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.

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. In this study, we show that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1a L strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbt L mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ∼32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1a L strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1a L strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the cka RIP strain, which carries the disruption of the catalytic subunit of casein kinase II. In the cka RIP strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop. In Neurospora, Drosophila, and mammals, the positive elements are all heterodimeric complexes, consisting of two PER-ARNT-SIM (PAS) domain-containing transcriptional factors that bind to the cis-elements in the promoter of the negative elements to activate their transcription. On the other hand, the negative elements repress their own transcription by inhibiting the activity of the positive elements through their physical interactions. It is unclear how negative elements inhibit the activity of positive elements to close the circadian negative feedback loops. Since the identification of the Drosophila doubletime (dbt) gene, which encodes for a casein kinase I (CKI) homolog, it has become clear that post-translational protein phosphorylation is essential for the function of circadian clocks Price et al. 1998). Despite the evolutionary distance, remarkable conservation of post-translational regulation exists among different eukaryotic systems from fungi to human (see Discussion;Liu 2005;Heintzen and Liu 2006).In the filamentous fungus Neurospora crassa, the core circadian negative feedback loop consists of four essen-

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...

Blue light regulates many molecular and physiological activities in a large number of organisms. In Neurospora crassa, a eukaryotic model system for studying blue-light responses, the transcription factor and blue-light photoreceptor WHITE COLLAR-1 (WC-1) and its partner WC-2 are central to blue-light sensing.

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Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. On the other hand, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. Here, by using whole-cell isotope labeling and quantitative mass spectrometry methods, we show that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially-first by a priming kinase, then by the FRQ-recruited casein kinases. We further show that the cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop.[Keywords: Circadian clock; Neurospora; protein kinase A; phosphorylation; casein kinase I] Supplemental material is available at http://www.genesdev.org. Cheng et al. 2001aCheng et al. , 2005. FFC represses the transcription of frq by inhibiting WCC activity through their physical interaction (Aronson et al. 1994;Merrow et al. 1997Merrow et al. , 2001Cheng et al. 2001aCheng et al. , 2003Denault et al. 2001;Froehlich et al. 2003;He et al. 2006). This circadian negative feedback loop generates the robust circadian rhythms of frq RNA and FRQ protein in constant darkness (DD) .Post-translational modification of clock proteins by phosphorylation plays essential roles in all circadian clocks (Price et al. 1998;Lowrey et al. 2000;Lin et al. 2002;Sathyanarayanan et al. 2004

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In Neurospora, the flavin adenine dinucleotide-containing protein WHITE COLLAR-1 is the blue-light photoreceptor for the circadian clock and other light responses. The putative chromophore-binding domain of WC-1, its light, oxygen, or voltage (LOV) domain, is similar to the LOV domains found in the plant phototropins, the Neurospora VIVID (VVD) protein, and the Arabidopsis FKF1 and its related proteins. Studies of the plant phototropins have identified 11 flavin-contacting residues that are also conserved in the LOV domains of WC-1, VVD, and FKF1. In this study, by mutating the putative WC-1 flavin-binding sites, we show that these sites are important for the light function of the protein, suggesting that the WC-1 LOV domain adapts a structure similar to that of the phototropin LOV domains. By creating a Neurospora strain in which the LOV domain of WC-1 is swapped with that of VVD, we show that the LOV domain of VVD partially replaces the function of the WC-1 LOV domain, suggesting that VVD is a wc-dependent photoreceptor in Neurospora. Furthermore, we show that the Neurospora strains containing a chimeric WC-1 protein with the LOV domain from FKF1 or phot1 can also sense light, suggesting that FKF1 and its related proteins are light sensors in Arabidopsis. Taken together, our data suggest that these LOV domains are structurally similar protein modules involved in blue-light sensing.