VIVID interacts with the WHITE COLLAR complex and FREQUENCY-interacting RNA helicase to alter light and clock responses in Neurospora

Abstract

The photoreceptor and PAS/LOV protein VIVID (VVD) modulates blue-light signaling and influences light and temperature responses of the circadian clock in Neurospora crassa. One of the main actions of VVD on the circadian clock is to influence circadian clock phase by regulating levels of the transcripts encoded by the central clock gene frequency (frq). How this regulation is achieved is unknown. Here we show that VVD interacts with complexes central for circadian clock and blue-light signaling, namely the WHITE-COLLAR complex (WCC) and FREQUENCY-interacting RNA helicase (FRH), a component that complexes with FRQ to mediate negative feedback control in Neurospora. VVD interacts with FRH in the absence of WCC and FRQ but does not seem to control the exosome-mediated negative feedback loop. Instead, VVD acts to modulate the transcriptional activity of the WCC.

Fig. 1.

VVD controls frq RNA levels. (A) Northern blots showing frq transcript levels in vvd⁺ (WT) or vvd^ko strains with or without an ectopic insertion of a QA-inducible vvd gene. Cultures were grown in LL for 24 h in the presence or absence of the inducer QA before transfer to DD, and samples were harvested after 24 h in LL (time point 0) or at the indicated times in DD. (B) As in A, but Northern blots were hybridized with a probe detecting the vvd transcript. Loading controls and quantitative analysis are shown in Fig. S1.

Fig. 2.

VVD interacts with FRH. (A) An antibody against FRH immunoprecipitates VVD in a Co-IP assay. Protein extracts from vvd^myc, vvd^ko, wc-2^ko, and frq^ko strains grown for 30 min in LL were incubated with FRH antibody. An FRH antiserum immunoprecipitates VVD (bottom lane). As expected, FRH immunoprecipitates FRQ in WT and vvd^ko strains but not in the frq^ko and wc-2^ko control strains. (B) VVD can interact with FRH in a strain (127-11) that lacks a functional WCC and FFC (SI Materials and Methods) but contains an ectopic qa-2–driven copy of the vvd gene [qa-vvd^myc (127-11)]. Western blots of total (input) extracts (top two lanes), extracts immunoprecipitated with FRH antiserum (middle two lanes), or unimmunized mouse serum (MS) (bottom two lanes) were probed with either FRH or MYC antiserum to detect FRH and VVD, respectively.

Fig. 3.

VVD represses frq transcription. (A) Northern blots showing frq transcript levels in WT (top two lanes) and vvd^ko strains (lanes three, four, and five from top) in cultures grown in LL for 24 h (time point 0) or for different lengths of time (h) in DD. Cultures were grown in the presence (+) or absence (−) of the transcriptional inhibitor thiolutin. Thiolutin was added either 1 h before (−1 h) or immediately after (+0 h) liquid cultures were transferred from light to dark. Ethidium bromide–stained ribosomal RNA was used to control for loading of Northern blots. (B) Quantitative analysis of Northern blots (shown in A) of untreated WT (●) and untreated (▲) and thiolutin-treated (immediately after the light-to-dark transfer) (△) vvd^ko strains. Within each experiment maximum frq RNA levels were set to 100%.

Fig. 4.

VVD is both a cytoplasmic and nuclear protein. (A) Total (T), nuclear (N), or cytoplasmic (C) extracts were prepared from Neurospora tissue grown for 24 h in DD (0) or with exposure to LL for the indicated times (h). Western blots were probed with an MYC antibody to detect VVD^MYC. The amido black-stained membrane serves as a loading control. (B) Graph showing the percent ratio of nuclear to cytoplasmic signal using the Western blot data shown in A. (C) Neurospora extracts of strains expressing GFP-tagged VVD (under ccg-1 promoter control) grown for 24 h in DD or 4 h in LL. Western blots were probed with a GFP antibody to detect VVD^GFP. CP, cytoplasmic protein; NP, nuclear protein. (D) Subcellular localization of VVD^GFP (under ccg-1 promoter control) in Neurospora conidiospores fixed at time points DD24 and LL4. Each subpanel shows confocal images of fluorescence from DAPI-stained spores (Upper Left), fluorescence from the GFP signal (Upper Right), an overlay of both (Lower Right), and corresponding bright-field image (Lower Left). (Scale bar, 1 μm.)

Fig. 5.

VVD interacts with the WCC in the light and in the dark. (A) Protein extracts from vvd^myc, vvd^ko, and wc-1^ko strains were incubated with WC-1 antibody. WC-1 immunoprecipitates VVD in the vvd^myc strain but not in a vvd^ko or wc-1^ko strain. (B) WC-1 antibody does not cross-react with VVD. QA-inducible VVD was expressed in a vvd^ko and a vvd^ko, wc-1^ko background, and protein extracts coimmunoprecipitated with WC-1 antibody. No VVD^MYC was immunoprecipitated in a vvd^ko, wc-1^ko background. (C) Control to show that QA-inducible VVD^MYC is stably expressed in the absence of WC-1. (D) A WC-1 antiserum immunoprecipitates QA-induced VVD^MYC in DD in a wc-1⁺ strain (qa-vvd^myc). WC-1 antiserum fails to immunoprecipitate VVD^MYC in a strain (127-11) that lacks WC-1 but expresses QA-induced VVD^MYC at levels similar to the control strain qa-vvd^myc. (E) A small proportion of VVD is found in sucrose gradient fractions containing WC-1, WC-2, and FRH. Western blot (top five lanes) and graph (Lower) showing the densitometric analysis of VVD^MYC, WC-1, WC-2, and FRH from protein extracts size-fractionated on a 10–35% sucrose gradient. Fraction 1 corresponds to low molecular weights and fraction 16 to higher molecular weights. Asterisks denote unspecific signals. For densitometry the maximum signal for each protein was set to 100%.

Fig. 6.

Model of VVD's action in negative feedback regulation of the WCC. (A) In the dark WC-1 and WC-2 heterodimerize to form a WCC^D. Upon light exposure a multimeric WCC complex, WCC^L, forms (11, 14) that mediates transcription of light-induced genes (e.g., vvd). VVD binds WC-1 to inhibit the efficient formation of WCC^L, resulting in a decrease of light-induced transcription. Without VVD the equilibrium between light and dark complexes is shifted toward WCC^L, resulting in increased transcription of frq in the light. Upon transfer to DD and without VVD, the increased activity of WCC^L results in prolonged activation of frq transcription (possibly via its proximal LRE), whereas in the WT FRQ-mediated phosphorylation of WCC^D leads to rapid inactivation of frq transcription. WCC^L and WCC^D have different preferences for proximal and distal promoter elements, with WCC^D preferentially engaged at the distal clock box where negative feedback regulation by FRQ takes place (11). (B) Simplified schematic of the frq RNA profile after light induction and in constant darkness in WT (thick line) and in a vvd^KO strain (thin line). The gray area depicts the difference in frq activation between the two strains and is a result of a change in equilibrium between WCC^D and WCC^L. Prolonged activation in the dark leads to a characteristic phase delay in the onset of frq transcript oscillations and overt circadian rhythmicity.