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 immunoprecipitates 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 transcript. 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). Henceforth all When frq transcription was allowed to proceed to the light dark references to VVD protein levels are based on VVDMYC expres- boundary before thiolutin was added, the kinetics of frq transcript decline in a vvdko strain were no longer slowed and re- sion. Lysates from the vvd myc-tagged strain exposed to 30 min of fi LL were incubated with either FRH or FRQ antiserum before sembled that of an untreated WT strain (compare fth lane with probing the blotted immunoprecipitates with MYC antiserum to top and third lanes of Fig. 3A). These data show that VVD targets frq transcription. test for the presence of VVD. Unfortunately, the FRQ antiserum fi proved too unspecific in our Co-IP experiments, so we were unable This conclusion was con rmed by an experiment in which we placed frq under the control of the QA-inducible promoter. The to judge whether VVD interacts with FRQ. However, when we ko ko ko fi qa-frq construct was integrated into a frq strain or a frq vvd used FRH antiserum, VVD was speci cally immunoprecipitated ko ko ko ko double-mutant strain to generate qa-frq (frq ) and qa-frq (frq (Fig. 2A). No signal was detected in frq and vvd strains that KO fi vvd ) strains, respectively. In analogy to our QA-inducible lacked a tagged copy of the vvd gene, indicating that the identi ed MYC signal is VVDMYC and not an unspecific signal. VVD expression system described above, this experiment To test whether a functional WCC or FFC is necessary for the allowed us to uncouple frq expression from its normal light- interaction of VVD with FRH, we exploited the QA-inducible induced transcriptional regulation and study the reduction in frq system as outlined above and expressed a qa-vvdmyc in a back- transcript levels in a controlled manner after release from the in- ground in which the wc-1, wc-2, frq, and vvd genes were deleted ducer. If VVD targets frq transcription, replacing the frq promoter – myc with the QA promoter should result in a similar drop in frq tran- (strain 127 11) (Fig. 2B). In this qa-vvd (127-11) strain, in which ko ko KO only the central clock gene frh remains intact, VVD still interacts script levels in both the qa-frq (frq ) and qa-frq (frq vvd ) with FRH, indicating that neither a functional FFC nor WCC is strains after release from the inducer. On the other hand, a post- necessary for the interaction. The interaction occurs at both dawn transcriptional action of VVD on frq levels should result in a dif- ko KO and dusk transitions (Fig. S2B). As expected, extracts show a sig- ference (i.e., delay) in frq mRNA turnover in qa-frq (frq vvd ) ko nificant depletion of FRH after immunodepletion (top two lanes strains as compared with qa-frq (frq ). After release from the in- ko ko KO in Fig. S2A). However, no significant depletion of VVD is seen, ducer, frq levels in qa-frq (frq ) and qa-frq (frq vvd ) strains suggesting that only a small fraction of total VVD interacts were followed for a period of 8 h in DD, and we observed no sig- with FRH. nificant difference in the decline of frq transcript kinetics in the two strains. There is some variability in QA-induced frq levels imme- VVD Affects the Transcriptional Limb of FFC-Mediated Negative diately following release from the inducer, but the kinetics of the Feedback. Next, we investigated the mechanism by which VVD decline of frq are similar in all strains and conditions tested (Fig. mediates frq RNA turnover. Two distinct pathways that regulate S3C). These data therefore support the conclusion that VVD levels of frq message have been described. First, a negative feed- influences frq RNA at the level of transcription and not via the back loop involving FRQ and FRH is important for rhythmic exosome-mediated function of the FFC. Interestingly, the pres- down-regulation of frq at the level of transcription (10, 11). Second, ence or absence of light had no significant influence on frq turn-

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009474107 Hunt et al. Downloaded by guest on September 26, 2021 a consequence of the drastic increase in VVD levels during exposure to light. However, when expressed as the ratio of nuclear to cytoplasmic VVD levels (Fig. 4B), our data suggested that nuclear entry of VVD may be light regulated. To gather independent information on VVD’s localization within the cell, we generated carboxyl-terminal fusions of GFP to VVD using plasmids (30) in which gene expression is controlled by the strong Neurospora ccg-1 promoter. The ccg-1–driven vvd-gfp construct is capable of rescuing the carotenoid and clock phenotypes of a vvdko strain (Fig. S4 A and C), and levels of VVDGFP are somewhat higher than in a strain that expresses vvd under its own promoter (Fig. S4B). Cell fractionation of the GFP-tagged VVD strains confirmed results obtained earlier with myc-tagged VVD (Fig. 4A) that VVD is localized both in the cytoplasm and nucleus (Fig. 4C). For confocal microscopy, we harvested conidia (that usually contain one to three nuclei) from strains kept in DD for 24 h and from strains kept in LL for 4 h. In a small number of conidia the nuclear localization of VVDGFP can be seen clearly (Fig. 4D), again confirming the results we obtained with our cellular extracts. As expected, conidia obtained from the 1H-GFP fusion strain also show nuclear localization for 1HGFP (Fig. S4D, Bottom). In contrast, we never saw any nuclear signal above background in nuclei from WT strains that were not tagged with GFP (Fig. S4D, Top and Middle). Because published data suggest that GFP alone is cytoplasmic and does not localize to the nucleus (31) the low percentage of nuclei that fluoresce in vvd gfp-tagged PHYSIOLOGY strains suggests that nuclear accumulation or entry of VVD may be highly dynamic. In conclusion, our combined data confirmed that a substantial proportion of VVD localizes to the nucleus.

Fig. 3. VVD represses frq transcription. (A) Northern blots showing frq transcript levels in WT (top two lanes) and vvdko 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) (△) vvdko strains. Within each experiment maximum frq RNA levels were set to 100%.

over, suggesting that light was not required for the activation of VVD (compare Fig. S3 A and B).

VVD Localizes to Both the Cytoplasm and the Nucleus. Because our data showed that VVD represses transcription, we explored VVD’s localization. VVD is reported to localize to the cytoplasm with no Fig. 4. VVD is both a cytoplasmic and nuclear protein. (A) Total (T), nuclear evidence for nuclear localization (18). However, the use of an MYC (N), or cytoplasmic (C) extracts were prepared from Neurospora tissue grown antibody to detect myc-tagged VVD reduces the threshold at which for 24 h in DD (0) or with exposure to LL for the indicated times (h). Western VVD can be detected (21). We prepared nuclear and cytoplasmic blots were probed with an MYC antibody to detect VVDMYC. The amido black- extracts from Neurospora cultures grown in DD and different time stained membrane serves as a loading control. (B) Graph showing the percent points following transfer to LL (Fig. 4). In agreement with previous ratio of nuclear to cytoplasmic signal using the Western blot data shown in A. results, we did not detect VVD in extracts from Neurospora tissue (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 grown in extensive periods of darkness. In contrast, after 30 min in GFP LL, significant amounts of VVD were seen in both the total extracts with a GFP antibody to detect VVD . CP, cytoplasmic protein; NP, nuclear protein. (D) Subcellular localization of VVDGFP (under ccg-1 promoter control) and the cytoplasmic fraction (Fig. 4A). Upon light exposure a faint in Neurospora conidiospores fixed at time points DD24 and LL4. Each subpanel signal also is detectable in nuclear extracts, and this signal becomes shows confocal images of fluorescence from DAPI-stained spores (Upper Left), stronger with increasing time in LL. (Fig. 4A). We could not rule fluorescence from the GFP signal (Upper Right), an overlay of both (Lower out the possibility that the increase in nuclear VVD is simply Right), and corresponding bright-field image (Lower Left). (Scale bar, 1 μm.)

Hunt et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 VVD Interacts with the WCC. The nuclear localization of VVD and VVD’s interaction with FRH led us to test whether VVD represses frq transcription by binding to the WCC. Such an activity would be consistent with the observation that VVD interacts with FRH, because FRH mediates the phosphorylation and inactivation of WCC (15, 32, 33). Moreover, VVD has been shown to influence WC-1 phosphorylation and thus WCC activity (16, 23). We performed Co-IP experiments in which Neu- rospora whole-cell lysates were incubated with WC-1 antiserum before probing for the presence of VVD. As can be seen in Fig. 5, VVD was immunoprecipitated in both LL (Fig. 5 A and B) and DD (Fig. 5D). Because the PAS/LOV domains of VVD and WC-1 are similar, it was important to control for the specificity of the WC-1–VVD interaction. Therefore we created strains in which vvdmyc could be expressed in a wc-1ko background. Because vvd expression is dependent on WC-1, a QA-driven vvd gene was expressed to uncouple VVD expression from WC-1 expression. The qa-vvd construct was integrated into a vvdko or a vvdko wc-1ko double mutant to generate qa-vvdmyc (vvdko) and qa-vvdmyc (vvdko wc-1KO) strains, respectively. In these strains VVDMYC is expressed at comparable levels, although VVD levels appeared slightly lower in the qa-vvdmyc (vvdko wc-1KO) strain (Fig. 5C). In these experiments VVD was immunoprecipitated only in strains that express WC-1 protein (Fig. 5B). In another control experiment using qa- vvdmyc (WT) and qa-vvdmyc (127-11) strains, we observed that VVD is immunoprecipitated only in the qa-vvdmyc (WT) strain (Fig. 5D). If VVD interacts with WC-1 via the WCC, we expected VVD also to interact with WC-2. However, our efforts to test this interaction by Co-IP were hampered by unspecific cross-inter- actions of the WC-2 and MYC antisera with VVDMYC and WC- 2, respectively. To test whether VVD must be signaling active for the interaction with WC-1, we tested two mutant strains that lack light-induced activity: vvdC71S, which is biologically inactive because of its in- ability to homodimerize (26), and vvdC108A, a strain in which the formation of a cysteinyl adduct that is critical for light signaling is impaired (18) (Fig. S2C). Both mutant proteins interacted with Fig. 5. VVD interacts with the WCC in the light and in the dark. (A) Protein WC-1, suggesting that VVD does not need to be in a signaling- myc ko ko active state to interact with WC-1. extracts from vvd , vvd , and wc-1 strains were incubated with WC-1 antibody. WC-1 immunoprecipitates VVD in the vvdmyc strain but not in To investigate further whether VVD is part of higher molecular a vvdko or wc-1ko strain. (B) WC-1 antibody does not cross-react with VVD. QA- weight complexes, as suggested by our immunoprecipitation inducible VVD was expressed in a vvdko and a vvdko, wc-1ko background, and experiments, we performed sucrose gradient experiments (Fig. protein extracts coimmunoprecipitated with WC-1 antibody. No VVDMYC was 5E). In agreement with previous results, we detected WC-2 in two immunoprecipitated in a vvdko, wc-1ko background. (C) Control to show that QA-inducible VVDMYC is stably expressed in the absence of WC-1. (D) A WC-1 peaks reported to be about 60 kDa and 200 kDa in size and found MYC + WC-1 to co-migrate with the higher molecular peak identified for antiserum immunoprecipitates QA-induced VVD in DD in a wc-1 strain (qa-vvdmyc). WC-1 antiserum fails to immunoprecipitate VVDMYC in a strain WC-2 (33). FRH migrated in a single peak that overlaps both WC- (127-11) that lacks WC-1 but expresses QA-induced VVDMYC at levels similar to MYC 1 and WC-2. The broad peak of VVD spanning the fractions the control strain qa-vvdmyc.(E) A small proportion of VVD is found in sucrose where monomeric WC-2 migrates is consistent with VVD forming gradient fractions containing WC-1, WC-2, and FRH. Western blot (top five a homodimer, as was suggested recently (24–26). The presence of lanes) and graph (Lower) showing the densitometric analysis of VVDMYC, WC- VVD in fractions that contain the much higher molecular weight 1, WC-2, and FRH from protein extracts size-fractionated on a 10–35% sucrose complexes of WC-1, WC-2, and FRH is consistent with our Co-IP gradient. Fraction 1 corresponds to low molecular weights and fraction 16 to higher molecular weights. Asterisks denote unspecific signals. For densi- data that showed an interaction of VVD with WC-1 and FRH. tometry the maximum signal for each protein was set to 100%. Does VVD interact with DNA-bound WCC? We tested this possibility in EMSA (Fig. S5). Nuclear proteins (LL 4 h) were extracted from vvdko, vvdmyc or vvdgfp strains and incubated with Discussion fi a previously identi ed proximal light-responsive element (pLRE) We have shown that VVD interacts with central components of the located in the frq promoter (12). In line with published results (12, Neurospora circadian clock and with components of blue-light 34, 35), the free probe was caught in a high molecular weight signaling to inhibit the transcriptional activity of the WCC at dawn complex when incubated with the nuclear extracts, and the com- and dusk. The model shown in Fig. 6 depicts how VVD may exert plex was supershifted when incubated with WC-2 antiserum (Fig. its function at the dawn and dusk transitions. We know that WC-1 S5). However, we observed no supershift when extracts were in- and WC-2 form multimers that can bind to proximal and distal cubated with either MYC antibody (Fig. S5A) or GFP antibody GATN cis elements in the frq promoter (12). The complex formed (Fig. S5B), suggesting that VVD binds free rather than DNA- in the dark (WCCD) is faster migrating (i.e., smaller) than the one bound WC-1. formed in the light (WCCL). The latter mediates transcription of

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1009474107 Hunt et al. Downloaded by guest on September 26, 2021 necessarily weak. This situation is reminiscent of the observation that only very small amounts of FRQ seem to interact with the WCC (15). Similarly, the observation that FRQ does not appear to be part of the promoter-bound WCC (12) resembles our observation that antisera against VVDMYC or VVDGFP do not supershift the WCC. However, it is possible that these antibodies are less efficient in EMSA experiments or that a DNA-bound interaction is too weak or transient to be detected by EMSA; therefore, we cannot rule out a DNA-bound interaction at this stage. In- terestingly, VVD mutants that are thought to be defecive in pho- tosignaling still can interact with WC-1 and FRH, suggesting that the repressive functions of VVD in light signaling may occur as part of a complex, with light-activation of VVD affecting the confor- mation of the entire complex. VVD’s role in repressing light responses aside, there is evidence that VVD promotes some aspects of light signaling (18), and a more recent microarray study Fig. 6. Model of VVD’s action in negative feedback regulation of the WCC. (A) suggests that VVD has a role in modulating late light responses in In the dark WC-1 and WC-2 heterodimerize to form a WCCD. Upon light ex- Neurospora (22). It therefore is likely that VVD may have func- posure a multimeric WCC complex, WCCL, forms (11, 14) that mediates tran- tions that do not require its interaction with the WCC or FFC. scription of light-induced genes (e.g., vvd). VVD binds WC-1 to inhibit the Finally, our data show that VVD can interact with FRH in the L efficient formation of WCC , resulting in a decrease of light-induced tran- absence of a functional FFC or WCC, and it is tempting to scription. Without VVD the equilibrium between light and dark complexes is speculate that FRH is the primary platform upon which the shifted toward WCCL, resulting in increased transcription of frq in the light. Upon transfer to DD and without VVD, the increased activity of WCCL results in various complexes assemble. prolonged activation of frq transcription (possibly via its proximal LRE), whereas in the WT FRQ-mediated phosphorylation of WCCD leadstorapid Materials and Methods inactivation of frq transcription. WCCL and WCCD have different preferences Plasmids and Strains. Strain 54–3(bd, a) was used as the WT in all experi- for proximal and distal promoter elements, with WCCD preferentially engaged ments. A detailed description of how strains were created can be found in SI at the distal clock box where negative feedback regulation by FRQ takes place Materials and Methods. All strains were verified using PCR or Southern blot PHYSIOLOGY (11). (B) Simplified schematic of the frq RNA profile after light induction and in analysis. Homokaryons were generated as previously described (37). constant darkness in WT (thick line) and in a vvdKO strain (thin line). The gray area depicts the difference in frq activation between the two strains and is Growth Conditions. Race tube and liquid culture experiments were carried out a result of a change in equilibrium between WCCD and WCCL. Prolonged ac- in Sanyo MLR-350 light- and temperature-controlled chambers as described in tivation in the dark leads to a characteristic phase delay in the onset of frq SI Materials and Methods. For transcription inhibitor experiments, thiolutin transcript oscillations and overt circadian rhythmicity. (Tocris Biosciences) was added at a final concentration of 12 μg/mL. Control samples were treated with DMSO.

light-induced genes, such as vvd (12, 35). Light induction of frq is Co-IP. Co-IP experiments and subsequent Western blot analysis were per- regulated mainly through the proximal light-responsive element formed as described in SI Materials and Methods. (LRE), whereas the distal element, although light responsive, is necessary for sustained rhythmicity of frq in DD (12). We have Cell Fractionation and EMSA. Cellular fractions were isolated essentially as shown here that VVD binds WC-1 both in LL and upon release previously described (38), and details are given in SI Materials and Methods. For EMSA, the frq proximal LRE oligonucleotides (Eurofins MWG Operon) into DD. It thus seems likely that VVD disrupts the efficient for- L sequences described previously (12) were used to make the dsDNA probe. mation of WCC . This disruption would result in a decrease of Binding conditions and gel electrophoresis are described in SI Materials light-induced transcription in LL as well as effective disruption in and Methods. DD of the WCCL that is present at the light–dark transition. In the D L absence of VVD, the equilibrium between WCC and WCC Sucrose Gradients. Protein extracts were obtained using standard protein L would be shifted toward WCC , resulting in increased transcrip- extraction buffer as described previously (30), and sucrose gradient experi- tion of frq in the light as well as during the first hours in the dark. ments were carried out as detailed in S1 Materials and Methods. However, because frq regulates its own transcription via its own gene product, down-regulation of frq in DD is not simply a function RNA Extraction and Northern Blot Analysis. RNA was extracted using the Qiagen of levels and activity of WCC but depends on the level and activity RNeasy mini kit according to the manufacturer’s instructions. Northern blot of FRQ. analysis was carried out as previously described (20) and as detailed in S1 Materials and Methods. Because it is known that (i) FRQ cannot repress its own transcription effectively in the light (36), (ii) the light-induction of frq is Confocal Microscopy. Conidia were fixed in 4% formaldehyde for 1.5 h, controlled largely by the proximal LRE (12), and (iii)WCCL is washed in distilled H2O, incubated in 50 μg/mL DAPI (Sigma) for 10 min at indicative of the light-activated state of WCC (12, 35), it seems room temperature, and then washed and resuspended in 25% glycerol. plausible that FRQ cannot repress its own transcription effectively Details for image collection are given in S1 Materials and Methods. via WCCL but exerts its main repressive activity on WCCD.This possibility could provide an alternative explanation of why frq ACKNOWLEDGMENTS. We thank Sue Crosthwaite (University of Manches- takes longer to decline in a vvdko strain, because the majority of ter, Manchester, UK) for helpful suggestions and critical reading of the L manuscript and Jay Dunlap, Jennifer Loros (Dartmouth Medical School, WCC would be predicted to be in the form of WCC . Hanover, NH), and Yi Liu (University of Texas Southwestern Medical Center, Our experiments suggest that only a small amount of VVD is in Dallas, TX) for the generous gift of FRQ WC-1, WC-2, and FRH antisera. We a complex with WC-1. Because VVD’s impact on light resetting of especially thank Jane Kott for help with the microscopy. Microscopes used in the circadian clock and photoadaptation is quite profound, one this study were purchased with grants from the Biotechnology and Bio- logical Sciences Research Council, the Wellcome Trust, and the University would expect that a larger fraction of VVD must complex with the of Manchester Strategic Fund. This work was supported by Grants BB/ FFC and WCC to achieve its function in these processes. Conse- D00988X/1 and BB/F012055/1 from the Biotechnology and Biological Scien- quently, we favor the idea that the interaction is short-lived but not ces Research Council to C.H.

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