Blue light regulates many physiological processes in fungi, but their photoreceptors are not known. In Neurospora crassa, all light responses depend on the Per-Arnt-Sim (PAS) domain-containing transcription factor white collar-1 (wc-1). By removing the WC-1 light, oxygen, or voltage domain, a specialized PAS domain that binds flavin mononucleotide in plant phototropins, we show that light responses are abolished, including light entrainment of the circadian clock. However, the WC-1-mediated dark activation of frq remains normal in this mutant, and the circadian clock can be entrained by temperature. Furthermore, we demonstrate that the purified Neurospora WC-1-WC-2 protein complex is associated with stoichiometric amounts of the chromophore flavin-adenine dinucleotide. Together, these observations suggest that WC-1 is the blue-light photoreceptor for the circadian clock and other light responses in Neurospora.

Interlocked feedback loops may represent a common feature among the regulatory systems controlling circadian rhythms. The Neurospora circadian feedback loops involve white collar-1 (wc-1), wc-2, and frequency ( frq) genes. We show that WC-1 and WC-2 proteins activate the transcription of frq gene, whereas FRQ protein plays dual roles: repressing its own transcription, probably by interacting with the WC-1͞WC-2 complex, and activating the expression of both WC proteins. Thus, they form two interlocked feedback loops: one negative and one positive. We establish the physiological significance of the interlocked positive feedback loops by showing that the levels of WC-1 and WC-2 determine the robustness and stability of the clock. Our data demonstrate that with WC-1 being the limiting factor in the WC-1͞WC-2 complex, the greater the levels of WC-1 and WC-2, the higher the level of the FRQ oscillation and the more robust the overt rhythms. Our data also show that, despite considerable changes in the levels of WC-1, WC-2, and FRQ, the period of the clock has been limited to a small range, suggesting that the interlocked circadian feedback loops are also important for determining the circadian period length of the clock. In eukaryotic and certain prokaryotic organisms, circadian clocks are responsible for controlling a wide variety of physiological, behavioral, cellular, and biochemical activities. At the molecular level, a common theme of various circadian oscillators is a network of positive and negative elements that form the core of the oscillators that establish the negative feedback loops generating the basic circadian rhythmicity (1). In a simple view, every oscillator has both positive and negative elements to comprise the feedback loop. The positive elements of the loop activate the expression of the negative elements, whereas the negative elements feedback to block their own activation by the positive elements. The identified positive elements in Neurospora, Drosophila, and mammals are all PAS domain-containing transcription factors (2-7). These factors form heterodimeric complexes and activate the transcription of the negative elements in each system, and the protein products of these negative elements feedback to inhibit their own expression (8)(9)(10)(11)(12).Recently, studies in Neurospora, Drosophila, and mammals have significantly furthered our view of the negative feedback nature of the circadian oscillator with the identification of interlocked feedback loops (13-15). In each system, the negative elements of the oscillator have been found to activate the expression of one of the positive elements. Thus, the negative and the positive elements form another positive feedback loop interlocked with the negative feedback loop. The similarity of such an arrangement in different clock systems suggests that it may be a common aspect in the eukaryotic circadian oscillators. However, evidence to support the physiological significance of the positive feedback loops is still lacking.In the Neurospora frq-wc based ...

ObjectiveNeutrophils are prominent components of solid tumours and exhibit distinct phenotypes in different tumour microenvironments. However, the nature, regulation, function and clinical relevance of neutrophils in human gastric cancer (GC) are presently unknown.DesignFlow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 105 patients with GC. Kaplan-Meier plots for overall survival were performed using the log-rank test. Neutrophils and T cells were isolated, stimulated and/or cultured for in vitro and in vivo regulation and function assays.ResultsPatients with GC showed a significantly higher neutrophil infiltration in tumours. These tumour-infiltrating neutrophils showed an activated CD54+ phenotype and expressed high level immunosuppressive molecule programmed death-ligand 1 (PD-L1). Neutrophils activated by tumours prolonged their lifespan and strongly expressed PD-L1 proteins with similar phenotype to their status in GC, and significant correlations were found between the levels of PD-L1 and CD54 on tumour-infiltrating neutrophils. Moreover, these PD-L1+ neutrophils in tumours were associated with disease progression and reduced GC patient survival. Tumour-derived GM-CSF activated neutrophils and induced neutrophil PD-L1 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signalling pathway. The activated PD-L1+ neutrophils effectively suppressed normal T-cell immunity in vitro and contributed to the growth and progression of human GC in vivo; the effect could be reversed by blocking PD-L1 on these neutrophils.ConclusionsOur results illuminate a novel mechanism of PD-L1 expression on tumour-activated neutrophils in GC, and also provide functional evidence for these novel GM-CSF-PD-L1 pathways to prevent, and to treat this immune tolerance feature of GC.

The eukaryotic circadian oscillators consist of autoregulatory negative-feedback loops. FRQ, WC-1, and WC-2 are three known components of the negative-feedback loop of the Neurospora circadian oscillator. FRQ represses its own transcription by interacting with the WC-1/WC-2 complex and inhibiting WC's role in transcriptional activation. Here we show that all FRQ associates with FRH, an essential DEAD box-containing RNA helicase in Neurospora. The budding yeast homolog of FRH, Dob1p/Mtr4p, is a cofactor of exosome, an important regulator of RNA metabolism in eukaryotes. Down-regulation of FRH by inducible expression of a hairpin RNA leads to low levels of FRQ but high levels of frq RNA and the abolishment of circadian rhythmicities. FRH is associated with the WC complex and this interaction is maintained in a frq null strain. Disruption of the FRQ-FRH complex by deleting a domain in FRQ eliminates the FRQ-WC interaction, suggesting that FRH mediates the interaction between FRQ and the WC complex. These data demonstrate that FRH is an essential component in the circadian negative-feedback loop and reveal an unexpected role of an RNA helicase in regulating gene transcription. Endogenous circadian (daily) clocks control a wide variety of physiological and molecular activities in most eukaryotic and some prokaryotic organisms. At the molecular level, autoregulatory negative-feedback loops composed of positive and negative elements form the core circadian oscillators (Dunlap 1999;King and Takahashi 2000;Reppert and Weaver 2001;Young and Kay 2001). The rhythmic activation of transcription of the negative elements by the positive elements is thought to be the main basis for the generation of the endogenous rhythmicity.In the Neurospora frequency (frq)-white collar (wc)-based circadian negative-feedback loop, a heterodimeric complex formed by WC-1 and WC-2 (two PAS domaincontaining transcription factors) acts as the positive element and activates the transcription of frq by binding to its promoter (Crosthwaite et al. 1997;Cheng et al. 2001b;Loros and Dunlap 2001;Froehlich et al. 2003). FRQ proteins (large FRQ [lFRQ] and small FRQ [sFRQ] resulting from alternative translation initiation) form homodimeric complexes and function as the negative elements in the loop by repressing their own transcription (Aronson et al. 1994a;Garceau et al. 1997;Liu et al. 1997;Cheng et al. 2001a). To close the negative-feedback loop, FRQ forms a complex with the WC proteins and prevents WC from binding to the frq promoter and activating frq transcription (Cheng et al. 2001a;Denault et al. 2001;Merrow et al. 2001;Froehlich et al. 2003). In strains lacking a functional FRQ protein, the negativefeedback loop is impaired, resulting in high frq mRNA levels (Aronson et al. 1994a;Merrow et al. 1997;. How FRQ inhibits the activity of WC complex is unclear. In frq null strains, in addition to their loss of circadian rhythmicities, less conidia and aerial hyphae are produced than a wild-type strain (Aronson et al. 1994b), suggesting that FRQ has funct...

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The frequency (frq) gene, the central component of the frq-based circadian negative feedback loop, regulates various aspects of the circadian clock in Neurospora. However, the biochemical function of its protein products, FRQ, is poorly understood. In this study, we demonstrated that the most conserved region of FRQ forms a coiled-coil domain. FRQ interacts with itself in vivo, and the deletion of the coiled-coil region results in loss of the interaction. Point mutations, which are designed to disrupt the coiled-coil structure, weaken or completely abolish the FRQ self-association and lead to the arrhythmicity of the overt rhythm. Mutations of the FRQ coiled-coil that inhibit self-association also prevent its interaction with two other key components of the Neurospora circadian clock, namely WC-1 and WC-2, the two PAS domain-containing transcription factors. Taken together, these data strongly suggest that the formation of the FRQ±FRQ and FRQ±WC complexes is essential for the function of the Neurospora clock.

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Circadian clocks control a wide variety of physiological, behavioral, cellular, and biochemical activities in most eukaryotic and certain prokaryotic organisms. At the molecular level, circadian oscillators consist of autoregulatory negative feedback loops, in which the positive elements of the loop activate the transcription of the negative elements, whereas the negative elements feedback to block their own transcriptional activation by interacting with the positive elements (Aronson et al.