Kinases and Circadian Clocks PER Goes It Alone

AbstractIn the January 30, 2004, issue of Molecular Cell, Nawathean and Rosbash describe how kinases cooperate to create a small but active pool of PER that can localize to the nucleus and act on its own in a transcriptional feedback loop underlying the Drosophila circadian clock

Carlene Allen Raper 1925-2019

The field of fungal genetics and biology lost one of its founding anchors with the death of Carlene Raper on September 5, 201

Phosphorylation of the Neurospora Clock Protein FREQUENCY Determines its Degradation Rate and Strongly Influences the Period Length of the Circadian Clock

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (\u3e30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock

A recommendation for naming proteins in Neurospora

The issue of gene product names is important in as much as it promotes consistency within the literature and promotes accessibility of the Neurospora literature to readers more familiar with other organisms

How Temperature Changes Reset a Circadian Oscillator

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

The Clock affecting 1 mutation of Neurospora is a recurrence of the frq\u3csup\u3e7\u3c/sup\u3e mutation7

The clock affecting-1 (cla-1) mutation of Neurospora crassa increases the period and decreases temperature compensation of the circadian rhythm, and was thought to define an uncloned gene with a possible role in the Neurospora clock. This defect, thought to be due to a translocation, was associated with a slow growth rate and a period of about 27 h at 25cla-1 and found the growth rate and period defects to be due to linked independent mutations. The translocation was not the cause of the long period. The csp-1 mutation, present in the original cla-1 strain, had a period shortening effect, thus cla-1 strains lacking csp-1 had a period length similar to that of frequency7 (frq7). The cla-1 period defect mapped to the frq locus, and sequencing of frq revealed cla-1 to be a re-isolation of frq7

Alternative Use of DNA Binding Domains by the Neurospora White Collar Complex Dictates Circadian Regulation and Light Responses

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\u27s 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

Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus Fumigatus

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 effi- ciency 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. fu- migatus 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 to- gether, 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

Neurospora WC-1 Recruits SWI/SNF to Remodel Frequency and Initiate a Circadian Cycle

In the negative feedback loop comprising the Neurospora circadian oscillator, the White Collar Complex (WCC) formed from White Collar-1 (WC-1) and White Collar-2 (WC-2) drives transcription of the circadian pacemaker gene frequency (frq). Although FRQ-dependent repression of WCC has been extensively studied, the mechanism by which the WCC initiates a circadian cycle remains elusive. Structure/function analysis of WC-1 eliminated domains previously thought to transactivate frq expression but instead identified amino acids 100–200 as essential for frq circadian expression. A proteomics-based search for coactivators with WCC uncovered the SWI/SNF (SWItch/Sucrose NonFermentable) complex: SWI/SNF interacts with WCC in vivo and in vitro, binds to the Clock box in the frq promoter, and is required both for circadian remodeling of nucleosomes at frq and for rhythmic frq expression; interestingly, SWI/SNF is not required for light-induced frq expression. These data suggest a model in which WC-1 recruits SWI/SNF to remodel and loop chromatin at frq, thereby activating frq expression to initiate the circadian cycle

The Fast-Evolving phy-2 Gene Modulates Sexual Development in Response to Light in the Model Fungus Neurospora crassa

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