A HAD family phosphatase CSP-6 regulates the circadian output pathway in Neurospora crassa.

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.

period-1 encodes an ATP-dependent RNA helicase that influences nutritional compensation of the Neurospora circadian clock.

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.

Analysis of clock-regulated genes in Neurospora reveals widespread posttranscriptional control of metabolic potential.

Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.

The Neurospora Transcription Factor ADV-1 Transduces Light Signals and Temporal Information to Control Rhythmic Expression of Genes Involved in Cell Fusion.

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.

Circadian control sheds light on fungal bioluminescence.

Bioluminescence, the creation and emission of light by organisms, affords insight into the lives of organisms doing it. Luminous living things are widespread and access diverse mechanisms to generate and control luminescence [1-5]. Among the least studied bioluminescent organisms are phylogenetically rare fungi-only 71 species, all within the ∼ 9,000 fungi of the temperate and tropical Agaricales order-are reported from among ∼ 100,000 described fungal species [6, 7]. All require oxygen [8] and energy (NADH or NADPH) for bioluminescence and are reported to emit green light (λmax 530 nm) continuously, implying a metabolic function for bioluminescence, perhaps as a byproduct of oxidative metabolism in lignin degradation. Here, however, we report that bioluminescence from the mycelium of Neonothopanus gardneri is controlled by a temperature-compensated circadian clock, the result of cycles in content/activity of the luciferase, reductase, and luciferin that comprise the luminescent system. Because regulation implies an adaptive function for bioluminescence, a controversial question for more than two millennia [8-15], we examined interactions between luminescent fungi and insects [16]. Prosthetic acrylic resin "mushrooms," internally illuminated by a green LED emitting light similar to the bioluminescence, attract staphilinid rove beetles (coleopterans), as well as hemipterans (true bugs), dipterans (flies), and hymenopterans (wasps and ants), at numbers far greater than dark control traps. Thus, circadian control may optimize energy use for when bioluminescence is most visible, attracting insects that can in turn help in spore dispersal, thereby benefitting fungi growing under the forest canopy, where wind flow is greatly reduced.