C-Terminal Region of the UV-B Photoreceptor UVR8 Initiates Signaling Through Interaction with the COP1 Protein

C-terminal region of the UV-B photoreceptor UVR8 initiates signaling through interaction with the COP1 protein

Catherine Cloix, Eirini Kaiserli1, Monika Heilmann, Katherine J. Baxter, Bobby A. Brown, Andrew O’Hara, Brian O. Smith, John M. Christie, and Gareth I. Jenkins2

Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom

Edited by Anthony R. Cashmore, University of Pennsylvania, Philadelphia, PA, and approved August 21, 2012 (received for review June 27, 2012) UV-B light initiates photomorphogenic responses in plants. Arabi- UVR8 is a seven-bladed β-propeller protein (8, 10, 11). UVR8 dopsis UV RESISTANCE LOCUS8 (UVR8) specifically mediates these exists as a homodimer in plants and in vitro, which rapidly disso- responses by functioning as a UV-B photoreceptor. UV-B exposure ciates to form monomers following exposure to low doses of UV-B converts UVR8 from a dimer to a monomer, stimulates the rapid (10–12). Recent elucidation of the crystal structure of UVR8 accumulation of UVR8 in the nucleus, where it binds to chromatin, shows that the dimer is maintained by salt-bridge interactions and induces interaction of UVR8 with CONSTITUTIVELY PHOTO- between specific charged amino acids at the dimer interface (10, MORPHOGENIC1 (COP1), which functions with UVR8 to control pho- 11). UV-B is perceived by specific excitonically coupled trypto- tomorphogenic UV-B responses. Although the crystal structure of phans that are adjacent to salt-bridging arginine residues, and UVR8 reveals the basis of photoreception, it does not show how hence photoreception results in breakage of salt bridges leading to UVR8 initiates signaling through interaction with COP1. Here we monomerization. Photoreception leads to the rapid nuclear ac- report that a region of 27 amino acids from the C terminus of cumulation of UVR8 (13) and interaction with the COP1 protein UVR8 (C27) mediates the interaction with COP1. The C27 region is (5, 12). Although necessary for UVR8 function, nuclear localiza- necessary for UVR8 function in the regulation of gene expression tion alone is insufficient to cause expression of target genes; UV-B and hypocotyl growth suppression in Arabidopsis. However, UVR8 exposure is still needed to activate UVR8 in the nucleus (13). lacking C27 still undergoes UV-B–induced monomerization in both COP1 and UVR8 regulate essentially the same set of genes (5, 14) yeast and plant protein extracts, accumulates in the nucleus in re- and this positive regulatory function contrasts with the role of sponse to UV-B, and interacts with chromatin at the UVR8-regulated COP1 as a negative regulator of photomorphogenesis in dark- ELONGATED HYPOCOTYL5 (HY5) gene. The UV-B–dependent inter- grown seedlings (15). In UV-B responses, COP1 is required for the action of UVR8 and COP1 is reproduced in yeast cells and we show stimulation of HY5 gene expression (14), whereas in dark-grown that C27 is both necessary and sufficient for the interaction of UVR8 seedlings COP1 acts as an E3 ubiquitin ligase to promote the de- with the WD40 domain of COP1. Furthermore, we show that C27 struction of HY5 (16). There is no evidence that COP1 acts as an interacts in yeast with the REPRESSOR OF UV-B PHOTOMORPHO- E3 ubiquitin ligase in UV-B photomorphogenic responses, al- GENESIS proteins, RUP1 and RUP2, which are negative regulators though in principle it could act to degrade an unidentified negative of UVR8 function. Hence the C27 region has a key role in UVR8 regulator of the responses. The RUP1 and RUP2 proteins nega- function. tively regulate UV-B photomorphogenic responses and interact directly with UVR8, but COP1 is required for their UV-B–induced V-B wavelengths (280–315 nm) are a minor component of expression, along with UVR8, rather than their degradation (17). Usunlight but have a major impact on living organisms. The The recent determination of UVR8 structure combined with damaging effects of UV-B are well documented, but plants rarely mutational analysis explains how UVR8 acts in photoreception (10, show signs of UV-damage despite constant exposure to sunlight. 11). However, these studies do not show how UVR8 interacts with This is because plants have evolved effective means of protection COP1 to initiate signaling, which is key to understanding UVR8 against UV-B, including the deposition of UV-absorbing phenolic function. Here we identify the region of UVR8 that interacts with compounds in the outer tissues and the production of efficient COP1 and show that it has a crucial role in UVR8 function in vivo. antioxidant and DNA repair systems (1–4). These UV-protective mechanisms are stimulated by low doses of UV-B through dif- Results ferential gene expression. Moreover, low levels of UV-B regulate C-Terminal 27-Amino-Acid Region of UVR8 Is Required for Function in uvr8 other responses in plants, including the suppression of hypocotyl Plants. In a previous mutant screen (6) we isolated several A uvr8-2 extension (5). Thus, in plants, UV-B acts as a key regulatory signal alleles (Fig. S1 ). One allele ( ) has a premature stop codon that initiates photomorphogenic responses and promotes survival. and lacks 40 amino acids at the C terminus of the protein. The uvr8-2 B The low dose, photomorphogenic responses to UV-B are truncated protein is detectable in plants (Fig. S1 ) but the CHS mediated by the photoreceptor UVR8 (3–7). UVR8 acts spe- mutant fails to induce UVR8-regulated transcripts in re- C cifically in UV-B to regulate over 100 genes, many of which are sponse to UV-B exposure (Fig. S1 ). A 27-amino-acid region from involved in UV protection (5, 6). Arabidopsis uvr8 mutant plants are highly sensitive to UV-B because they fail to express UV- protective genes (6, 8). Among the genes regulated by UVR8 is Author contributions: G.I.J. designed research; C.C., E.K., M.H., K.J.B., B.A.B., and A.O. performed research; C.C., E.K., M.H., B.O.S., J.M.C., and G.I.J. analyzed data; and G.I.J. that encoding the ELONGATED HYPOCOTYL 5 (HY5) wrote the paper. transcription factor, which mediates most, if not all, gene ex- The authors declare no conflict of interest. pression responses initiated by UVR8 (6, 7). UVR8 interacts This article is a PNAS Direct Submission. HY5 with chromatin via histones, in particular H2B (9) at the 1Present address: Chory Laboratory, Plant Biology, The Salk Institute for Biological Stud- gene (6, 9) and a number of other UVR8-regulated genes (9), ies, La Jolla, CA 92037. which raises the possibility that UVR8 promotes recruitment or 2To whom correspondence should be addressed. E-mail: [email protected]. activation of transcription factors and/or chromatin remodeling This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. proteins that regulate target genes such as HY5. 1073/pnas.1210898109/-/DCSupplemental.

16366–16370 | PNAS | October 2, 2012 | vol. 109 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1210898109 Downloaded by guest on October 1, 2021 the C terminus of UVR8 (C27; amino acids 397–423) contains required for function, it is not required for UVR8 to bind to a number of amino acids that are highly conserved among UVR8 chromatin at a target gene locus. proteins from different plant species (boxed in Fig. S2), but not UV-B promotes the conversion of UVR8 from a dimer to present in sequences related to UVR8, such as eukaryotic RCC1 a monomer in vitro (10), in plants and when it is expressed het- (8). The fact that these amino acids are lacking in the uvr8-2 mu- erologously in yeast (12). Fig. 2B shows the effect of UV-B illu- tant led us to investigate their role in UVR8 function. mination on HA-tagged UVR8 in protein extracts of yeast. UVR8 lacking specifically the C27 region, with a translational Following UV-B illumination of the extracts, proteins were re- GFP fusion at the N terminus was expressed in uvr8-1 null mutant solved by native gel electrophoresis and HA–UVR8 was detected plants under the control of the native UVR8 promoter (Fig. 1A). by an anti-HA antibody. The HA–UVR8 dimer is present before The level of expression of the GFP–ΔC27UVR8 fusion in trans- illumination and the monomer is detectable after 5 min illumination genic plants (Fig. 1B) was similar to that of a wild-type GFP– with a relatively low fluence rate of UV-B. Longer exposures gen- UVR8 fusion that was shown previously to complement the im- erate increasing amounts of the monomer while the amount of di- paired UV-B induction of gene expression in uvr8-1 (13) (Fig. 1C). mer decreases. The same result is observed with yeast expressing However, as shown in Fig. 1C,GFP–ΔC27UVR8 fails to com- HA–ΔC27UVR8. plement uvr8-1 in that no UV-B induction of UVR8-regulated UVR8 monomerization is observed when plant extracts are HY5 transcripts is observed in three independent transgenic lines. exposed specifically to UV-B wavelengths (Fig. S4) and analyzed Moreover, uvr8-1 plants transformed with GFP–ΔC27UVR8 are by SDS/PAGE without boiling to denature the samples (12). The highly sensitive to UV-B, similar to the uvr8-1 mutant (Fig. S3). UVR8 dimer is very resistant to SDS when not boiled (10–12) and Therefore, the C27 region is required for UVR8 function in vivo. only dissociates in the absence of UV-B under low pH (10) or high To further examine the role of the C27 region in UVR8 function salt (11). As shown in Fig. 2C, before UV-B illumination of – –Δ we measured the suppression of hypocotyl extension in response to extracts, GFP UVR8 and GFP C27UVR8 were present pre- UV-B, which is mediated by UVR8 (5). Whereas uvr8-1 plants dominantly as the dimer, whereas following UV-B exposure the expressing GFP–UVR8 exhibit hypocotyl suppression in UV-B, monomer increased in abundance. These experiments show that similar to wild-type, those expressing GFP–ΔC27UVR8 have the C27 region of UVR8 is not required for the primary effect of similar hypocotyl length to uvr8-1 (Fig. 1D), demonstrating that UV-B exposure on the protein in vivo, namely conversion from the C27 region is required for UVR8 to mediate the response. a dimer to a monomer. –Δ Hypocotyl growth suppression by UV-B additionally requires Although GFP C27UVR8 is not functional, it nevertheless

D uvr8-1 still accumulates in the nucleus in response to UV-B illumination PLANT BIOLOGY HY5/HYH (Fig. 1 ), so the lack of response in plants and – in uvr8-1 expressing GFP–ΔC27UVR8 may result from impaired of plants. In previous experiments, we showed that GFP UVR8 is HY5/HYH gene expression (Fig. 1C) (7). detectable in about half of the nuclei of plants not exposed to UV- B but that UV-B stimulates a rapid increase in both the fraction of fl fl C27 Region of UVR8 Is Not Required for Interaction with Chromatin, nuclei showing GFP uorescence and the brightness of the uo- D UV-B–Dependent Monomerization, or Nuclear Accumulation. GFP– rescence (13). A very similar response is shown in Fig. 2 for –Δ fi fl UVR8 (and native UVR8) binds to chromatin at several genes it GFP C27UVR8. Quanti cation of GFP uorescence in nuclei fi regulates, including the HY5 gene (9). Chromatin immunopre- identi ed by DAPI staining shows that UV-B exposure increases –Δ ∼ cipitation with an anti-GFP antibody demonstrates that both the fraction of nuclei containing GFP C27UVR8 from 60% to E GFP–ΔC27UVR8 and GFP–UVR8 associate with chromatin over 90% (Fig. 2 ). containing the HY5 promoter region but not the control ACTIN2 C27 Region of UVR8 Is Required for Interaction with COP1 in Plants gene (Fig. 2A). Therefore, although the C27 region of UVR8 is and Yeast. UVR8 interacts with COP1 in a UV-B–dependent manner in plants (5). Here the interaction was tested by immunoprecipitation of GFP–UVR8 and analysis of the immunopre- A C GFP- C27UVR8 GFP- cipitate for the presence of COP1 using an anti-COP1 antibody. Ler uvr8-1 4.3 11.2 7.2 UVR8 As shown in Fig. 3A, for plants expressing GFP–UVR8, COP1 is - + - + - + - + - + - + UV-B eGFP UVR8 C27 HY5 not detectable in the immunoprecipitate obtained from dark- UVR8pro treated plants but is present following UV-B exposure, indicating ACTIN2 interaction with GFP–UVR8. In contrast, COP1 was not de- B D tectable in immunoprecipitates of GFP–ΔC27UVR8 from either GFP- dark-treated or UV-B–exposed plants. Therefore, the C27 re- GFP- C27UVR8 UVR8 4.3 11.2 7.2 uvr8-1 gion of UVR8 is required for interaction with COP1 in plants. kDa GFP- To facilitate further study of the interaction between UVR8 83 UVR8 and COP1, experiments were undertaken in yeast. The interaction in yeast is specific to UV-B wavelengths and is not rbcL mediated by the yeast DNA damage signaling pathway (Fig. S5). 46 As shown in Fig. 3B, deletion of the C27 region prevents in- Fig. 1. The GFP–ΔC27UVR8 fusion does not functionally complement teraction of UVR8 with COP1, consistent with the results transgenic uvr8-1 plants. (A) The GFP–ΔC27UVR8 fusion protein lacking obtained in plants. The lack of interaction cannot be explained UVR8 amino acids 397–423 coupled to the UVR8 promoter. (B) Western blot by a failure of the yeast cells to express the proteins, as each A of whole cell extracts from uvr8-1 plants expressing UVR8pro:GFP–UVR8 or protein was detectable (Fig. S6 ). The C27 region alone inter- –Δ UVR8pro:GFP C27UVR8 (lines 4.3, 11.2, and 7.2) probed with anti-GFP an- acts with intact COP1 but the UV-B dependence is lost; in- tibody. Ponceau staining of Rubisco large subunit (rbcL) is shown as a load- teraction is seen in both darkness and UV-B. Intact UVR8 ing control. (C) RT-PCR assays of HY5 and control ACTIN2 transcripts in Ler, interacts with the WD40 region of COP1 (the C-terminal 341 –Δ uvr8-1, uvr8-1/UVR8pro:GFP C27UVR8 (lines 4.3, 11.2, and 7.2) and uvr8-1/ – −2 −1 amino acids) in a UV-B dependent manner. The C27 region UVR8pro:GFP–UVR8 (line 6.2) (13) plants grown under 20 μmol m s white − μ −2 −1 + interacts with the WD40 region of COP1, but again not in a UV- light ( ) and exposed to 3 mol m s broadband UV-B for 4 h ( ). (D) – – Hypocotyl lengths (±SE, n = 10) for 4-d-old wild-type Ler, wild-type Ws, uvr8- B dependent manner. These results show that a UV-B de-

1, uvr8-1/UVR8pro:GFP–UVR8, uvr8-1/UVR8pro:GFP–ΔC27UVR8 (line 4.3), and pendent interaction requires intact UVR8, whereas intact COP1 hy5-ks50 hyh (Ws background) plants grown in 1.5 μmol m−2 s−1 white light is not sufﬁcient, consistent with UVR8 acting as the UV-B (−UV-B) supplemented with 1.5 μmol m−2 s−1 narrowband UV-B (+UV-B). photoreceptor for the response. We further conclude that the

Cloix et al. PNAS | October 2, 2012 | vol. 109 | no. 40 | 16367 Downloaded by guest on October 1, 2021 initiated by interaction with COP1, is poorly understood. The A B HA-UVR8 HA- C27UVR8

GFP-UVR8 GFP- C27UVR8 - - - - data presented here show that the C27 region of UVR8 is key to kDa 0.5 5 10 30 0.5 5 10 30 min 146 understanding UVR8 signaling. The C27 region is not present in IN IN Mock Mock GFP GFP GFP D structurally related proteins such as RCC1 and is not present in HY5pro 66 Arabidopsis ACTIN2 M other proteins, including those with moderate sequence similarity to UVR8. However, the C27 region contains stretches of amino acids that are highly conserved in UVR8 C GFP-UVR8 GFP- C27UVR8 D E kDa - + - + UV-B sequences from various plant species, including lower plants, 100 175 -UV-B consistent with its importance in UVR8 function. Both the uvr8- D 2 83 allele, which lacks the C-terminal 40 amino acids of the pro- M 50 tein, and transgenic uvr8-1 plants expressing the GFP– +UV-B 47 rbcL %GFP/DAPI 0 -UV-B +UV-B

Fig. 2. The C27 region of UVR8 is not required for chromatin association, UV- A GFP-UVR8 GFP- C27UVR8 B–induced monomerization or nuclear accumulation. (A) Chromatin immu- - + - + UV-B noprecipitation assays of DNA associated with GFP–UVR8 or GFP–ΔC27UVR8. IN IP IN IP IN IP IN IP PCR of the HY5 promoter (−331 to +23) and control ACTIN2 DNA from uvr8-1/

UVR8pro:GFP–UVR8 and uvr8-1/UVR8pro:GFP–ΔC27UVR8 (line 11.2) plants − − − − COP1 grown under 20 μmol m 2 s 1 white light and exposed to 3 μmol m 2 s 1 broadband UV-B for 4 h. IN, input DNA before immunoprecipitation; GFP, DNA GFP-UVR8 immunoprecipitated by anti-GFP antibody; mock, no antibody control. (B) Western blots of whole cell extracts from yeast expressing HA-UVR8 (Left)or HA-ΔC27UVR8 (Right) probed with anti-HA antibody. The yeast extracts were − − B UV-B C UV-B treated (+) or not (−)with1μmol m 2 s 1 narrowband UV-B for the times BD AD - + BD AD - + shown. Samples were run on an 8% native gel. The UVR8 dimer (D) and monomer (M) are indicated. (C) Western blot of whole cell extracts from uvr8- p53 Ant T p53 Ant T 1/UVR8 :GFP–UVR8 and uvr8-1/UVR8 :GFP–ΔC27UVR8 (line 11.2) plants pro pro - - probed with anti-GFP antibody. Extracts were treated (+) or not (−) with 4 μmol - - m−2 s−1 narrowband UV-B for 30 min before SDS-loading buffer was added. UVR8 RUP1 Samples were then run on a 7.5% SDS/PAGE gel without boiling. Ponceau UVR8 RUP2 staining of Rubisco large subunit (rbcL) is shown as a loading control. The UVR8 UVR8 COP1 fl dimer (D) and monomer (M) are indicated. (D) Confocal images of GFP uo- C27UVR8 COP1 C27UVR8 RUP1 rescence in leaf epidermal tissue of uvr8-1/UVR8pro:GFP–ΔC27UVR8 (line 11.2) − − plants grown under 20 μmol m 2 s 1 white light (−UV-B) and exposed to 3 μmol C27-UVR8 COP1 C27UVR8 RUP2 m−2 s−1 broadband UV-B for 4 h (+UV-B). (Scale bars, 20 μm.) (E) Percentage of UVR8 WD40 C27-UVR8 RUP1 nuclei identified by DAPI fluorescence in uvr8-1/UVR8pro:GFP–ΔC27UVR8 (line − − 11.2) plants showing colocalization of GFP fluorescence under 20 μmol m 2 s 1 − − C27-UVR8 WD40 C27-UVR8 RUP2 white light (−UV-B) and following exposure to 3 μmol m 2 s 1 broadband UV-B for 4 h (+UV-B). Data are the mean ± SE (n = 20 images). D - UV-B + UV-B -1 -2 -3 -1 -2 -3 C27 region of UVR8 is both necessary and sufficient for in- BD AD 1 10 10 10 1 10 10 10 teraction with COP1 via its WD40 domain. p53 Ant T

C27 Region of UVR8 Mediates Interaction with RUP1 and RUP2. RUP1 - - and RUP2 negatively regulate UVR8 function in plants and in- UVR8 COP1 teract directly with UVR8 in the presence and absence of UV-B (17). Because RUP1 and RUP2 are small WD40 proteins and UVR8 RUP1 C27 interacts with the WD40 domain of COP1, we tested UVR8 RUP2 whether C27 interacts with RUP1 and RUP2. As shown in Fig. 3C, UVR8 interacts with RUP1 and RUP2 in the presence or Fig. 3. The C27 region of UVR8 is necessary and sufﬁcient for interaction with absence of UV-B in yeast, although examination of a dilution both the WD40 region of COP1 and RUP proteins. (A) The ΔC27UVR8 protein series indicates that the interaction is stronger in the presence of does not interact with COP1 in plants. Whole cell extracts were obtained from D UV-B (Fig. 3 ). Moreover, taking into account the similar levels uvr8-1/UVR8pro:GFP–UVR8 and uvr8-1/UVR8pro:GFP–ΔC27UVR8 (line 4.3) − − of expression of COP1, RUP1, and RUP2 in the yeast cells (Fig. plants treated (+) or not (−) with 3 μmol m 2 s 1 narrowband UV-B. Coim- S6B), the interaction between UVR8 and COP1 in UV-B munoprecipitation assays were carried out under the same illumination con- μ appears to be at least as strong as between UVR8 and the RUP ditions. Input samples (15 g, IN) and eluates (IP) were fractionated by SDS/ PAGE and Western blots were probed with anti-COP1 and anti-GFP antibodies. proteins (Fig. 3D). However, UVR8 lacking C27 fails to interact C (B) Yeast two-hybrid plasmids containing a DNA binding domain (BD) and an with either RUP1 or RUP2 (Fig. 3 ), although the proteins were activation domain (AD) fused to the proteins indicated were cotransformed in expressed in the cells (Fig. S6A). C27 alone interacts with both yeast, which were then spotted on fully selective media plates. All colonies RUP1 and RUP2. Therefore, C27 is both necessary and sufﬁ- grew on nonselective media (not shown). As controls, yeast were cotrans- cient to mediate an interaction between UVR8 and the RUP formed with plasmids containing mammalian p53 and antigen T (positive proteins that is independent of UV-B. control) or no inserts (−, negative control). ΔC27UVR8 lacks amino acids 397– 423 of UVR8 (Fig. 1A). Construct C27–UVR8 contains only these 27 amino acids Discussion of UVR8. WD40 corresponds to the 341 C-terminal amino acids of COP1 (334– 675). Yeast were left to grow in darkness (−) or under 0.1 μmol m−2 s−1 nar- Although recent research has established the physiological im- rowband UV-B (+). (C) Yeast two-hybrid assay undertaken as in B with RUP1 portance of UVR8 and the structural basis of its action as and RUP2. (D) Yeast two-hybrid assay undertaken as in B except that yeast a photoreceptor, the mechanism of UVR8 signaling, which is were spotted onto plates in a serial dilution.

16368 | www.pnas.org/cgi/doi/10.1073/pnas.1210898109 Cloix et al. Downloaded by guest on October 1, 2021 ΔC27UVR8 fusion show no detectable UV-B induction of HY5 COP1 interacts with various other components involved in or CHS transcripts. It is known that HY5, sometimes acting re- regulating photomorphogenesis, including the cry1 photoreceptor dundantly with the closely related HYH (HY5 HOMOLOG) (19, 20), SPA proteins (21), CULLIN4, and DAMAGED DNA transcription factor (7, 18), has a key role in mediating UVR8- BINDING PROTEIN1 (DDB1) (22, 23). Hence UVR8 could regulated gene expression (5, 6), and hence the loss of HY5 possibly interact with other photomorphogenic proteins via its expression in plants lacking C27 will impair responses. Thus, association with COP1 or influence the interaction of COP1 with uvr8-1 plants expressing GFP–ΔC27UVR8 are both highly sen- other components. For instance, the interaction of COP1 with sitive to UV-B, likely because of a general absence of UVR8- UVR8 might reduce its availability to act as an E3 ubiquitin ligase regulated gene expression, and impaired in the suppression of in association with other proteins (23). Moreover, interaction with hypocotyl growth by UV-B, similar to uvr8-1 (5, 6, 8) and hy5 (6) UVR8 following UV-B exposure could possibly inhibit the E3 or hy5 hyh (7) mutants. ubiquitin ligase activity of COP1 and contribute to the light in- The loss of function of GFP–ΔC27UVR8 is not because de- activation of COP1, which is not fully understood (15). Thus, letion of the C-terminal region of UVR8 leads to instability and further research is required to establish both how the interaction of degradation of the protein, as the mutant protein is expressed in UVR8 with COP1 leads to transcriptional regulation in photo- both uvr8-2 and transgenic GFP–ΔC27UVR8 plants. The non- morphogenic UV-B responses and how it impacts on the function functional GFP–ΔC27UVR8 protein still forms a dimer, as in the of COP1 in other photomorphogenic responses. wild-type, indicating that the C27 region is not required for in- RUP1 and RUP2 are WD40 repeat proteins, like COP1 (17), teraction between UVR8 molecules. Moreover, the GFP– and the data presented here indicate that they interact with the ΔC27UVR8 protein is converted to a monomer following UV-B C27 region of UVR8, which also binds COP1. Hence, in addition exposure in both yeast and plants, and so the primary effect of UV- to its key role in binding COP1 to initiate signaling, the C27 region B on the protein is not prevented by the C27 deletion. Similar is important in the regulation of UVR8 activity through its ability findings are obtained with purified trypsin-treated UVR8 lacking to interact with other WD40 proteins. Although the interaction of 40 amino acids at the C terminus (10). Furthermore, the GFP– RUP1 and RUP2 with C27 is stimulated by UV-B in yeast, both ΔC27UVR8 protein accumulates in the nucleus of plants in re- proteins bind to UVR8 in the absence of UV-B, in contrast to sponse to UV-B and interacts with chromatin at the HY5 gene COP1, whose interaction is dependent on UV-B. As discussed similar to wild-type UVR8. Thus, the C27 region is not required for above, COP1 is unable to access C27 in the UVR8 dimer. The nuclear accumulation of UVR8 and its interaction with histones. much smaller size of the RUP proteins does not explain their The data presented here show that the C27 region is required for stronger interaction with the C27 region in darkness, because the PLANT BIOLOGY the interaction of UVR8 with COP1, which is pivotal to photo- WD40 region of COP1 alone, which is similar in size to the RUP – B morphogenic UV-B responses. The ΔC27UVR8 protein fails to proteins, shows a UV-B dependent interaction with C27 (Fig. 3 ). interact with COP1 in either yeast or plants. Although COP1 is Hence, there are likely differences in the primary sequence or required for the induction of gene expression by UVR8, it is not structure of the RUPs compared with the WD40 region of COP1 required for either monomerization (12) or the interaction of that enable them to interact with the C27 region in dimeric UVR8. UVR8 with chromatin (5), consistent with the findings reported It is not clear how the RUP proteins repress UV-B photomor- here for plants expressing GFP–ΔC27UVR8. The observation that phogenic responses in vivo. The data presented here suggest that the GFP–ΔC27UVR8 protein accumulates in the nucleus in re- COP1 and the RUP proteins have similar strength of interaction sponse to UV-B indicates that this process also does not require with C27 in yeast in the presence of UV-B, so the RUPs may not COP1. The experiments in yeast show that the C27 region is both impair interaction of COP1 with UVR8 in plants by having greater binding affinities for C27. Because the RUPs are UV-B induced, necessary and sufficient for interaction with the WD40 region of an increase in their abundance relative to COP1 may limit binding COP1. Holm et al. (18) identified a motif in HY5 and several other of COP1 to C27. However, more detailed study of the amounts, proteins that is responsible for interaction with the WD40 region availability, and binding affinities of the proteins in planta is of COP1. This motif (VPE/D-hydrophobic residue-G with several needed to understand the mechanism of negative regulation. upstream negatively charged residues) is partly conserved in the C27 region of UVR8 (VPDETG with one upstream glutamate Methods residue) and could be involved in the interaction with COP1. Plant Materials, Treatments, and Assays. Wild-type Arabidopsis thaliana There is no reason to suppose that yeast itself contains a pho- ecotypes Landsberg erecta (Ler) and Wassilewskija (Ws), the uvr8-1 mutant toreceptor that would mediate the UV-B–dependent interaction (Ler background) (8) and hy5-ks50 hyh double mutant (Ws background) (18) between UVR8 and COP1. Yeast is very unlikely to have a protein were used in these experiments. All transgenic lines were produced in the that could substitute as a plant UV-B photoreceptor and the ac- uvr8-1 mutant. Details of seed origin, production of the GFP–ΔC27UVR8 tivation of DNA damage signaling is not responsible for initiating construct (UVR8 lacking amino acids 397–423), plant transformation, and the interaction between UVR8 and COP1. This interaction is growth conditions are given in SI Methods. Plants were exposed to either induced specifically by UV-B wavelengths and requires intact broadband UV-B (spectrum in Fig. S7A) or narrowband UV-B (spectrum in Fig. S7B), as indicated in Fig. S7 legends. UVR8 acting as the photoreceptor. Intact UVR8 is able to me- – RT-PCR experiments, ChIP assays, and GFP–UVR8 subcellular localization diate a UV-B dependent interaction with the WD40 region of analyses were undertaken as described in refs. 7, 9, and 13, respectively. COP1 alone, whereas intact COP1 is unable to mediate a UV-B- dependent interaction with the C27 region alone, and thus C27 UVR8 Dimer/Monomer Analysis. UVR8 fused to the HA epitope was expressed binds to COP1 (and specifically to the WD40 domain) in both in yeast and whole cell extracts were prepared as described in SI Methods. − − darkness and following UV-B exposure. It is interesting that the Aliquots of the extracts were exposed to 1 μmol m 2 s 1 narrowband UV-B. C27 region alone binds constitutively to COP1, whereas intact Proteins were fractionated on a 8% (wt/vol) native PAGE gel and Western UVR8 binds to COP1 only following UV-B exposure. It is possible blots probed with anti-HA antibody (Cell Signaling Technology; 2367). that the C27 region is shielded from COP1 in intact UVR8, which is Plants were grown on agar plates containing half-strength Murashige μ −2 −1 in the dimeric form in darkness, and that UV-B–induced mono- and Skoog (MS) salts under 100 mol m s constant white light at 21 °C merization, together with associated conformational changes, for 10 d then put in darkness for 16 h. Arabidopsis whole cell extracts were prepared as described (13) and monomerization of UVR8 in the extracts was exposes the C27 region to COP1. The crystal structure of UVR8 examined essentially as described (12). Whole cell extracts were kept on ice − − (10, 11) does not include the C-terminal 40 amino acids and hence in the absence or presence of 4 μmol m 2 s 1 narrowband UV-B for 30 min. it is important to determine where the C27 region resides in the Four times loading buffer [250 mM Tris·HClpH6.8,2%(wt/vol)SDS,20% protein and whether its position changes after UV-B exposure. (vol/vol) β-mercaptoethanol, 40% (vol/vol) glycerol, 0.5% bromophenol

Cloix et al. PNAS | October 2, 2012 | vol. 109 | no. 40 | 16369 Downloaded by guest on October 1, 2021 blue] was added to the samples. The proteins were loaded on a 7.5% (wt/ microcolumn was equilibrated using 200 μL high-salt lysis buffer [450 mM vol) SDS/PAGE gel without boiling. Western blots were incubated with anti- NaCl, 1% Triton (vol/vol), 50 mM Tris·HCl pH 8, 5 mM PMSF, protease GFP antibody as described (13). The data shown are representative of at inhibitors, Complete Mini, 11836153001; Roche]. The lysate was applied onto least three independent experiments. the column and left to run through. After ﬁve washes with 200 μLofhigh- salt lysis buffer and one wash with 300 mM NaCl, Tris·HCl pH 7.5, 20 μLof Yeast Two-Hybrid Assay. Proteins were cloned as fusions with either the DNA elution buffer (0.1 M triethylamine pH 11.8, 0.1% Triton X-100) was applied binding domain or activation domain of the yeast transcription factor GAL4 onto the column and left for 5 min at room temperature. An extra 50 μLof (primers used for generating fusion proteins are listed in Table S1). Vectors containing the fusions were transformed into yeast and interaction between elution buffer was added and the eluate was collected in a tube containing 3 μ proteins was assayed by growth on fully selective medium for 3 d at 30 °C L of 1 M Mes, pH 3 for neutralization. The eluates were analyzed by SDS/ − − either in darkness or under 0.1 μmol m 2 s 1 narrowband UV-B. Controls for PAGE gel and Western blot using antibodies anti-GFP or anti-COP1 (kindly autoactivation are shown in Fig. S8. The data presented are representative provided by Nam-Hai Chua, The Rockefeller University, New York) (24). Data of at least three independent experiments. Full details are given in are representative of at least three experiments. SI Methods. Full details of all materials and methods are given in SI Methods.

Coimmunoprecipitation of UVR8 and COP1. Plants were grown on agar plates ACKNOWLEDGMENTS. We thank Dr. Roman Ulm for sharing data on UVR8 − − containing half-strength MS salts under 100 μmol m 2 s 1 constant white monomerization prior to publication and guidance with deﬁning the COP1 light at 21 °C for 12 d then put in darkness for 16 h. The plants were treated WD40 domain, Drs. In-Cheol Jang and Nam-Hai Chua for sending the COP1 − − for 3 h with 3 μmol m 2 s 1 narrowband UV-B. Arabidopsis whole cell antibody, Jane Findlay for technical assistance, and Jane Forrester for extracts were prepared as described in ref. 13 in the absence or presence of 3 preliminary experiments with the RUP proteins. This research was supported − − μmol m 2 s 1 narrowband UV-B. The coimmunoprecipitation assays were by grants from the UK Biotechnology and Biological Sciences Research carried out in the same conditions. Samples were incubated for 30 min on ice Council and The Leverhulme Trust. E.K. was supported by a University of with 50 μL anti-GFP microbeads (μMacs, 130–091-370; Miltenyi Biotec). The Glasgow doctoral studentship.

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