Qiyang He

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation

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.

Functional conservation of light, oxygen, or voltage domains in light sensing

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 Neurosporastrains 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.

WHITE COLLAR-1, a multifunctional Neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2

WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors, are the positive elements of the circadian feedback loops in Neurospora. In addition, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. Recently, we identified WC-1 as the blue light photoreceptor responsible for these light responses. In this study, we show that the formation of the FRQ-WC complex in vivo, a step critical in closing the circadian negative feedback loop, requires WC-1. In addition, we show that WC-1 negatively regulates the expression ofwc-2 at the level of the transcription, forming another interacting loop. In a wc-1 mutant, we demonstrate that there is alternative protein initiation of WC-1, and the requirements of WC-1 for the light induction of frq and other genes differ significantly, suggesting the existence of different WC complexes in the cell. Consistent with this interpretation, our results show that there are at least two different types of WC-1/WC-2 complexes in vivo, and that the larger WC-1/WC-2 complex contains more than one WC-1 molecule. Using a series ofwc-1 mutants, we show that the WC-1 PASC domain and its C-terminal region are essential for the formation of the WC-1/WC-2 complex. Functional analyses reveal that the DNA-binding domain of WC-1 is required only for the activation of frq in the dark and not for the light function of the protein, confirming that WC-1 is a multifunctional protein with separable protein domains.

Light-independent Phosphorylation of WHITE COLLAR-1 Regulates Its Function in the Neurospora Circadian Negative Feedback Loop

Phosphorylation is a major regulatory mechanism controlling circadian clocks. In the Neurospora circadian clock, the PER-ARNT-SIM (PAS) domain-containing transcription factor, WHITE COLLAR (WC)-1, acts both as the blue light photoreceptor of the clock and as a positive element in the circadian negative feedback loop in constant darkness, by activating the transcription of the frequency (frq) gene. To understand the role of WC-1 phosphorylation, five in vivo WC-1 phosphorylation sites, located immediately downstream of the WC-1 zinc finger DNA binding domain, were identified by tandem mass spectrometry using biochemically purified endogenous WC-1 protein. Mutations of these phosphorylation sites suggest that they are major WC-1 phosphorylation sites under constant conditions but are not responsible for the light-induced hyperphosphorylation of WC-1. Although phosphorylation of these sites does not affect the light function of WC-1, strains carrying mutations of these sites show short period, low amplitude, or arrhythmic conidiation rhythms in constant darkness. Furthermore, normal or slightly higher levels of frq mRNA and FRQ proteins were observed in a mutant strain containing mutations of all five sites despite its low WC-1 levels. Together, these data suggest that phosphorylation of these sites negatively regulates the function of WC-1 in the circadian negative feedback loop and is important for the function of the Neurospora circadian clock.

Photoreception in Neurospora: a tale of two White Collar proteins.

Neurospora crassa is the best-understood fungal organism in terms of the mechanism of light responses. All known Neurospora photoresponses are mediated by blue light. Two Per-Arnt-Simdomain containing transcription factors, WHITE COLLAR-1 (WC-1) and WC-2, are essential components for almost all light responses. Recently, WC-1was shown to be a blue-light photoreceptor. How light affects the DNA binding of the WC proteins to the promoter of the circadian clock gene frequency was also demonstrated. These studies established a mechanism that explains the light responses mediated by activation of transcription. The purpose of this review is to summarize the findings of recent studies on the molecular mechanism of photoreception in Neurospora.

Phosphorylation of FREQUENCY Protein by Casein Kinase II Is Necessary for the Function of the Neurospora Circadian Clock

ABSTRACT FREQUENCY (FRQ), a key component of the Neurospora circadian clock, is progressively phosphorylated after its synthesis. Previously, we identified casein kinase II (CKII) as a kinase that phosphorylates FRQ. Disruption of the catalytic subunit of CKII abolishes the clock function; it also causes severe defects in growth and development. To further establish the role of CKII in clock function, one of the CKII regulatory subunit genes, ckb1, was disrupted in Neurospora. In the ckb1 mutant strain, FRQ proteins are hypophosphorylated and more stable than in the wild-type strain, and circadian rhythms of conidiation and FRQ protein oscillation were observed to have long periods but low amplitudes. These data suggest that phosphorylation of FRQ by CKII regulates FRQ stability and the function of the circadian feedback loop. In addition, mutations of several putative CKII phosphorylation sites of FRQ led to hypophosphorylation of FRQ and long-period rhythms. Both CKA and CKB1 proteins are found in the cytoplasm and in the nucleus, but their expressions and localization are not controlled by the clock. Finally, disruption of a Neurospora casein kinase I (CKI) gene, ck-1b, showed that it is not required for clock function despite its important role in growth and developmental processes. Together, these data indicate that CKII is an important component of the Neurospora circadian clock.

The Ccr4-Not protein complex regulates the phase of the neurospora circadian clock by controlling WHITE COLLAR protein stability and activity

Background: The stability and activity of WCC is important for Neurospora circadian clock function. Results: Not1 is a WC-interacting protein. Down-regulation of not1 and ccr4 result in low WC levels and delayed circadian phases. Conclusion: The Ccr4-Not complex regulates the Neurospora clock by controlling WCC stability and activity. Significance: This study identifies the Ccr4-Not complex as a new factor in the Neurospora circadian clock. In the Neurospora circadian negative feedback loop, WHITE COLLAR 1 (WC-1) and WC-2 form the WC complex that activates frequency (frq) transcription. Here we show that Not1 is a WC-interacting protein and is important for maintaining WC levels. The not1 transcript displays a circadian oscillation with a similar phase as frq. Down-regulation of not1 leads to low levels of WC-1 and WC-2 and a delayed circadian phase as a result of increased protein degradation and increased WC activity. Protein purification of Not1 shows that it is part of the Neurospora Ccr4-Not complex. ccr4 is a clock-controlled gene and is regulated directly by the WC complex. Down-regulation of ccr4 results in a phase delay and period lengthening of the clock. Together, our findings suggest that the Ccr4-Not complex participates in the Neurospora clock function by interacting with and regulating the WC complex.