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1009474107.Full.Pdf VIVID interacts with the WHITE COLLAR complex and FREQUENCY-interacting RNA helicase to alter light and clock responses in Neurospora Suzanne M. Hunt, Seona Thompson, Mark Elvin, and Christian Heintzen1 Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Edited* by Jay C. Dunlap, Dartmouth Medical School, Hanover, NH, and approved August 10, 2010 (received for review June 30, 2010) The photoreceptor and PAS/LOV protein VIVID (VVD) modulates dawn and dusk, VVD affects light resetting and entrainment of the blue-light signaling and influences light and temperature responses circadian clock (16, 20). Finally, VVD plays a role in maintaining of the circadian clock in Neurospora crassa. One of the main actions of the correct timing of clock-controlled output pathways at different VVD onthe circadian clock is to influence circadian clockphase by reg- temperatures (21). ulating levels of the transcripts encoded by the central clock gene Outside the circadian system VVD plays a key role in photo- frequency (frq). How this regulation is achieved is unknown. Here we adaptation (18, 22, 23). VVD transiently dimerizes in a light- show that VVD interacts with complexes central for circadian clock dependent manner (24–26), and this conformational change may and blue-light signaling, namely the WHITE-COLLAR complex (WCC) relay light signals to downstream targets. However, despite the and FREQUENCY-interacting RNA helicase (FRH), a component that significance of VVD for aligning the Neurospora clock with the complexes with FRQ to mediate negative feedback control in Neu- external day, we still do not know how VVD accomplishes these rospora. VVD interacts with FRH in the absence of WCC and FRQ but activities at the molecular level. No interaction partners of VVD does not seem to control the exosome-mediated negative feedback fi loop. Instead, VVD acts to modulate the transcriptional activity of (except VVD itself) have been identi ed, and consequently ’ the WCC. a mechanistic understanding of VVD s activities is lacking. Here we show that VVD interacts with clock components WC-1 and PHYSIOLOGY blue light | entrainment | photoreceptor | phase | circadian FRH. Our data suggest that VVD acts in the nucleus as a FRH- dependent corepressor of WCC. ight, in addition to providing an energy source for many life Results Lforms on Earth, acts as a signal that may trigger development or frq serve as a repetitive cue that marks the passing of external time. VVD Regulates Transcript Levels at Dusk. Molecular and physi- fl External time cues are used by cellular timers such as circadian ological data have shown that VVD in uences clock resetting at ’ clocks to lock their periods to that of the external day. The process dawn and dusk. At dusk VVD s impact on molecular events is ev- of period locking is called “entrainment” and ensures that cellular ident when comparing frq mRNA levels of WT and vvd-knockout ko ko and behavioral activities happen at times of day when their adap- (vvd ) strains. The frq transcript remains elevated longer in vvd tive value is highest (1–3). Blue light plays a central role in the strains than in the WT, with a delay of about 4 h in reaching basal entrainment of circadian clocks. Indeed blue-light photoreceptors levels (top two lanes in Fig. 1A) (16, 20). To obtain more direct and circadian clocks may have coevolved from a mechanism that proof of VVD’s role in the regulation of frq transcript levels, we originally served to detect (photoreceptor) and avoid (timer) created a strain in which a quinic acid (QA)-inducible copy of an harmful radiation (4–6). Our understanding of the molecular bases myc epitope-tagged vvd gene (qa-vvdmyc) was inserted at the his-3 of circadian clocks and their responses to light has improved dra- locus. The qa-vvdmyc construct was integrated into WT or vvdko matically during the last decade or so, and the eukaryotic model strains, and these strains are referred henceforth to as qa-vvdmyc organism Neurospora crassa has become one of the best-studied myc KO – (WT) and qa-vvd (vvd ), respectively. By using this strategy, we systems for understanding both processes (7 9). were able to uncouple vvd expression from its normal light regu- The key components of the Neurospora circadian clock are the lation (Fig. 1B and Fig. S1). Indeed, the ectopic expression of VVD products of the white collar (wc-1 and wc-2), frequency (frq), and induced by the addition of QA in qa-vvdmyc (vvdko) restores the frq-interacting helicase (frh) genes (4, 10, 11). The blue-light pho- − toreceptor WC-1, and its interaction partner WC-2, form the normal decline in frq levels (compare the QA and +QA samples transcriptionally and photoactive WHITE COLLAR complex in Fig. 1A, fourth panel) and accelerates frq degradation in qa- myc − (WCC) that activates frq expression (4, 12). FRQ protein, in turn, vvd (WT) beyond that seen in a normal WT (compare the QA complexes with FRH to form an FRQ-FRH complex (FFC) that and +QA samples in the first and third lanes in Fig. 1A and see represses WCC activity (9, 11). Thus, photoreception and tem- quantification of data in Fig. S1A). The observation that frq levels poral organization of gene expression are linked via the WCC (4, are somewhat lower in QA medium was expected, because full 12–14). Hyperphosphorylated WCC is transcriptionally less active, expression of frq is dependent on glucose (27). Taken together, and repression of WCC by FRQ occurs via FRQ-mediated phos- these data illustrate an inverse correlation between frq transcript phorylation of WCC by Casein kinase 1 and 2 (CK1 and 2) (14, 15). levels and VVD protein. A second feedback loop that acts to repress WCC activity involves the product of the vivid (vvd) gene (16). Like WC-1, VVD is a PAS/LOV protein and blue-light photoreceptor; however, Author contributions: S.M.H. and C.H. designed research; S.M.H., S.T., and M.E. performed unlike WC-1, its presence is not essential for circadian rhythmicity research; S.M.H., S.T., M.E., and C.H. analyzed data; and C.H. wrote the paper. in constant darkness (DD) (16–19). Nevertheless, VVD has im- The authors declare no conflict of interest. portant roles within the Neurospora circadian system. Without *This Direct Submission article had a prearranged editor. VVD the organism is more sensitive to light, resulting in the rapid 1To whom correspondence should be addressed. E-mail: christian.heintzen@manchester. breakdown of circadian organization in continuous illumination, ac.uk. whereas in the presence of VVD temporal rhythmicity is main- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tained in constant light (LL). By influencing clock resetting at both 1073/pnas.1009474107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1009474107 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Fig. 2. VVD interacts with FRH. (A) An antibody against FRH immunopre- cipitates VVD in a Co-IP assay. Protein extracts from vvdmyc, vvdko, wc-2ko, and frqko 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 vvdko strains but not in the frqko and wc-2ko 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-vvdmyc (127-11)]. Western blots Fig. 1. VVD controls frq RNA levels. (A) Northern blots showing frq transcript of total (input) extracts (top two lanes), extracts immunoprecipitated with levels in vvd+ (WT) or vvdko strains with or without an ectopic insertion of FRH antiserum (middle two lanes), or unimmunized mouse serum (MS) a QA-inducible vvd gene. Cultures were grown in LL for 24 h in the presence (bottom two lanes) were probed with either FRH or MYC antiserum to de- or absence of the inducer QA before transfer to DD, and samples were har- tect FRH and VVD, respectively. vested after 24 h in LL (time point 0) or at the indicated times in DD. (B)Asin A, but Northern blots were hybridized with a probe detecting the vvd tran- script. Loading controls and quantitative analysis are shown in Fig. S1. the FFC also functions at the posttranscriptional level to control frq mRNA degradation via the exosome (28). If VVD influences frq transcription, inhibition of transcription VVD Interacts with FRH. Because the FFC plays a key role in frq should abolish the differences in frq levels that exist between WT negative feedback (11, 28, 29), it was possible that VVD directly and vvdko strains. To test this possibility, we used the transcrip- modulates the activity of this complex to influence frq levels at tional inhibitor thiolutin (28) to inhibit transcription 1 h before or dusk. To test whether VVD interacts with the FFC, we performed coimmunoprecipitation (Co-IP) experiments on Neurospora directly after the transfer of Neurospora cultures from light-to- whole-cell lysates using FRH or FRQ antisera, respectively. To dark (Fig. 3). When transcription was inhibited before the light-to- dark transition, we saw no or very little frq transcript present in facilitate detection of VVD, we used a strain that expresses MYC- ko tagged VVD in a vvdko background. We have shown previously that either WT or vvd strains, suggesting effective repression of transcription by the drug (second and fourth lanes in Fig. 3A). this strain rescues all known vvd mutant phenotypes, thus dem- – onstrating that VVDMYC is fully functional (21).
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