriptional regulation of clock-controlled genes, ccgs, by the heterodimer in the positive arm, once believed to be the absolute driver of changes in expression of ccgs and of output, appears to be just the first step in the pathway of circadian control over cellular output.
Study of Neurospora, a model system evolutionarily related to animals and sharing a circadian system having nearly identical regulatory architecture to that of animals, has advanced our understanding ...of all circadian rhythms. Work on the molecular bases of the Oscillator began in Neurospora before any clock genes were cloned and provided the second example of a clock gene, frq, as well as the first direct experimental proof that the core of the Oscillator was built around a transcriptional translational negative feedback loop (TTFL). Proof that FRQ was a clock component provided the basis for understanding how light resets the clock, and this in turn provided the generally accepted understanding for how light resets all animal and fungal clocks. Experiments probing the mechanism of light resetting led to the first identification of a heterodimeric transcriptional activator as the positive element in a circadian feedback loop, and to the general description of the fungal/animal clock as a single step TTFL. The common means through which DNA damage impacts the Oscillator in fungi and animals was first described in Neurospora. Lastly, the systematic study of Output was pioneered in Neurospora, providing the vocabulary and conceptual framework for understanding how Output works in all cells. This model system has contributed to the current appreciation of the role of Intrinsic Disorder in clock proteins and to the documentation of the essential roles of protein post‐translational modification, as distinct from turnover, in building a circadian clock.
Neurospora crassa, a genetically tractable model system, has informed the study of animal clocks. Within the oscillator core is a heterodimeric PAS‐domain transcription factor that activates expression of proteins that enter a negative element complex capable of blocking positive element activity. The heterodimer also activates clock‐controlled genes, leading to rhythmic cascades of gene expression within the cell. Light resets clock phase by changing the amount of the negative element complex.
Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed intrinsically disordered proteins (IDPs), demonstrates that flexibility in structure ...can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein FREQUENCY (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to FREQUENCY-Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via a pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.
Display omitted
•Helicase function of FRH is not essential for Neurospora clock•FFC is in the N-terminal end of FRH•FRQ is an IDP•FRH acts nonenzymatically to support the IDP FRQ
Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted ...mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen.
The capacity for biological timekeeping arose at least three times through evolution, in prokaryotic cyanobacteria, in cells that evolved into higher plants, and within the group of organisms that ...eventually became the fungi and the animals.
is a tractable model system for understanding the molecular bases of circadian rhythms in the last of these groups, and is perhaps the most intensively studied circadian cell type. Rhythmic processes described in fungi include growth rate, stress responses, developmental capacity, and sporulation, as well as much of metabolism; fungi use clocks to anticipate daily environmental changes. A negative feedback loop comprises the core of the circadian system in fungi and animals. In
, the best studied fungal model, it is driven by two transcription factors, WC-1 and WC-2, that form the White Collar Complex (WCC). WCC elicits expression of the
gene. FRQ complexes with other proteins, physically interacts with the WCC, and reduces its activity; the kinetics of these processes is strongly influenced by progressive phosphorylation of FRQ. When FRQ becomes sufficiently phosphorylated that it loses the ability to influence WCC activity, the circadian cycle starts again. Environmental cycles of light and temperature influence
and FRQ expression and thereby reset the internal circadian clocks. The molecular basis of circadian output is also becoming understood. Taken together, molecular explanations are emerging for all the canonical circadian properties, providing a molecular and regulatory framework that may be extended to many members of the fungal and animal kingdoms, including humans.
Compensation is a defining principle of a true circadian clock, where its approximately 24-hour period length is relatively unchanged across environmental conditions. Known compensation effectors ...directly regulate core clock factors to buffer the oscillator's period length from variables in the environment. Temperature Compensation mechanisms have been experimentally addressed across circadian model systems, but much less is known about the related process of Nutritional Compensation, where circadian period length is maintained across physiologically relevant nutrient levels. Using the filamentous fungus Neurospora crassa, we performed a genetic screen under glucose and amino acid starvation conditions to identify new regulators of Nutritional Compensation. Our screen uncovered 16 novel mutants, and together with 4 mutants characterized in prior work, a model emerges where Nutritional Compensation of the fungal clock is achieved at the levels of transcription, chromatin regulation, and mRNA stability. However, eukaryotic circadian Nutritional Compensation is completely unstudied outside of Neurospora. To test for conservation in cultured human cells, we selected top hits from our fungal genetic screen, performed siRNA knockdown experiments of the mammalian orthologs, and characterized the cell lines with respect to compensation. We find that the wild-type mammalian clock is also compensated across a large range of external glucose concentrations, as observed in Neurospora, and that knocking down the mammalian orthologs of the Neurospora compensation-associated genes CPSF6 or SETD2 in human cells also results in nutrient-dependent period length changes. We conclude that, like Temperature Compensation, Nutritional Compensation is a conserved circadian process in fungal and mammalian clocks and that it may share common molecular determinants.
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.
Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate ...and control luminescence 1–5. Among the least studied bioluminescent organisms are phylogenetically rare fungi—only 71 species, all within the ∼9,000 fungi of the temperate and tropical Agaricales order—are reported from among ∼100,000 described fungal species 6, 7. All require oxygen 8 and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia 8–15, we examined interactions between luminescent fungi and insects 16. Prosthetic acrylic resin “mushrooms,” internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.
•Bioluminescence in N. gardneri, a basidiomycete, is regulated by the circadian clock•Luciferin, reductase, and luciferase, which together make light, all peak at night•Prosthetic LED-illuminated acrylic mushrooms can be used to study insect behavior•Insects that can disperse fungal spores are attracted to light at night
Oliveira et al. report that bioluminescence in Neonothopanus gardneri is regulated by the circadian clock. Nighttime light attracts insects that can disperse spores, a benefit to fungi living under the forest canopy, where winds are reduced.
Significance Circadian clocks regulate gene expression levels to allow an organism to anticipate environmental conditions. These clocks reside in all the major branches of life and confer a ...competitive advantage to the organisms that maintain them. The clock in the fungus Neurospora crassa is an excellent model for basic understanding of core circadian architecture as well as for filamentous fungi. Here, we identify genes whose expression is clock regulated; indeed, as much as 40% of the transcriptome may be clock regulated, broadly directing daytime catabolism and nighttime growth. Both transcriptional control and posttranscriptional regulation play major roles in control of cycling transcripts such that DNA binding of transcription factors alone appears insufficient to set the phase of circadian transcription.
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 ( ccg s) 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 ccg s. 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.
Defining necessary circadian clock elements
The circadian clock in organisms as diverse as fungi and humans have a rather similar structure: Timing depends on daily cycles of transcription in ...circuits in which feedback loops control the timing of oscillations. A critical role has been ascribed to negative elements, which lead to inhibition of their own transcription, and to degradation of these elements, which is signaled by phosphorylation events. However, Larrando
et al.
show that in the fungus
Neurospora
, after manipulations that prevent phosphorylation-signaled degradation of the negative element FREQUENCY (FRQ), rhythms still persist (see the Perspective by Kramer). They suggest a model in which other phosphorylation events on Frq (of which there are over 100) must have critical roles in controlling the clock, independent of negative element degradation.
Science
, this issue
10.1126/science.1257277
; see also p.
476
Control of negative circadian elements in the simple fungus
Neurospora
goes beyond targeted proteolysis.
Also see Perspective by
Kramer
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 Δ
fwd-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, Δ
fwd-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.
Distinct roles for FRQ phosphorylation and degradation in the clock.
White Collar-1 and -2 (WC-1 and WC-2) activate
frq
expression and FRQ (with FRH and CK1) later inhibit expression. FRQ phosphorylation affects interactions with WC-1/WC-2, reducing inhibition. By influencing these key interactions, FRQ phosphorylations determine the rate at which core clock events, those within the clock face, occur. After key phosphorylations close the loop, degradation-related events need not affect circadian period.
The mechanistic basis of eukaryotic circadian oscillators in model systems as diverse as
Neurospora
,
Drosophila
, and mammalian cells is thought to be a transcription-and-translation–based negative feedback loop, wherein progressive and controlled phosphorylation of one or more negative elements ultimately elicits their own proteasome-mediated degradation, thereby releasing negative feedback and determining circadian period length. The
Neurospora crassa
circadian negative element FREQUENCY (FRQ) exemplifies such proteins; it is progressively phosphorylated at more than 100 sites, and strains bearing alleles of
frq
with anomalous phosphorylation display abnormal stability of FRQ that is well correlated with altered periods or apparent arrhythmicity. Unexpectedly, we unveiled normal circadian oscillations that reflect the allelic state of
frq
but that persist in the absence of typical degradation of FRQ. This manifest uncoupling of negative element turnover from circadian period length determination is not consistent with the consensus eukaryotic circadian model.
PDF
