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In theNeurosporacircadian system, the White Collar Complex (WCC) drives expression of the principal circadian negative arm componentfrequency(frq). FRQ interacts with FRH (FRQ-interacting helicase) and CK-1 forming a stable complex that represses its own expression by inhibiting WCC. In this study, a genetic screen identified a gene, designated asbrd-8, that encodes a conserved auxiliary subunit of the NuA4 histone acetylation complex. Loss ofbrd-8reduces H4 acetylation and RNA polymerase (Pol) II occupancy atfrqand 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 ofbrd-8, bye-1, histone hH2Az, 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 new auxiliary elements of the fungal NuA4 complex having homology to mammalian components, which along with conventional NuA4 subunits, are required for timely and dynamicfrqexpression and thereby a normal and persistent circadian rhythm.

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 efficacy of ten different fluorescent protein tags were compared in a constant context of genomic and intracellular location; 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.

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.

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 mammalian cells, we selected the top two hits from our fungal genetic screen, performed siRNA knockdown experiments of the mammalian homologs, 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 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.

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.

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 Ca2+ 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 Ca2+-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 Ca2+ 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.

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

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

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

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.

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.

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. Fil: Hughes, Michael E.. Washington University School Of Medicine In St. Louis; Estados Unidos Fil: Abruzzi, Katherine C.. Brandeis University; Estados Unidos Fil: Allada, Ravi. Northwestern University; Estados Unidos Fil: Anafi, Ron. University of Pennsylvania; Estados Unidos Fil: Arpat, Alaaddin Bulak. Universite de Lausanne; Suiza Fil: Asher, Gad. Weizmann Institute Of Science Israel; Israel Fil: Baldi, Pierre. University of California at Irvine; Estados Unidos Fil: de Bekker, Charissa. University Of Central Florida; Estados Unidos Fil: Bell Pedersen, Deborah. Texas A&M University; Estados Unidos Fil: Blau, Justin. University of New York; Estados Unidos Fil: Brown, Steve. Universitat Zurich; Suiza Fil: Ceriani, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Chen, Zheng. University Of Texas Health Science Center At Houston; Estados Unidos Fil: Chiu, Joanna C.. University of California at Davis; Estados Unidos Fil: Cox, Juergen. Max Planck Institute Of Biochemistry; Alemania Fil: Crowell, Alexander M.. Geisel School Of Medicine At Dartmouth; Estados Unidos Fil: DeBruyne, Jason P.. Morehouse School Of Medicine; Estados Unidos Fil: Dijk, Derk-Jan. University Of Surrey; Reino Unido Fil: DiTacchio, Luciano. University Of Kansas Medical Center; Estados Unidos Fil: Doyle, Francis J.. Harvard University; Estados Unidos Fil: Duffield, Giles E.. University of Notre Dame; Estados Unidos Fil: Dunlap, Jay C.. Geisel School Of Medicine At Dartmouth; Estados Unidos Fil: Eckel Mahan, Kristin. University Of Texas Medical School At Houston; Estados Unidos Fil: Esser, Karyn A.. University Of Florida College Of Medicine; Estados Unidos Fil: FitzGerald, Garret A.. University of Pennsylvania; Estados Unidos Fil: Forger, Daniel B.. University Of Michigan; Estados Unidos Fil: Francey, Lauren J.. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Fu, Ying Hui. University of California; Estados Unidos Fil: Gachon, Frédéric. Nestlé Institute Of Health Sciences; Suiza Fil: Gatfield, David. Universite de Lausanne; Suiza Fil: de Goede, Paul. University Of Amsterdam; Países Bajos Fil: Golden, Susan S.. University of California at San Diego; Estados Unidos Fil: Green, Carla. University of Texas; Estados Unidos Fil: Harer, John. University of Duke; Estados Unidos Fil: Harmer, Stacey. University of California at Davis; Estados Unidos Fil: Haspel, Jeff. Washington University; Estados Unidos Fil: Hastings, Michael H.. The Medical Research Council Laboratory Of Molecular Biology; Reino Unido Fil: Herzel, Hanspeter. Charité Universitätsmedizin Berlin; Alemania Fil: Herzog, Erik D.. Washington University in St. Louis; Estados Unidos Fil: Hoffmann, Christy. Washington University in St. Louis; Estados Unidos Fil: Hong, Christian. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Hughey, Jacob J.. Vanderbilt University School Of Medicine; Estados Unidos Fil: Hurley, Jennifer M.. Rensselaer Polytechnic Institute; Estados Unidos Fil: de la Iglesia, Daniel Horacio. University Of Washington; Estados Unidos Fil: Johnson, Carl. Vanderbilt University; Estados Unidos Fil: Kay, Steve A.. University of California at San Diego; Estados Unidos Fil: Koike, Nobuya. Kyoto Prefectural University Of Medicine; Japón Fil: Kornacker, Karl. Ohio State University; Estados Unidos Fil: Kramer, Achim. Charité Universitätsmedizin Berlin; Alemania Fil: Lamia, Katja. The Scripps Research Institute; Estados Unidos Fil: Leise, Tanya. Amherst College; Estados Unidos Fil: Lewis, Scott A.. Washington University; Estados Unidos Fil: Li, Jiajia. Washington University; Estados Unidos Fil: Li, Xiaodong. Wuhan University; China Fil: Liu, Andrew C.. The University Of Memphis; Estados Unidos Fil: Loros, Jennifer J.. Geisel School Of Medicine At Dartmouth; Estados Unidos Fil: Martino, Tami A.. University of Guelph; Canadá Fil: Menet, Jerome S.. Texas A&M University; Estados Unidos Fil: Merrow, Martha. Ludwig Maximilians Universitat; Alemania Fil: Millar, Andrew J.. University of Edinburgh; Reino Unido Fil: Mockler, Todd. Donald Danforth Plant Science Center; Estados Unidos Fil: Naef, Felix. Ecole Polytechnique Federale de Lausanne; Suiza Fil: Nagoshi, Emi. Universidad de Ginebra; Suiza Fil: Nitabach, Michael N.. University of Yale. School of Medicine; Estados Unidos Fil: Olmedo, Maria. Universidad de Sevilla; España Fil: Nusinow, Dmitri A.. Donald Danforth Plant Science Center; Estados Unidos Fil: Ptácek, Louis J.. University of California; Estados Unidos Fil: Rand, David. University of Warwick; Reino Unido Fil: Reddy, Akhilesh B.. The Francis Crick Institute; Reino Unido Fil: Robles, Maria S.. Ludwig Maximilians Universitat; Alemania Fil: Roenneberg, Till. Ludwig Maximilians Universitat; Alemania Fil: Rosbash, Michael. Brandeis University; Estados Unidos Fil: Ruben, Marc D.. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Rund, Samuel S.C.. University of Edinburgh; Reino Unido Fil: Sancar, Aziz. University of North Carolina; Estados Unidos Fil: Sassone Corsi, Paolo. University of California at Irvine; Estados Unidos Fil: Sehgal, Amita. University of Pennsylvania; Estados Unidos Fil: Sherrill Mix, Scott. University of Pennsylvania; Estados Unidos Fil: Skene, Debra J.. University Of Surrey; Reino Unido Fil: Storch, Kai Florian. Mcgill University, Douglas Mental Health University Institute; Canadá Fil: Takahashi, Joseph S.. Ut Southwestern Medical Center; Estados Unidos Fil: Ueda, Hiroki R.. The University of Tokyo; Japón Fil: Wang, Han. Soochow University; China Fil: Weitz, Charles. Harvard Medical School; Estados Unidos Fil: Westermark, P. O.. Leibniz Institute For Farm Animal Biology; Alemania Fil: Wijnen, Herman. University of Southampton; Reino Unido Fil: Xu, Ying. Soochow University; China Fil: Wu, Gang. Cincinnati Children's Hospital Medical Center; Estados Unidos Fil: Yoo, Seung Hee. University of Texas; Estados Unidos Fil: Young, Michael. The Rockefeller University; Estados Unidos Fil: Zhang, Eric Erquan. National Institute Of Biological Sciences, Beijing; China Fil: Zielinski, Tomasz. University of Edinburgh; Reino Unido Fil: Hogenesch, John B.. Cincinnati Children's Hospital Medical Center; Estados Unidos

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.

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 near 24-h 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.

MotivationIdentification of constitutive reference genes is critical for analysis of gene expression. Large numbers of high throughput time series expression data are available, but current methods for identifying invariant expression are not tailored for time series. Identification of reference genes from these data sets can benefit from methods which incorporate the additional information they provide.ResultsHere we show that we can improve identification of invariant expression from time series by modelling the time component of the data. We implement the Prediction Interval Ranking Score (PIRS) software, which screens high throughput time series data and provides a ranked list of reference candidates. We expect that PIRS will improve the quality of gene expression analysis by allowing researchers to identify the best reference genes for their system from publicly available time series.AvailabilityPIRS can be downloaded and installed with dependencies using ‘pip install pirs’ and Python code and documentation is available for download at https://github.com/aleccrowell/PIRS.Contactalexander.m.crowell@gmail.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.

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.

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.

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.

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.

Neurospora crassa is an important model organism for filamentous fungi as well as for circadian biology and photobiology. Although the community-accumulated tool set for the molecular analysis of Neurospora is extensive, two components are missing: (1) dependable reference genes whose level of expression are relatively constant across light/dark cycles and as a function of time of day and (2) a catalog of primers specifically designed for real-time PCR (RT-PCR). To address the first of these we have identified genes that are optimal for use as reference genes in RT-PCR across a wide range of expression levels; the mRNA/transcripts from these genes have potential for use as reference noncycling transcripts outside of Neurospora. In addition, we have generated a genome-wide set of RT-PCR primers, thereby streamlining the analysis of gene expression. In validation studies these primers successfully identified target mRNAs arising from 70% (34 of 49) of all tested genes and from all (28) of the moderately to highly expressed tested genes.

Summary 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 (λ max 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.

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

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

The fungus Neurospora crassa constitutes an important model system extensively used in chronobiology. Several studies have addressed how environmental cues, such as light, can reset or synchronize a circadian system. By means of an optimized firefly luciferase reporter gene and a controllable lighting system, we show that Neurospora can display molecular circadian rhythms in dim light when cultures receive bright light prior to entering dim light conditions. We refer to this behavior as the “bright to dim oscillatory response” (BDOR). The bright light treatment can be applied up to 76 h prior to dim exposure, and it can be as short as 15 min in duration. We have characterized this response in respect to the duration of the light pulse, the time of the light pulse before dim, the intensity of dim light, and the oscillation dynamics in dim light. Although the molecular mechanism that drives the BDOR remains obscure, these findings suggest that a long-term memory of bright light exists as part of the circadian molecular components. It is important to consider the ecological significance of such dim light responses in respect to how organisms naturally maintain their timing mechanism in moonlight.

The role of the frq gene in the Neurospora crassa circadian rhythm has been widely studied, but technical limitations have hindered a thorough analysis of frq circadian expression waveform. Through our experiments, we have shown an improved precision in defining Neurospora’s circadian rhythm kinetics using a codon optimized firefly luciferase gene reporter linked to a frq promoter. In vivo examination of this real-time reporter has allowed for a better understanding of the relationship of the light responsive elements of the frq promoter to its circadian feedback components. We provide a detailed phase response curve showing the phase shifts induced by a light pulse applied at different points of the circadian cycle. Using the frq-luc reporter, we have found that a 12-h light:12-h dark cycle (12L:12D) results in a luciferase expression waveform that is more complex and higher in amplitude than that seen in free-running conditions of constant darkness (DD). When using a lighting regime more consistent with solar timing, rather than a square wave pattern, one observes a circadian waveform that is smoother, lower in amplitude, and different in phasing. Using dim light in place of darkness in these experiments also affects the resulting waveform and phasing. Our experiments illustrate Neurospora’s circadian kinetics in greater detail than previous methods, providing further insight into the complex underlying biochemical, genetic, and physiological mechanisms underpinning the circadian oscillator.

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

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

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.

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.

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

Circadian clocks organize our inner physiology with respect to the external world providing life with the ability to anticipate and thereby better prepare for major fluctuations in its environment. Circadian systems are widely represented in nearly all major branches of life except archaebacteria, and within the eukaryotes the filamentous fungus Neurospora crassa has served for nearly half a century as a durable model organism for uncovering the basic circadian physiology and molecular biology. Studies using Neurospora have clarified our fundamental understanding of the clock as nested positive and negative feedback loops regulated through transcriptional and post-transcriptional processes. These feedback loops are centered on a limited number of proteins that form molecular complexes, and their regulation provides a physical explanation for nearly all clock properties. This review will introduce the basics of circadian rhythms, the model filamentous fungus Neurospora crassa, and provide an overview of the molecular components and regulation of the circadian clock.

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

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.

Many physiological processes exhibit diurnal variations that persist even in the absence of environmental timing cues. These rhythmic changes in physiology that operate to anticipate the needs of the organism and show a periodicity of about 24 hours are called circadian rhythms. Circadian rhythms are present in central and peripheral tissues and generated by an intracellular oscillating timing mechanism known as the circadian oscillator or molecular clock (Duguay and Cermakian, 2009; Kohsaka and Bass, 2007; Yang et al., 2007). The mammalian molecular clock relies on a transcriptional-translational feedback loop in which BMAL1 and CLOCK bind as heterodimers to regulatory elements on target genes, stimulating their transcription (Darlington et al., 1998; Ripperger and Schibler, 2006). Among those genes are the repressor proteins PERIOD (PER) and CRYPTOCHROME (CRY) families. Once PER and CRY have reached a critical concentration and are appropriately modified, they inhibit the activity of BMAL1-CLOCK, thereby repressing their own transcription and that of the other BMAL1-CLOCK target genes. Following additional modifications, PER and CRY are degraded, and the cycle begins again (Dardente and Cermakian, 2007; Dibner et al., 2010; Dunlap, 1999; Griffin Jr. et al., 1999; Lee et al., 2001). A second interconnecting feedback loop involving orphan nuclear receptors of the Rev-erb and ROR families regulates the levels of expression of BMAL1, CLOCK, and CRY1. In this secondary loop, ROR members activate both BMAL1 and Rev-erbα expression, while in turn, Rev-erbα represses transcription of both itself and BMAL1, closing the loop (Akashi and Takumi, 2005; Duguay and Cermakian, 2009; Liu et al., 2008; Sato et al., 2004). These nuclear receptors are not essential for circadian rhythms but are thought to improve the robustness of the clock, affecting its period length and phase-shifting properties (Akashi and Takumi, 2005; Liu et al., 2008). In addition, these nuclear receptors are thought to be links between circadian rhythms and metabolism (Duguay and Cermakian, 2009; Schmutz et al., 2010). Several lines of evidence show a strong interplay between metabolism and circadian rhythms (Duguay and Cermakian, 2009; Eckel-Mahan and Sassone-Corsi, 2009; Froy, 2010). Transcriptome profiling studies revealed that several key genes involved in metabolism are rhythmically expressed (Panda et al., 2002; Yang et al., 2006). Mice devoid of a functional circadian oscillator develop metabolic syndrome, and mutations or ablations of clock genes are often associated with metabolic problems (Eckel-Mahan and Sassone-Corsi, 2009; Green et al., 2008; Turek et al., 2005). Conversely, the dominance of feeding cycles as a zeitgeber (time cue) for peripheral clocks implies that metabolic signals impact the clock machinery itself (Arble et al., 2009; Damiola et al., 2000; Eckel-Mahan and Sassone-Corsi, 2009). Glucose and lipid metabolism in the liver and adipose tissue exhibit circadian variations due, at least in part, to circadian expression of key enzymes in these pathways (Lamia et al., 2008; Panda et al., 2002; So et al., 2009; Zvonic et al., 2006). The expression of these genes is regulated by several pathways including nuclear receptor signaling (Christian et al., 2006; Laitinen et al., 2005; Lau et al., 2008; Sonoda et al., 2008), and interestingly, a number of nuclear receptors are themselves expressed in a circadian manner. Thus, in addition to Rev-erbα, the expression of peroxisome proliferator–activated receptors (PPARs), estrogen-related receptors (ERRs), and thyroid hormone receptors (TRs) is subject to circadian control in liver, muscle, and adipose tissues (Yang et al., 2006). Furthermore, the ability of nuclear receptors to stimulate the expression of metabolic genes depends on the recruitment of coactivators whose activity and/or expression not only varies in response to environmental stimuli but that are also expressed in a circadian manner (Asher et al., 2008; Liu et al., 2007; Nakahata et al., 2008; Panda et al., 2002). For example, PGC1α and PGC1β, which are coactivators for ERRs and PPARs, are responsible for the activation of catabolic gene expression in metabolic tissues (Handschin and Spiegelman, 2006). The expression of these 2 cofactors is increased following exercise and stress or in cold temperatures, but it is also subject to circadian control (Handschin and Spiegelman, 2006; Liu et al., 2007). RIP140 is a corepressor for many catabolic genes and antagonizes the positive effects of PGC1 coactivators (Christian et al., 2006; Hallberg et al., 2008; White et al., 2008). Thus, catabolism is increased in mice devoid of RIP140, and they are lean and protected against diet-induced obesity and insulin resistance (Leonardsson et al., 2004). On the other hand, RIP140 functions as a coactivator for a number of genes involved in triglyceride synthesis (Herzog et al., 2007), growth factors of the EGF family (Nautiyal et al., 2010), and inflammatory cytokines (Zschiedrich et al., 2008). Transcriptome profiling has shown that RIP140 mRNA is subject to circadian oscillations in the liver (Hughes et al., 2009; Panda et al., 2002), and we have previously noted alterations in the daily patterns of rest/activity in RIP140-null mice kept in normal 12-hour light/12-hour dark conditions (Hudson-Davies et al., 2008). In view of these observations, we have examined the relationship between this metabolic cofactor and the molecular clock. We have found that RIP140 is not only subject to circadian gene expression but also functions as a positive regulator of the molecular clock by potentiating the activity of retinoid-related orphan receptor α (RORA).

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

Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA - 1 ( MAT1 - 1 - 1 ), SmtA - 2 ( MAT1 - 1 - 2 ), and SmtA - 3 ( MAT1 - 1 - 3 ), contained in the MAT1 - 1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora . Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA - 2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA - 1 and SmtA - 3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA - 1 and SmtA - 2 . Both genes are involved in the regulation of gene expression, including that of pheromone genes.

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

The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC). Repression of WCC's transcriptional activity by FRQ via negative feedback is indispensable for clock function. An unbiased genetic screen that targeted mutants with defects in negative feedback regulation yielded a fully viable arrhythmic strain bearing a novel allele of FRQ-interacting RNA helicase (frh), an essential gene that encodes a putative exosome component protein. In the allele, frhR806H, clock function is completely disturbed, while roles of FRQ-interacting RNA helicase (FRH) essential for viability are left intact. FRHR806H still interacts with FRQ, but interaction between the FRQ–FRHR806H complex (FFC) and WCC is severely affected. Phosphorylation of WC-1 is reduced in the mutant leading to constantly elevated WCC activity, which breaks the negative feedback loop. WCC levels are considerably reduced in the mutant, especially those of WC-1, consistent both with loss of positive feedback (FRQ-dependent WC-1 stabilization) and with a reduced level of the FRQ-mediated WCC phosphorylation that leads to high WCC activity accompanied by rapid transcription-associated turnover. FRH overexpression promotes WC-1 accumulation, confirming that FRH together with FRQ plays a role in WC-1 stabilization. Identification of a viable allele of frh, displaying virtually complete loss of both negative and positive circadian feedback, positions FRH as a core component of the central oscillator that is permissive for rhythmicity but appears not to modulate periodicity. Moreover, the results suggest that there are clock-specific roles for FRH that are distinct from the predicted essential exosome-associated functions for the protein.