Mechanism of the Neurospora circadian clock, a FREQUENCY-centric view.
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ABSTRACT:
SUBMITTER: Cha J
PROVIDER: S-EPMC4303299 | biostudies-other | 2015 Jan
REPOSITORIES: biostudies-other
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Project description:Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.
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Project description:Rhythmic activation and repression of clock gene transcription is essential for the functions of eukaryotic circadian clocks. In the Neurospora circadian oscillator, frequency (frq) transcription requires the WHITE COLLAR (WC) complex. Here, we show that the transcriptional corepressor regulation of conidiation-1 (RCO-1) is essential for clock function by regulating frq transcription. In rco-1 mutants, both overt and molecular rhythms are abolished, frq mRNA levels are constantly high, and WC binding to the frq promoter is dramatically reduced. Surprisingly, frq mRNA levels were constantly high in the rco-1 wc double mutants, indicating that RCO-1 suppresses WC-independent transcription and promotes WC complex binding to the frq promoter. Furthermore, RCO-1 is required for maintaining normal chromatin structure at the frq locus. Deletion of H3K36 methyltransferase su(var)3-9-enhancer-of-zeste-trithorax-2 (SET-2) or the chromatin remodeling factor CHD-1 leads to WC-independent frq transcription and loss of overt rhythms. Together, our results uncover a previously unexpected regulatory mechanism for clock gene transcription.
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Project description:Rhythmic activation and repression of the frequency (frq) gene are essential for normal function of the Neurospora circadian clock. WHITE COLLAR (WC) complex, the positive element of the Neurospora circadian system, is responsible for stimulation of frq transcription. We report that a C2H2 finger domain-containing protein IEC-1 and its associated chromatin remodeling complex INO80 play important roles in normal Neurospora circadian clock function. In iec-1KO strains, circadian rhythms are abolished, and the frq transcript levels are increased compared to that of the wild-type strain. Similar results are observed in mutant strains of the INO80 subunits. Furthermore, ChIP data show that recruitment of the INO80 complex to the frq promoter is IEC-1-dependent. WC-mediated transcription of frq contributes to the rhythmic binding of the INO80 complex at the frq promoter. As demonstrated by ChIP analysis, the INO80 complex is required for the re-establishment of the dense chromatin environment at the frq promoter. In addition, WC-independent frq transcription is present in ino80 mutants. Altogether, our data indicate that the INO80 complex suppresses frq transcription by re-assembling the suppressive mechanisms at the frq promoter after transcription of frq.
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Project description:The frequency (frq) gene of Neurospora crassa has long been considered essential to the function of this organism's circadian rhythm. Increasingly, deciphering the coupling of core oscillator genes such as frq to the output pathways of the circadian rhythm has become a major focus of circadian research. To address this coupling it is critical to have a reporter of circadian activity that can deliver high resolution spatial and temporal information about the dynamics of core oscillatory proteins such as FRQ. However, due to the difficulty of studying the expression of circadian rhythm genes in aerobic N. crassa cultures, little is known about the dynamics of this gene under physiologically realistic conditions. To address these issues we report a fluorescent fusion to the frq gene using a codon optimized version of the mCherry gene. To trace the expression and accumulation of FRQ-mCherryNC (FRQ-mCh) during the circadian rhythm, growing vegetative hyphae were scanned every hour under confocal microscopy (100x). Fluorescence of FRQ-mCh was detected only at the growing edge of the colony, and located in the cytoplasm and nuclei of vegetative hyphae for a distance of approximately 150-200microm from the apices of leading hyphae. When driven by the frq promoter, apparently there was also a second FRQ entrance into the nucleus during the circadian cycle; however the second entrance had a lower accumulation level than the first entrance. Thus this fluorescent fusion protein has proven useful in tracking the spatial dynamics of the frq protein and has indicated that the dynamics of the FRQ protein's nuclear trafficking may be more complex than previously realized.
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Project description:The fungus Neurospora crassa is a model system for investigating the mechanism of circadian rhythmicity, and the core of its circadian oscillator is thought to be a transcription/translation feedback loop involving the products of the frq (frequency), wc-1 (white-collar-1) and wc-2 (white-collar-2) genes. Several reports of rhythmicity in frq and wc null mutants have raised questions about how central the FRQ/WC loop is to the circadian system of Neurospora. Several research groups have attempted to answer this question by looking for entrainment of the conidiation banding rhythm in frq null mutants. Because the frq mutants are blind to light and cannot be entrained to light/dark cycles, these groups have used symmetric temperature cycles of equal-duration cool and warm phases to entrain the rhythm. Under these conditions, the direct effects of temperature on conidiation (masking effects) can compromise observations of the endogenous rhythm. I have reexamined this question by using short heat pulses to clearly separate masking from endogenous rhythms, and I have assayed entrainment in both frq and wc-1 null mutants. I found similar patterns of entrainment in the wild type and both mutant strains. Strong masking effects were found in the frq mutant but not in the wc-1 mutant. I conclude that a rapidly damping temperature-entrainable oscillator is present in the null mutants. A single temperature-entrainable oscillator may drive the conidiation rhythm in all strains, and additional properties such as light sensitivity and temperature compensation may be conferred by the intact FRQ/WC loop in the WT strain.
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