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Circadian rhythms are ∼24-h biological oscillations that enable organisms to anticipate daily environmental cycles, so that they may designate appropriate day/night functions that align with these changes. The molecular clock in animals and fungi consists of a transcription-translation feedback loop, the plant clock is comprised of multiple interlocking feedback-loops, and the cyanobacterial clock is driven by a phosphorylation cycle involving three main proteins. Despite the divergent core clock mechanisms across these systems, all circadian clocks are able to buffer period length against changes in the ambient growth environment, such as temperature and nutrients. This defining capability, termed compensation, is critical to proper timekeeping, yet the underlying mechanism(s) remain elusive. Here we examine the known players in, and the current models for, compensation across five circadian systems. While compensation models across these systems are not yet unified, common themes exist across them, including regulation via temperature-dependent changes in post-translational modifications., (© 2024 Wiley Periodicals LLC.)

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) have been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is mainly controlled by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Circadian clocks temporally coordinate daily organismal biology over the 24-h cycle. Their molecular design, preserved between fungi and animals, is based on a core-oscillator composed of a one-step transcriptional-translational-negative-feedback-loop (TTFL). To test whether this evolutionarily conserved TTFL architecture is the only plausible way for achieving a functional circadian clock, we adopted a transcriptional rewiring approach, artificially co-opting regulators of the circadian output pathways into the core-oscillator. Herein we describe one of these semi-synthetic clocks which maintains all basic circadian features but, notably, it also exhibits new attributes such as a "lights-on timer" logic, where clock phase is fixed at the end of the night. Our findings indicate that fundamental circadian properties such as period, phase and temperature compensation are differentially regulated by transcriptional and posttranslational aspects of the clockworks., (© 2024. The Author(s).)

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins F requency ( F RQ), the F RQ-interacting RNA helicase (FRH), and C asein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks., Competing Interests: DT, NM, BB, SC, JD, AB, BC No competing interests declared, (© 2023, Tariq, Maurici et al.)

Some cell biological studies of Neurospora crassa have been limited by the rapid rates of hyphal growth and fusion. In this study, we investigated the causative mutation in the standard C24 allele of for (FGSC #9) and assayed the growth and circadian phenotype of the for strain under different nutritional conditions. We show that the for strain can be maintained as metabolically active single cells for 2 days before its growth advances into branched mycelia. This culturing system offers the potential to advance subcellular dynamic research and to facilitate greater understanding of N. crassa in the early developmental stages., Competing Interests: Competing Interests The authors declare no competing interests.

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency ( frq ) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) has been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is controlled mainly by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses.

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

In the Neurospora circadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm component frequency (frq). FRQ interacts with FRH (FRQ-interacting RNA helicase) and CKI, forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated as brd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss of brd-8 reduces H4 acetylation and RNA polymerase (Pol) II occupancy at frq and other known circadian genes, and leads to a long circadian period, delayed phase, and defective overt circadian output at some temperatures. In addition to strongly associating with the NuA4 histone acetyltransferase complex, BRD-8 is also found complexed with the transcription elongation regulator BYE-1. Expression of brd-8, bye-1, histone h2a.z, and several NuA4 subunits is controlled by the circadian clock, indicating that the molecular clock both regulates the basic chromatin status and is regulated by changes in chromatin. Taken together, our data identify auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamic frq expression and thereby a normal and persistent circadian rhythm., (© 2023. The Author(s).)

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ) binds with FRH (FRQ-interacting RNA helicase) and Casein Kinase 1 (CK1) to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar Complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, FFC-WCC was assessed in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially WT period, indicating that the binding strength between the positive and negative elements in the feedback loop is required for the clock but is not a determinant of the period length.

In the negative feedback loop driving the Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites., Competing Interests: Conflicts of interest None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Genetics Society of America.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kelliher et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The brightness and photostability of ten different fluorescent protein tags were compared in a well-controlled system; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

The transcription-translation negative feedback loops underlying animal and fungal circadian clocks are remarkably similar in their molecular regulatory architecture and, although much is understood about their central mechanism, little is known about the spatiotemporal dynamics of the gene products involved. A common feature of these circadian oscillators is a significant temporal delay between rhythmic accumulation of clock messenger RNAs (mRNAs) encoding negative arm proteins, for example, frq in Neurospora and Per1-3 in mammals, and the appearance of the clock protein complexes assembled from the proteins they encode. Here, we report use of single-molecule RNA fluorescence in situ hybridization (smFISH) to show that the fraction of nuclei actively transcribing the clock gene frq changes in a circadian manner, and that these mRNAs cycle in abundance with fewer than five transcripts per nucleus at any time. Spatial point patterning statistics reveal that frq is spatially clustered near nuclei in a time of day-dependent manner and that clustering requires an RNA-binding protein, PRD-2 (PERIOD-2), recently shown also to bind to mRNA encoding another core clock component, casein kinase 1. An intrinsically disordered protein, PRD-2 displays behavior in vivo and in vitro consistent with participation in biomolecular condensates. These data are consistent with a role for phase-separating RNA-binding proteins in spatiotemporally organizing clock mRNAs to facilitate local translation and assembly of clock protein complexes.

Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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

Circadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. In Neurospora crassa , negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock's positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 ( period-2 ) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1 prd-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I ., Competing Interests: CK, RL, QX, CB, JL, JD No competing interests declared, (© 2020, Kelliher et al.)

Introduction: The circadian circuit, a roughly 24 h molecular feedback loop, or clock, is conserved from bacteria to animals and allows for enhanced organismal survival by facilitating the anticipation of the day/night cycle. With circadian regulation reportedly impacting as high as 80% of protein coding genes in higher eukaryotes, the protein-based circadian clock broadly regulates physiology and behavior. Due to the extensive interconnection between the clock and other cellular systems, chronic disruption of these molecular rhythms leads to a decrease in organismal fitness as well as an increase of disease rates in humans. Importantly, recent research has demonstrated that proteins comprising the circadian clock network display a significant amount of intrinsic disorder., Main Body: In this work, we focus on the extent of intrinsic disorder in the circadian clock and its potential mechanistic role in circadian timing. We highlight the conservation of disorder by quantifying the extent of computationally-predicted protein disorder in the core clock of the key eukaryotic circadian model organisms Drosophila melanogaster, Neurospora crassa, and Mus musculus. We further examine previously published work, as well as feature novel experimental evidence, demonstrating that the core negative arm circadian period drivers FREQUENCY (Neurospora crassa) and PERIOD-2 (PER2) (Mus musculus), possess biochemical characteristics of intrinsically disordered proteins. Finally, we discuss the potential contributions of the inherent biophysical principals of intrinsically disordered proteins that may explain the vital mechanistic roles they play in the clock to drive their broad evolutionary conservation in circadian timekeeping., Conclusion: The pervasive conservation of disorder amongst the clock in the crown eukaryotes suggests that disorder is essential for optimal circadian timing from fungi to animals, providing vital homeostatic cellular maintenance and coordinating organismal physiology across phylogenetic kingdoms. Video abstract.

The circadian clock broadly governs immune cell function, leading to time-of-day differences in inflammatory responses and subsequently, pathogen clearance. However, the effect of inflammatory signals on circadian machinery is poorly understood. We found that in bone marrow-derived macrophages, some host-derived pro-inflammatory cytokines, e.g., IFN-γ or TNF-α, and pathogen-associated molecular patterns, e.g., LPS or Pam3Csk4, suppress the amplitude in oscillations of circadian negative feedback arm clock components such as PER2, and when examined, specific combinations of these immune-related signals suppressed the amplitude of these oscillations to a greater degree in both bone marrow-derived and peritoneal macrophages. At the transcript level, multiple components of the circadian clock were affected in different ways by pro-inflammatory stimulus, including Per2 and Nr1d1 . This suppressive effect on PER2 did not arise from nor correlate with cell death or clock resetting. Suppression of the clock by IFN-γ was dependent on its cognate receptor; however, pharmacological inhibition of the canonical JAK/STAT and MEK pathways did not hinder suppression, suggesting a mechanism involving a non-canonical pathway. In contrast, anti-inflammatory signals such as IL-4 and dexamethasone enhanced the expression of PER2 protein and Per2 mRNA. Our results suggest that the circadian system in macrophages can differentially respond to pro- and anti-inflammatory signals in their microenvironments., (Copyright © 2020 Chen, Fuller, Dunlap and Loros.)

Work on the filamentous fungus Neurospora crassa has contributed to or pioneered many aspects of research on circadian clock mechanism, a process that is functionally conserved across eukaryotes. Biochemical assays of the fungal circadian clock typically involve growth in liquid medium where Neurospora forms a spherical ball of submerged mycelium. Here, we revive a method for dispersed growth of Neurospora in batch culture using polyacrylic acid as an additive to the medium. We demonstrate that dispersed growth cultures utilize more carbon than mycelial balls, but nonetheless retain a functional circadian clock. This culturing method is suited for use in circadian experiments where uniform exposure to nutrients and/or increased biomass is required., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

In the negative feedback loop driving fungal and animal circadian oscillators, negative elements (FREQUENCY [FRQ], PERIODS [PERs], and CRYPTOCHROMES [CRYs]) are understood to inhibit their own expression, in part by promoting the phosphorylation of their heterodimeric transcriptional activators (e.g., White Collar-1 [WC-1]-WC-2 [White Collar complex; WCC] and BMAL1/Circadian Locomotor Output Cycles Kaput [CLOCK]). However, correlations between heterodimer activity and phosphorylation are weak, contradictions exist, and mechanistic details are almost wholly lacking. We report mapping of 80 phosphosites on WC-1 and 15 on WC-2 and elucidation of the time-of-day-specific code, requiring both a group of phosphoevents on WC-1 and two distinct clusters on WC-2, that governs circadian repression, leading to feedback loop closure. Combinatorial control via phosphorylation also governs rhythmic WCC binding to the promoters of clock-controlled genes mediating the essential first step in circadian output, a group encoding both transcription factors and signaling proteins. These data provide a basic mechanistic understanding for fundamental events underlying circadian negative feedback and output, key aspects of circadian biology., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Motivation: Decreasing costs are making it feasible to perform time series proteomics and genomics experiments with more replicates and higher resolution than ever before. With more replicates and time points, proteome and genome-wide patterns of expression are more readily discernible. These larger experiments require more batches exacerbating batch effects and increasing the number of bias trends. In the case of proteomics, where methods frequently result in missing data this increasing scale is also decreasing the number of peptides observed in all samples. The sources of batch effects and missing data are incompletely understood necessitating novel techniques., Results: Here we show that by exploiting the structure of time series experiments, it is possible to accurately and reproducibly model and remove batch effects. We implement Learning and Imputation for Mass-spec Bias Reduction (LIMBR) software, which builds on previous block-based models of batch effects and includes features specific to time series and circadian studies. To aid in the analysis of time series proteomics experiments, which are often plagued with missing data points, we also integrate an imputation system. By building LIMBR for imputation and time series tailored bias modeling into one straightforward software package, we expect that the quality and ease of large-scale proteomics and genomics time series experiments will be significantly increased., Availability and Implementation: Python code and documentation is available for download at https://github.com/aleccrowell/LIMBR and LIMBR can be downloaded and installed with dependencies using 'pip install limbr'., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We report the application of a recently proposed approach for modeling biological systems using a maximum entropy production rate principle in lieu of having in vivo rate constants. The method is applied in four steps: (1) a new ordinary differential equation (ODE) based optimization approach based on Marcelin's 1910 mass action equation is used to obtain the maximum entropy distribution; (2) the predicted metabolite concentrations are compared to those generally expected from experiments using a loss function from which post-translational regulation of enzymes is inferred; (3) the system is re-optimized with the inferred regulation from which rate constants are determined from the metabolite concentrations and reaction fluxes; and finally (4) a full ODE-based, mass action simulation with rate parameters and allosteric regulation is obtained. From the last step, the power characteristics and resistance of each reaction can be determined. The method is applied to the central metabolism of Neurospora crassa and the flow of material through the three competing pathways of upper glycolysis, the non-oxidative pentose phosphate pathway, and the oxidative pentose phosphate pathway are evaluated as a function of the NADP/NADPH ratio. It is predicted that regulation of phosphofructokinase (PFK) and flow through the pentose phosphate pathway are essential for preventing an extreme level of fructose 1,6-bisphophate accumulation. Such an extreme level of fructose 1,6-bisphophate would otherwise result in a glassy cytoplasm with limited diffusion, dramatically decreasing the entropy and energy production rate and, consequently, biological competitiveness., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest.

Regulatable promoters are important genetic tools, particularly for assigning function to essential and redundant genes. They can also be used to control the expression of enzymes that influence metabolic flux or protein secretion, thereby optimizing product yield in bioindustry. This review will focus on regulatable systems for use in filamentous fungi, an important group of organisms whose members include key research models, devastating pathogens of plants and animals, and exploitable cell factories. Though we will begin by cataloging those promoters that are controlled by nutritional or chemical means, our primary focus will rest on those who can be controlled by a literal flip-of-the-switch: promoters of light-regulated genes. The vvd promoter of Neurospora will first serve as a paradigm for how light-driven systems can provide tight, robust, tunable, and temporal control of either autologous or heterologous fungal proteins. We will then discuss a theoretical approach to, and practical considerations for, the development of such promoters in other species. To this end, we have compiled genes from six previously published light-regulated transcriptomic studies to guide the search for suitable photoregulatable promoters in your fungus of interest.

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

Circadian clocks are ubiquitous in eukaryotic organisms where they are used to anticipate regularly occurring diurnal and seasonal environmental changes. Nevertheless, little is known regarding pathways connecting the core clock to its output pathways. Here, we report that the HAD family phosphatase CSP-6 is required for overt circadian clock output but not for the core oscillation. The loss of function Δcsp-6 deletion mutant is overtly arrhythmic on race tubes under free running conditions; however, reporter assays confirm that the FREQUENCY-WHITE COLLAR COMPLEX core circadian oscillator is functional, indicating a discrete block between oscillator and output. CSP-6 physically interacts with WHI-2, Δwhi-2 mutant phenotypes resemble Δcsp-6, and the CSP-6/WHI-2 complex physically interacts with WC-1, all suggesting that WC-1 is a direct target for CSP-6/WHI-2-mediated dephosphorylation and consistent with observed WC-1 hyperphosphorylation in Δcsp-6. To identify the source of the block to output, known clock-controlled transcription factors were screened for rhythmicity in Δcsp-6, identifying loss of circadian control of ADV-1, a direct target of WC-1, as responsible for the loss of overt rhythmicity. The CSP-6/WHI-2 complex thus participates in the clock output pathway by regulating WC-1 phosphorylation to promote proper transcriptional/translational activation of adv-1/ADV-1; these data establish an unexpected essential role for post-translational modification parallel to circadian transcriptional regulation in the early steps of circadian output.

Understanding the genetic basis of the switch from asexual to sexual lifestyles in response to sometimes rapid environmental changes is one of the major challenges in fungal ecology. Light appears to play a critical role in the asexual-sexual switch-but fungal genomes harbour diverse light sensors. Fungal opsins are homologous to bacterial green-light-sensory rhodopsins, and their organismal functions in fungi have not been well understood. Three of these opsin-like proteins were widely distributed across fungal genomes, but homologs of the Fusarium opsin-like protein CarO were present only in plant-associated fungi. Key amino acids, including potential retinal binding sites, functionally diverged on the phylogeny of opsins. This diversification of opsin-like proteins could be correlated with life history-associated differences among fungi in their expression and function during morphological development. In Neurospora crassa and related species, knockout of the opsin NOP-1 led to a phenotype in the regulation of the asexual-sexual switch, modulating response to both light and oxygen conditions. Sexual development commenced early in ∆nop-1 strains cultured in unsealed plates under constant blue and white light. Furthermore, comparative transcriptomics showed that the expression of nop-1 is light-dependent and that the ∆nop-1 strain abundantly expresses genes involved in oxidative stress response, genes enriched in NAD/NADP binding sites, genes with functions in proton transmembrane movement and catalase activity, and genes involved in the homeostasis of protons. Based on these observations, we contend that light and oxidative stress regulate the switch via light-responsive and ROS pathways in model fungus N. crassa and other fungi., (© 2017 John Wiley & Sons Ltd.)

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5' leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5' region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5' conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression. IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression., (Copyright © 2017 Ivanov et al.)

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

Light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. Toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor ADV-1 in Neurospora crassa A large proportion of ADV-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in development, metabolism, cell growth, and cell fusion. We show that ADV-1 is necessary for transducing light and/or temporal information to its immediate downstream targets, including controlling rhythms in genes critical to somatic cell fusion. However, while ADV-1 targets are altered in predictable ways in Δadv-1 cells in response to light, this is not always the case for rhythmic target gene expression. These data suggest that a complex regulatory network downstream of ADV-1 functions to generate distinct temporal dynamics of target gene expression relative to the central clock mechanism., (Copyright © 2017 Dekhang et al.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: The given strain of Aspergillus fumigatus under study varies across laboratories, ranging from a few widely used "standards," e.g., Af293 or CEA10, to locally acquired isolates that may be unique to one investigator. Since experiments concerning physiology or gene function are seldom replicated by others, i.e., in a different A. fumigatus background, the extent to which behavioral heterogeneity exists within the species is poorly understood. As a proxy for assessing such intraspecies variability, we analyzed the light response of 15 A. fumigatus isolates and observed striking quantitative and qualitative heterogeneity among them. The majority of the isolates fell into one of two seemingly mutually exclusive groups: (i) "photopigmenters" that robustly accumulate hyphal melanin in the light and (ii) "photoconidiators" that induce sporulation in the light. These two distinct responses were both governed by the same upstream blue light receptor, LreA, indicating that a specific protein's contribution can vary in a strain-dependent manner. Indeed, while LreA played no apparent role in regulating cell wall homeostasis in strain Af293, it was essential in that regard in strain CEA10. The manifest heterogeneity in the photoresponses led us to compare the virulence levels of selected isolates in a murine model; remarkably, the virulence did vary greatly, although not in a manner that correlated with their overt light response. Taken together, these data highlight the extent to which isolates of A. fumigatus can vary, with respect to both broad physiological characteristics (e.g., virulence and photoresponse) and specific protein functionality (e.g., LreA-dependent phenotypes)., Importance: The current picture of Aspergillus fumigatus biology is akin to a collage, patched together from data obtained from disparate "wild-type" strains. In a systematic assessment of 15 A. fumigatus isolates, we show that the species is highly heterogeneous with respect to its light response and virulence. Whereas some isolates accumulate pigments in light as previously reported with strain Af293, most induce sporulation which had not been previously observed. Other photoresponsive behaviors are also nonuniform, and phenotypes of identical gene deletants vary in a background-dependent manner. Moreover, the virulence of several selected isolates is highly variable in a mouse model and apparently does not track with any observed light response. Cumulatively, this work illuminates the fact that data obtained with a single A. fumigatus isolate are not necessarily predictive of the species as whole. Accordingly, researchers should be vigilant when making conclusions about their own work or when interpreting data from the literature., (Copyright © 2016 Fuller et al.)

Neurospora crassa is a model organism for the study of circadian clocks, molecular machineries that confer ∼24-hr rhythms to different processes at the cellular and organismal levels. The FREQUENCY (FRQ) protein is a central component of the Neurospora core clock, a transcription/translation negative feedback loop that controls genome-wide rhythmic gene expression. A genetic screen aimed at determining new components involved in the latter process identified regulation of conidiation 1 (rco-1), the ortholog of the Saccharomyces cerevisiae Tup1 corepressor, as affecting period length. By employing bioluminescent transcriptional and translational fusion reporters, we evaluated frq and FRQ expression levels in the rco-1 mutant background observing that, in contrast to prior reports, frq and FRQ expression are robustly rhythmic in the absence of RCO-1, although both amplitude and period length of the core clock are affected. Moreover, we detected a defect in metabolic compensation, such that high-glucose concentrations in the medium result in a significant decrease in period when RCO-1 is absent. Proteins physically interacting with RCO-1 were identified through co-immunoprecipitation and mass spectrometry; these include several components involved in chromatin remodeling and transcription, some of which, when absent, lead to a slight change in period. In the aggregate, these results indicate a dual role for RCO-1: although it is not essential for core-clock function, it regulates proper period and amplitude of core-clock dynamics and is also required for the rhythmic regulation of several clock-controlled genes., (Copyright © 2016 by the Genetics Society of America.)

In the Neurospora crassa circadian clock, a protein complex of frequency (FRQ), casein kinase 1a (CK1a), and the FRQ-interacting RNA Helicase (FRH) rhythmically represses gene expression by the white-collar complex (WCC). FRH crystal structures in several conformations and bound to ADP/RNA reveal differences between FRH and the yeast homolog Mtr4 that clarify the distinct role of FRH in the clock. The FRQ-interacting region at the FRH N-terminus has variable structure in the absence of FRQ A known mutation that disrupts circadian rhythms (R806H) resides in a positively charged surface of the KOW domain, far removed from the helicase core. We show that changes to other similarly located residues modulate interactions with the WCC and FRQ A V142G substitution near the N-terminus also alters FRQ and WCC binding to FRH, but produces an unusual short clock period. These data support the assertion that FRH helicase activity does not play an essential role in the clock, but rather FRH acts to mediate contacts among FRQ, CK1a and the WCC through interactions involving its N-terminus and KOW module., (© 2016 The Authors.)

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 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Rapid responses to changes in incident light are critical to the guidance of behavior and development in most species. Phytochrome light receptors in particular play key roles in bacterial physiology and plant development, but their functions and regulation are less well understood in fungi. Nevertheless, genome-wide expression measurements provide key information that can guide experiments that reveal how genes respond to environmental signals and clarify their role in development. We performed functional genomic and phenotypic analyses of the two phytochromes in Neurospora crassa, a fungal model adapted to a postfire environment that experiences dramatically variable light conditions. Expression of phy-1 and phy-2 was low in early sexual development and in the case of phy-2 increased in late sexual development. Under light stimulation, strains with the phytochromes deleted exhibited increased expression of sexual development-related genes. Moreover, under red light, the phy-2 knockout strain commenced sexual development early. In the evolution of phytochromes within ascomycetes, at least two duplications have occurred, and the faster-evolving phy-2 gene has frequently been lost. Additionally, the three key cysteine sites that are critical for bacterial and plant phytochrome function are not conserved within fungal phy-2 homologs. Through the action of phytochromes, transitions between asexual and sexual reproduction are modulated by light level and light quality, presumably as an adaptation for fast asexual growth and initiation of sexual reproduction of N. crassa in exposed postfire ecosystems., Importance: Environmental signals, including light, play critical roles in regulating fungal growth and pathogenicity, and balance of asexual and sexual reproduction is critical in fungal pathogens' incidence, virulence, and distribution. Red light sensing by phytochromes is well known to play critical roles in bacterial physiology and plant development. Homologs of phytochromes were first discovered in the fungal model Neurospora crassa and then subsequently in diverse other fungi, including many plant pathogens. Our study investigated the evolution of red light sensors in ascomycetes and confirmed-using the model fungus Neurospora crassa-their roles in modulating the asexual-sexual reproduction balance in fungi. Our findings also provide a key insight into one of the most poorly understood aspects of fungal biology, suggesting that further study of the function of phytochromes in fungi is critical to reveal the genetic basis of the asexual-sexual switch responsible for fungal growth and distribution, including diverse and destructive plant pathogens., (Copyright © 2016 Wang et al.)

Light plays an important role for most organisms on this planet, serving either as a source of energy or information for the adaptation of biological processes to specific times of day. The fungal kingdom is estimated to contain well over a million species, possibly 10-fold more, and it is estimated that a majority of the fungi respond to light, eliciting changes in several physiological characteristics including pathogenesis, development and secondary metabolism. Two model organisms for photobiological studies have taken centre-stage over the last few decades--Neurospora crassa and Aspergillus nidulans. In this review, we will first discuss our understanding of the light response in N. crassa, about which the most is known, and will then juxtapose N. crassa with A. nidulans, which, as will be described below, provides an excellent template for understanding photosensory cross-talk. Finally, we will end with a commentary on the variability of the light response among other relevant fungi, and how our molecular understanding in the aforementioned model organisms still provides a strong base for dissecting light responses in such species., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Visible light is a pervasive environmental signal that orients most organisms in space and time. For a fungus, the detection of light is facilitated by diverse classes of photoreceptor proteins, which in turn coordinate growth, spore dispersal, stress resistance, primary metabolism, and toxin production. We will first provide a discussion on signal input, focusing on recent insights into how fungal photoreceptors detect and transmit information at the biochemical and molecular levels. We will then pivot our discussion to how light influences fungal behaviors that are of industrial, agricultural, or even medical relevance. Because the light environment can be easily manipulated in many contexts, we will argue that understanding fungal photobiology is both an important basic and applied endeavor., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Mutants in the period-1 (prd-1) gene, characterized by a recessive allele, display a reduced growth rate and period lengthening of the developmental cycle controlled by the circadian clock. We refined the genetic location of prd-1 and used whole genome sequencing to find the mutation defining it, confirming the identity of prd-1 by rescuing the mutant circadian phenotype via transformation. PRD-1 is an RNA helicase whose orthologs, DDX5 [DEAD (Asp-Glu-Ala-Asp) Box Helicase 5] and DDX17 in humans and DBP2 (Dead Box Protein 2) in yeast, are implicated in various processes, including transcriptional regulation, elongation, and termination, ribosome biogenesis, and mRNA decay. Although prd-1 mutants display a long period (∼25 h) circadian developmental cycle, they interestingly display a WT period when the core circadian oscillator is tracked using a frq-luciferase transcriptional fusion under conditions of limiting nutritional carbon; the core oscillator in the prd-1 mutant strain runs with a long period under glucose-sufficient conditions. Thus, PRD-1 clearly impacts the circadian oscillator and is not only part of a metabolic oscillator ancillary to the core clock. PRD-1 is an essential protein, and its expression is neither light-regulated nor clock-regulated. However, it is transiently induced by glucose; in the presence of sufficient glucose, PRD-1 is in the nucleus until glucose runs out, which elicits its disappearance from the nucleus. Because circadian period length is carbon concentration-dependent, prd-1 may be formally viewed as a clock mutant with defective nutritional compensation of circadian period length.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)