The photomorphogenic repressors COP1 and DET1: 20 years later

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1 Review The photomorphogenic repressors and DET1: 20 years later On Sun Lau 1,2 and Xing Wang Deng 1 1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT , USA 2 Department of Biology, Stanford University, Stanford, CA , USA and DET1 are among the first repressors of photomorphogenesis to be identified, more than 20 years ago. Discovery of these repressors as conserved regulators of the ubiquitin-proteasome system has established protein degradation as a central theme in light signal transduction. is a RING E3 ubiquitin ligase that targets key regulators for degradation, and DET1 complexes with 0 and, which is proposed to aid in -mediated degradation. Recent studies have strengthened the role of as a major signaling center. DET1 is also emerging as a chromatin regulator in repressing gene expression. Here, we review current understanding on and DET1, with a focus on their role as part of two distinct, multimeric -based E3 ligases. COP/DET/FUS: from nine genetic loci to three protein complexes and DET1 are founding members of a group of genes termed the CONSTITUTIVE PHOTOMORPHOGENIC/ DE-ETIOLATED/FUSCA (COP/DET/FUS) [1,2]. These loci were identified through genetic screens in Arabidopsis (Arabidopsis thaliana) for seedling mutants that display light-grown phenotypes in darkness (COP/DET) or seeds that accumulate high levels of anthocyanin (FUS). Nine of the COP/DET/FUS loci were subsequently cloned, and they encode, DET1, 0 and COP9 signalsome (CSN) subunits 1 to 4, 7 and 8. These proteins were found to be important regulators in the ubiquitin ()-proteasome system and are conserved in other eukaryotes including humans. Besides photomorphogenesis, the COP/DET/ FUS proteins also play key roles in many other biological processes, as evident from the pleiotropic and lethal phenotypes displayed in their mutants in plants, and invertebrate and vertebrate animals [1 6]. Extensive biochemical and genetic studies have now defined the COP/DET/FUS proteins as constituents of three distinct protein complexes: the SUPPRESSOR OF PHYA-105 (SPA) complex, the CSN and the 0 DET1 (CDD) complex (Table 1)., which is a part of a 700 kda complex, is a RING E3 ligase that mediates the ubiquitination of a myriad of substrates for degradation by the proteasome [5]. CSN, which consists of six of the COP/ DET/FUS proteins, is a protease with eight distinct subunits that regulates all CULLIN-RING E3 ligases (CRL) through its NEDD8/RUB1 isopeptidase activity [3]. The CDD Corresponding author: Deng, X.W. (xingwang.deng@yale.edu). complex, which includes the remaining two members of the COP/DET/FUS protein group, consists of 0, DET1 and DAMAGED DNA BINDING PROTEIN 1 () [7,8]. It has been 20 years since the revelation of the molecular identity of the first gene from the COP/DET/FUS loci [9]. This study not only represented the beginning of the cloning and identification of a set of evolutionarily conserved regulators, but also led to a paradigm of light signal transduction signaling through targeted protein degradation. Here, we review our current understanding of and DET1 in Arabidopsis, focusing on the two -based E3 complexes they constitute. We begin with an update on the central role played by in the light signaling pathway and how regulates flowering and the circadian rhythm. We then discuss the functional unit of, the SPA complex, and the recent finding that it acts as a substrate receptor for and forms the multimeric SPA E3 ligase. Finally, we review the unorthodox DET1 E3 complex, CDD ligase, and present the latest discovery that DET1 plays a direct role in transcriptional repression. Owing to the scope of this article, readers interested in the CSN, which is expected to contribute to the function of and DET1 as a general regulator, are encouraged to consult these specific reviews [2,3,10,11]. : an E3 ubiquitin ligase at the heart of light signaling was the first cloned COP/DET/FUS locus and has been one of the best characterized among them [5,9]. In plants, the function of is closely tied to the light signaling pathway. acts as a central repressor in the pathway, where it promotes the ubiquitination and degradation of the positive regulators and is itself regulated by multiple photoreceptors (Figure 1). Besides seedling photomorphogenesis [5], research in the past five years has strengthened and expanded the role of in other processes, most of which are light-regulated responses. These processes include flowering [12 14], circadian rhythm [14], UV-B signaling [15,16], stomatal opening and development [17,18], shade avoidance response [19], plant defense [20], crosstalk between light and brassinosteroid (BR) signaling [21], cold acclimation response [22] and light-induced root elongation [23] in Arabidopsis, and juvenile adult phase change in rice (Oryza sativa) [24]. In many of these processes, the molecular involvement of is direct interaction and ubiquitination of the key /$ see front matter ß 2012 Elsevier Ltd. All rights reserved. Trends in Plant Science xx (2012)

2 Table 1. Components of the three COP/DET/FUS complexes and the core E3 ubiquitin ligase in Arabidopsis Symbol AGI code Full name Phenotype of dark-grown mutant seedlings Refs SPA complex a AT2G32950 CONSTITUTIVE PHOTOMORPHOGENIC 1 cop phenotype (light-grown like) b [9,93] SPA1 AT2G46340 SUPPRESSOR OF PHYA cop phenotype in the quadruple spa1 spa2 spa3 spa4 mutant; [72] SPA2 AT4G11110 SPA1-RELATED 2 milder cop phenotype in the triple and double mutants SPA3 AT3G15354 SPA1-RELATED 3 SPA4 AT1G53090 SPA1-RELATED 4 0 DET1 (CDD) complex 0 AT3G13550 CONSTITUTIVE PHOTOMORPHOGENIC 10 cop phenotype [82,94] DET1 AT4G10180 DE-ETIOLATED 1 cop phenotype [81,95] a AT4G05420 DAMAGED DNA BINDING PROTEIN 1A Wild-type phenotype, but enhances cop phenotype of det1 [8] b AT4G21100 DAMAGED DNA BINDING PROTEIN 1B Embryonic lethal COP9 signalsome (CSN) CSN1 AT3G61140 COP9 SIGNALOSOME SUBUNIT 1 cop phenotype [2] CSN2 AT2G26990 COP9 SIGNALOSOME SUBUNIT 2 cop phenotype CSN3 AT5G14250 COP9 SIGNALOSOME SUBUNIT 3 cop phenotype CSN4 AT5G42970 COP9 SIGNALOSOME SUBUNIT 4 cop phenotype CSN5a AT1G22920 COP9 SIGNALOSOME SUBUNIT 5a cop phenotype in the csn5a csn5b double mutant [2,96] CSN5b AT1G71230 COP9 SIGNALOSOME SUBUNIT 5b CSN6a AT5G56280 COP9 SIGNALOSOME SUBUNIT 6a cop phenotype in the csn6a csn6b double mutant [2,96] CSN6b AT4G26430 COP9 SIGNALOSOME SUBUNIT 6b CSN7 AT1G02090 COP9 SIGNALOSOME SUBUNIT 1 cop phenotype [2] CSN8 AT4G14110 COP9 SIGNALOSOME SUBUNIT 1 cop phenotype Core ligase c AT5G46210 CULLIN4 cop phenotype in knockdown mutants [79,85] a AT5G20570 RING-BOX 1a Not tested in loss of function mutant; weak de-etiolation [97] in overexpression lines b AT3G42830 RING-BOX 1b Not tested a The SPA complex is likely to be a tetramer of two s and two SPA proteins. b cop phenotype: mutants undergo constitutive photomorphogenesis (cop) in darkness, which includes the development of a short hypocotyl and open cotyledons. c For its component, please refer to the a/b entries under CDD complex. factors (Table 2). These studies have consolidated as a vital regulatory point in light signal transduction. In this section, we review the current state of in the light signaling pathway and how it specifically regulates flowering and the circadian clock. Light signaling pathway Light is an important environmental signal that controls many developmental processes in plants [25]. Central to these light-regulated processes is a light signaling pathway that connects light signals to gene expression. From the model of seedling photomorphogenesis, the COP/DET/FUS proteins were identified as negative regulators of light signaling. A major breakthrough in addressing how the COP/DET/FUS proteins control the developmental program was the discovery that is a E3 ligase that mediates the degradation of various photomorphogenesis-promoting transcription factors by the -proteasome system, with the other COP/DET/FUS proteins likely to aid the process [26 28]. In darkness, targets these transcription factors, including ELONGATED HYPOCOTYL 5 (), LONG AFTER FAR-RED LIGHT 1 (LAF1) and LONG HYPOCOT- YL IN FAR RED (HFR1) for ubiquitination and degradation, leading to suppression of photomorphogenesis (Figures 1a and 2a) [26,27,29 32]. Recent studies also highlight the importance of several B-box Zinc Finger proteins (BBXs), including BBX4/COL3 and BBX22/LZS1/STH3, and a BR-regulated GATA transcriptional factor, GATA2, in promoting photomorphogenesis, and they are also targeted by for degradation [21,33 35]. In the presence of light, activated photoreceptors repress function and allow the accumulation of the photomorphogenesis-promoting transcription factors, resulting in photomorphogenic development (Figures 1a and 2b) [5]. Plants have an elaborate set of photoreceptors that allow perception of a broad spectrum of light, ranging from UV-B to far-red ( nm) [25]. Among them, the far-red and red light-sensing phytochromes (phya and phyb), the blue light-sensing cryptochromes (cry1 and cry2) and the recently identified UV-B receptor, UVR8, are the major photoreceptors that regulate photomorphogenesis in response to specific wavelengths of light [25,36]. Remarkably, the signaling of all of these photoreceptors is mediated through (Figure 1). A well-known inhibitory mechanism of by visible light involves its export from the nucleus on light exposure, thus excluding its activity against nuclear-localized transcription factors (Figure 2b) [37]. However, the kinetics of the nuclear exclusion of is rather slow (approximately 24 h) and this strategy may represent only long-term suppression of under extended light conditions [5,38]. A more rapid mechanism for downregulation of by phytochromes and cryptochromes is believed to exist because changes in the transcriptome initiated by far-red, red and blue light can be observed within an hour [39 41]. In addition, a large fraction of these early response genes are 2

3 (a) Under visible light (b) Under UV-B Far-red light Red light UV-B Photoreceptors: phya phyb cry1 cry2 UVR8 Signaling center: E3 ligase Effectors:, HYH, LAF1, etc. Transcription factors CO Transcriptional regulator GI Light responses: Photomorphogenesis Flowering Circadian rhythm UV-B tolerance TRENDS in Plant Science Figure 1. at the center of light signal transduction. represents a signaling center that integrates signals from various photoreceptors and also controls many downstream light-regulated responses. (a) Phytochromes (phya and phyb) and cryptochromes (cry1 and cry2) are the major photoreceptors that perceive a wide spectrum of visible light. When activated, the photoreceptors act to suppress. is a repressor in light signal transduction and functions as a ubiquitin E3 ligase that ubiquitinates multiple light-response effectors for degradation. Thus, photoreceptor-mediated suppression of allows accumulation of the effectors, resulting in the specific light responses. (b) Under UV-B, however, acts as a positive regulator in the signaling pathway. Activated UVR8, the newly identified UV-B receptor, interacts directly with and together they promote the transcription of for UV-B tolerance. Note: solid lines between and other proteins denote that a demonstrated physical interaction is involved in regulation. See Table 2 for a complete list of substrates. CO, CONSTANS;, CONSTITUTIVE PHOTOMORPHOGENIC 1; cry1 and cry2, cryptochrome 1 and 2; GI, GIGANTEA;, ELONGATED HYPOCOTYL 5; HYH, -HOMOLOG; LAF1, LONG AFTER FAR-RED LIGHT 1; phya and phyb, phytochrome A and B; UV-B, ultraviolet-b radiation; UVR8, UV RESISTANCE LOCUS 8;, ubiquitin. direct targets of, a substrate [42]. Correspondingly, protein accumulates rapidly (within an hour) on far-red light treatment [43]. How phya and phyb might inhibit remains unknown, but recent studies have shown that signaling from cryptochromes and UVR8 involves direct interaction with and/or its binding partner SPA1. The direct interaction between and cryptochromes was first reported a decade ago and is important for blue light-mediated photomorphogenic response [44,45]. However, because the interaction is not light dependent, it was unclear how light-activated cryptochromes can regulate. Three recent studies have provided insights into this by showing that cry1 and cry2 interact with SPA1 in a blue light-dependent manner [46 48]. Although the detailed modes of action for cry1 and cry2 differ, both of the cryptochrome SPA interactions are believed to perturb the function of the SPA E3 complex and suppress its activity (see below and [49,50]) (Figure 2b). Contrary to its role in the visible light spectrum, is a positive regulator in UV-B light signaling (Figure 1b) [51]. On UV-B irradiation, UVR8 monomerizes and interacts with [15,36]. Through an unknown mechanism, they promote the expression of, which is responsible for the activation of a subset of UV-B induced genes [52]. In addition to acting as a signaling integrator, is involved in the negative feedback regulation of photoreceptors in light, where it promotes the degradation of phya, phyb and cry2 (Figure 1a) [53 55]. In the case of phya and phyb, is the E3 ligase that promotes their ubiquitination. Flowering Mutations in have long been known to affect flowering time [56]. Two recent studies have identified the molecular link that explains how controls flowering in response to photoperiod [12,13]. In Arabidopsis, the transition from the vegetative to the flowering stage is controlled by day length. The transcriptional regulator CONSTANS (CO) promotes flowering under long days (LDs), but not short days (SDs) [57]. CO functions by activating the transcription of FLOWERING LOCUS T (FT), which is a potent inducer of flowering [58]. CO is regulated transcriptionally by the circadian clock and posttranslationally by light, such that flowering is induced only when CO transcription at dusk coincides with the exposure to light (as in LD) when the CO protein is stabilized [59,60]. In darkness, CO is ubiquitinated and degraded through the -proteasome system, thus preventing flowering under SDs even in the presence of CO transcripts [60]. Two studies reported that is the E3 ligase responsible for the ubiquitination of CO during the night (Figure 1a) 3

4 Table 2. Targets of the E3 ubiquitin ligase Substrate AGI code Protein identity Processes involved Interaction with Refs AT5G11260 bzip transcription factor Photomorphogenesis, light signaling + [26,27] HYH AT3G17609 bzip transcription factor Photomorphogenesis, light signaling + [28] LAF1 AT4G25560 MYB transcription factor Photomorphogenesis, light signaling + [29] HFR1 AT1G02340 bhlh transcription factor Photomorphogenesis, light signaling + [30 32] BBX24/STO AT1G06040 B-box Zinc Finger protein Photomorphogenesis, light signaling + [69,98,99] BBX4/COL3 AT2G24790 B-box Zinc Finger protein Photomorphogenesis, light signaling + [34] BBX22/LZF1/STH3 AT1G78600 B-box Zinc Finger protein Photomorphogenesis, light signaling Through? [33,100] GATA2 AT2G45050 GATA transcription factor Photomorphogenesis, light and + [21] brassinosteroid crosstalk phya AT1G09570 Phytochrome Light perception + [53] phyb AT2G18790 Phytochrome Light perception + [55] CO/BBX1 AT5G15840 B-box Zinc Finger protein Flowering + [101,102] GI AT1G22770 Unknown protein Circadian rhythm and flowering Through ELF3 [14] SCAR1 AT2G34150 SCAR family member Root growth + [23] HRT AT5G43470 R protein Plant defense + [20] [12,13]. Mutants of cop1 flower early under SDs and the phenotypes are largely suppressed in the co mutant background., through its WD40 repeats domain, interacts physically with CO (discussed below), ubiquitinates CO in vitro and promotes CO degradation in vivo. In the presence of light, particularly blue and far-red, activity is inhibited, leading to accumulation of CO. Interestingly, SPA1, a component of the SPA complex, also interacts with CO and negatively affects CO stability [61]. Thus, plays a role in photoperiod perception by degrading CO at night, preventing flowering under SDs. Circadian rhythm The promotion of proteolysis of CO by, however, is only part of its regulatory mechanism in the control of flowering. Another recent study has suggested that can also modulate CO transcriptionally by regulating the flowering- and circadian clock-associated protein GIGAN- TEA (GI) (Figures 1a and Fig. 3) [14]. Importantly, it establishes that can play a role in modulating the circadian clock. First, the study demonstrates that the SD-insensitive early flowering phenotype of cop1 mutants can be partially rescued when the plants are entrained under a light dark cycle with a reduced environmental time period (e.g. from 24 h to 18 h), possibly coinciding with the circadian period of the mutant. This indicates that a circadian defect in the cop1 mutant contributes to its early flowering phenotype. Furthermore, has the ability to interact with EARLY FLOWERING 3 (ELF3), which allows to interact with GI in vivo and target GI for degradation (Figure 3a). GI is known to interact with the blue light-sensing F-box proteins FLA- VIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and ZEITLUPE (ZTL) in a blue light-dependent manner [62,63]. FKF1 and ZTL are regulators of flowering time and circadian rhythms, respectively, and their interaction with GI is important for their function. The blue light-stabilized FKF1 GI interaction targets CYCLING DOF FACTOR 1 (CDF1), a transcriptional repressor of CO, for degradation (Figure 3b) [64].The ZTL GI interaction also mediates the degradation of TIMING OF CAB EXPRESSION 1 (TOC1), a central clock component that has recently be shown to function as a transcriptional repressor of core circadian genes [65 67]. The ability of to regulate GI abundance enables it to function as a light-regulated switch for flowering and as a modulator of the circadian clock. The E3 machinery: a multimeric SPA ligase is a 76 kda RING E3 ligase and contains three protein protein interaction domains: an N-terminal RING-finger region, a coiled-coil domain and seven WD40 repeats at its C terminus [5]. The RING-finger motif mediates the interaction with -conjugating enzymes (E2s), and the coiled-coil region of allows the formation of homodimers or heterodimers with members of the SPA protein family (discussed below) [5,68]. The WD40 repeats function as the substrate-interaction domain [5,69]. Although possesses the interaction domains of a functional E3 ligase and can ubiquitinate targets on its own in vitro, it forms a protein complex of greater than 700 kda in vivo, indicating that function may require additional protein components [26,29]. In plants, the SPA family of proteins has been established as core components of the complex [26,68,70 72]. The SPA family consists of four members, SPA1 to SPA4, that each contain an N-terminal kinase-like domain, followed by a coiled-coil domain and, interestingly, a WD repeats domain that shares a high homology with (44% identity for SPA1) [72,73]. All four SPA family members interact with in vitro and in vivo, and the interaction is mediated through the respective coiled-coil regions [26,68,70]. Affinity purification of the SPA1 complex has demonstrated as its major constituent, further supporting the presence of the SPA complex [71]. The SPA family has overlapping but not entirely redundant functions in plants, and they exhibit distinct light- and organ-specific expression patterns, suggesting that spatiotemporal control of SPA complexes may be required for functional specificity [70,72,74]. Given their ability to homo- and heterodimerize and the approximate 440 kda size of the core complex, the SPA complexes are proposed to be heterogeneous in nature, with a 4

5 (a) In darkness (b) Under light - E3 ligase core SPA1 The SPA1 complex Transcription factor Degradation SPA1 -SPA1 E3 ligase E2~ cry1 receptor SPA1 The SPA1 complex Light Transcription factor cry1 SPA1 Nuclear export Cytoplasm Nucleus targets Photomorphogenesis Nucleus targets Photomorphogenesis TRENDS in Plant Science Figure 2. Mode of action of the SPA1 E3 ligase. (a) In darkness, is responsible for the proteasome-mediated degradation of transcription factors, such as, that promote photomorphogenesis. forms a tetrameric complex with members of the SPA family (two s and two SPAs) and interacts with the substrate. The WDXR motifs on either or SPA further mediate the interaction with the core, and the multimeric SPA1 E3 ligase is assembled. Activated E2s are recruited to the E3, resulting in the poly-ubiquitination of and its degradation. Photomorphogenesis is repressed. (b) Under light, is inhibited by the activated photoreceptors. In blue light, cry1 interacts with SPA1 and sequesters it from, thereby probably disrupting the SPA1 complex that is important for E3 function. also interacts with cry1 in a light-independent manner (not shown here). Visible light further promotes the nuclear export of for long-term suppression of. As a result of reduced activity, accumulates, binds to its targets and promotes photomorphogenesis. cry1, cryptochrome 1;, CONSTITUTIVE PHOTOMORPHOGENIC 1;, CULLIN4;, DAMAGED DNA BINDING PROTEIN 1;, ELONGATED HYPOCOTYL 5;, RING-BOX 1; SPA1, SUPPRESSOR OF PHYA-105 1;, ubiquitin. core tetramer of two proteins and combinations of two SPA proteins (Figure 2a) [70]. Genetic and biochemical evidence suggests that complex formation between and SPA proteins is important for function. and SPA1 interact genetically, and the quadruple spa mutant displays a striking constitutive photomorphogenic phenotype similar to strong cop1 mutants (Table 1) [26,72]. In spa mutants, at least three substrates, HFR1 and CO have been shown to accumulate at higher levels [26,61,70,75]. In addition, in vitro ubiquitination assays have shown that SPA1 can modulate the activity of, and at least in the case of LAF1, the coiled-coil region of SPA1 can enhance E3 activity at low concentration [26,29]. Finally, the identification of the blue-light dependent interaction between cryptochromes and SPAs, as discussed above, supports their importance in function [46 48]. Given that SPA proteins can also directly interact with, HFR1 and CO through their WD repeat domains [26,61,75], SPA proteins may contribute to function through substrate recruitment and/or enhancement of activity. Furthermore, they may also play a structural role in the formation of a SPA ligase (discussed below). A recent study of the relationship between and has led to a model of a multimeric E3 ligase in which the SPA complex acts as a substrate receptor of the E3 ligase (Figure 2a) [76]. The ligases belong to the multi-subunit CRL family, which consist of a CULLIN, a small RING finger protein named /ROC1 and a substrate receptor module [77]. CULLIN functions as a scaffold on which and the substrate receptor module dock at its C and N terminus, respectively. recruits E2s to the ligase, and the substrate receptor module mediates the interaction with substrates. For the ligases, the substrate receptor module consists of the core adaptor protein and a group of interchangeable DWD/DCAF/CDW proteins as substrate receptors. functions as a linker protein that connects the various DWD proteins to the core (Figure 2a). The DWD (for -binding WD40) proteins are a subset of WD40 motif-containing proteins that associate with through WDXR motifs. In plants, there are 85 and 78 putative DWDs in Arabidopsis and rice, respectively [78]. Interestingly, and the four SPAs are among the putative DWD proteins. Indeed, and the SPAs were shown to interact with the two Arabidopsis isoforms, a and b, in vitro, and the interactions were dependent on their WDXR motifs [76]. The in vivo interaction between and SPA1/3/4 with b was also demonstrated. In addition, associates with in vivo, and they interact genetically to repress photomorphogenesis [76,79]. These results suggest that the SPA complexes bind to with their WDXR motifs and act as substrate receptors in the formation of multimeric SPA ligases (Figure 2a). Given that is a functional E3 on its own in vitro, it is unclear why would function as a part of a multimeric ligase. One possible explanation is that the formation of the tetrameric SPA complex may mask the RING domain of and hinder its recruitment of E2s, thus requiring the activity of the ligase. Furthermore, the machinery 5

6 (a) Night? SPA GI ELF3 Degradation (b) Day (afternoon) cry1/2 receptor? SPA GI ELF3 FKF1 ZTL FKF1 ZTL Blue-light sensing F-box protein Blue-light sensing F-box protein FKF1 GI ZTL GI CDF1 Transcriptional repressor TOC1 Transcriptional repressor CDF1 Degradation TOC1 Degradation CDF1 Flowering Clock progression CO TOC1 Circadian genes CO Circadian genes Flowering control Clock control Flowering control Clock control TRENDS in Plant Science Figure 3. Model of regulation of GI for flowering time and circadian rhythm control. (a) At night,, through binding with ELF3, interacts and targets GI for degradation. ELF3 is also degraded in the process. In the absence of GI, two blue light-sensing F-box proteins, FKF1 and ZTL, which are involved in the control of flowering and circadian rhythm, respectively, fail to promote degradation of their respective substrates, CDF1 and TOC1. The two transcriptional repressors then repress CO, an activator of flowering, and circadian genes, respectively. also interacts physically with CO protein and targets its degradation (not shown). The involvement of SPAs in this process has not been investigated. (b) In the afternoon, transcription of the circadian-regulated GI, ELF3, FKF1 and ZTL starts to peak. Because is inhibited by the activated cryptochromes, GI accumulates. also induces the formation of FKF1 GI and ZTL GI complexes, and they mediate the degradation of CDF1 and TOC1, respectively, leading to transcription of CO and circadian genes. The FKF1 GI CDF1 complex also associates with the promoter of CO (not shown). cry1/2, cryptochrome 1 or 2; CDF1, CYCLING DOF FACTOR 1; CO, CONSTANS;, CONSTITUTIVE PHOTOMORPHOGENIC 1; ELF3, EARLY FLOWERING 3; FKF1, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1; GI, GIGANTEA; SPA1, SUPPRESSOR OF PHYA-105 1; TOC1, TIMING OF CAB EXPRESSION 1;, ubiquitin; ZTL, ZEITLUPE. may be a more robust E3 ligase in general that has evolved to function within. These recent studies on the E3 machinery in plants also demonstrated key differences between the composition of the E3 ligase in plants and animals. Like its counterpart, human can act as a substrate receptor for the -based ligase in the degradation of substrates, such as the proto-oncogenic transcription factor c- Jun [80]. However, in this A- hdet1 h ligase, the connection of h to A- is dependent on human DET1, which bridges h to. In plants, DET1 is not required for the interaction between and ; instead, and DET1 form distinct ligases [76]. It is interesting to note that h, as well as its homologs in mouse and chicken, has only one of the two conserved WDXR motifs found in plants. This might explain why h cannot directly associate with. DET1: a component of an unconventional ligase and a transcriptional regulator Although the biochemical function of and CSN started to emerge in the past decade, understanding the molecular roles of DET1 and 0, the two remaining COP/DET/FUS proteins, has been more challenging. DET1 has no recognizable domain besides two nuclear localization signals [81]. 0 is a novel type of -conjugating enzyme (E2) variant protein (UEV), which are E2-like proteins that lack E2 activity [82,83]. The discovery that DET1 and 0 form a complex with (the CDD complex), together with the recent establishment of as a core adaptor of the -based E3 ligase, has now linked 0 and DET1 to the E3 machinery [7,8,77,80]. DET1 was first found to complex with through affinity purification in tobacco (Nicotiana tabacum) cell lines [8]. Subsequently, the biochemical purification of 0 in cauliflower (Brassica oleracea) and gel-filtration studies in Arabidopsis demonstrated that 0 is another component of the CDD complex [7,76]. The CDD complex is also conserved in humans, where it exists as the hdet1 DDA1 (DDD) E2 complex [84]. The E2 denotes the canonical E2s of the human UBE2E family in the complex, of which 0 is the closest homolog in plants. Although UBE2Es are functional E2s, only the uncharged form of UBE2Es (not conjugated to ) was found binding to the complex. This suggests that some structural or biochemical feature of UBE2Es and 0 other than E2 activity is responsible for their recruitment to these complexes. Through, 0 and DET1 can connect to and form a multimeric CDD ligase (Figure 4) [79,85]. However, as mentioned above, the typical substrate receptors for ligases are DWD proteins containing WDXR motifs, through which they interact with 6

7 (a) Important for E3 activity of s? (b) Transcriptional repression? ~ SPA1 -SPA1 E3 ligase DDB2 E2 E2 ~ Enhance E3 activity? DET1 0 CDD E3 ligase??? 0? DET1 CCA1/ LHY Transcription factors Histone ubiquitination? Transcriptional repression TOC1 Enhance activity of chromatinregulating (s)? DDB2 E3 ligase TRENDS in Plant Science Figure 4. Model of two possible roles of the CDD E3 ligase. (a) CDD E3 ligase or DET1 has been implicated to be important for the activity of two ligases: SPA1 and DDB2. CDD may act through enhancing the E3 activities of other s (see text for details). (b) Because DET1 functions as a transcriptional corepressor, CDD may also play a role in transcriptional repression. DET1 was found to interact physically with two closely related transcription factors, CCA1 and LHY, bind to their targets, such as TOC1, and suppress their transcription. Possible mechanisms for CDD-mediated repression include histone ubiquitination and the enhancement of other ligases that interact with chromatin. CCA1, CIRCADIAN CLOCK ASSOCIATED 1; CDD, the 0 DET1 complex;, CONSTITUTIVE PHOTOMORPHOGENIC 1; 0, CONSTITUTIVE PHOTOMORPHOGENIC 10;, CULLIN4;, DAMAGED DNA BINDING PROTEIN 1; DDB2, DAMAGED DNA BINDING PROTEIN 2; DET1, DE-ETIOLATED 1;, ELONGATED HYPOCOTYL 5; LHY, LATE ELONGATED HYPOCOTYL 1;, RING- BOX 1; SPA1, SUPPRESSOR OF PHYA-105 1; TOC1, TIMING OF CAB EXPRESSION 1;, ubiquitin.. 0 and DET1 are not DWDs and lack the WDXR motif; therefore it remains unclear whether the CDD can function as a substrate receptor. Another possibility is that the CDD complex may act as an adaptor for other substrate receptor proteins, similar to the role previously discussed for human DET1 [80]. A recent structural study has shown that a novel motif, the H-box motif, found in some viral hijacker proteins and DWDs, can mediate or aid in their interaction with [86]. Interestingly, DET1 carries a H-box-like motif and may bind to through this mechanism [87]. Thus, DET1 may have evolved to hijack the machinery to target interacting proteins or modulate the activity of other ligases. Some evidence has suggested a role of the CDD complex in the regulation of -based ligases activity. This stemmed from the findings that 0 can enhance the E2 activity of a broad range of E2s and promote poly- chain formation by ch1 and ch13/uev1, two -chain forming E2s, in vitro [7,88]. Importantly, purified CDD complex, likely through its 0 subunit, enhanced the E3 activity of the core, as judged by increased auto-ubiquitination of [79]. Thus, it has been proposed that the CDD complex plays a role in activating ligases, which may help to explain why it is required for the SPA -mediated degradation of and other substrates (Figure 4a). A recent study of UV-C induced DNA damage responses in Arabidopsis is also consistent with this proposal [89]. In this study, DET1 was shown to be important for the autodegradation of a DWD substrate receptor DDB2 by during DNA repair (Figure 4a). It was further suggested that DET1 transiently associates with the DDB2 ligase and promotes DDB2 degradation. Nevertheless, the recombinant human DDD complex, without UBE2E, was shown to inhibit the activity of A ROC1 in vitro [84], which may be a direct result of the absence of UBE2E. Clearly, additional experiments, particularly in vitro ubiquitination assays with all the requisite protein components, are needed to address whether the CDD complex can regulate other ligases. Apart from our understanding of the CDD complex, DET1 has been implicated in chromatin regulation and has recently been shown to function as a transcriptional corepressor [90]. The first report on the involvement of DET1 in direct gene regulation came from a study in fruit flies [4]. The Drosophila DET1 homolog, ABO, localizes to the histone gene cluster on chromatin, specifically at the promoter regions of the histone genes. Because abo mutants have an elevated level of histone transcripts, Drosophila DET1 was proposed to act as a negative regulator of histone genes. In addition, both tomato (Solanum lycopersicum) and Arabidopsis DET1 s were shown to interact with the non-acetylated tails of histone H2B in the 7

8 context of the nucleosome, further suggesting a role of DET1 in chromatin modeling [91]. However, because DET1 has no DNA-binding domain and no DNA-binding activity of DET1 was detected, it was not known how DET1 regulates specific genes [81]. Our recent study has addressed this gap by showing that DET1 can interact physically with two closely related MYB transcription factors, CCA1 and LHY, and is recruited to their target genes (Figure 4b) [90]. CCA1 and LHY are core morning components of the plant circadian clock and function during the day to repress the evening genes, such as TOC1 [92]. We found that DET1 associates with the promoter of TOC1 and other evening genes in a CCA1/LHY-dependent manner and mediates their repression. Transcriptional reporter assays in yeast and in planta also demonstrated that DET1 can repress transcription when tethered to promoters. These results not only reveal the role of DET1 in the circadian clock, but also define a novel function of DET1 as a transcriptional corepressor. Given the pleiotropic phenotypes beyond circadian defects in the det1 mutants, DET1 may be recruited more broadly by other transcription factors in regulating diverse cellular processes. It has not been determined whether 0 and participate in the transcriptional repression activity of DET1, but considering that DET1 generally exists in vivo with the CDD complex, it is likely that they play a role [7,8]. Connecting the ligase to the transcriptional repression activity of DET1 may reveal the mechanism behind this process (Figure 4b). Concluding remarks and outlook The tremendous progress on the COP/DET/FUS proteins achieved in the past 20 years has shaped our understanding of how light controls gene expression and developmental programs. The pursuit of their molecular functions has also yielded general principles regarding these conserved proteins that regulate diverse processes. Building on these insights, recent studies have expanded and strengthened the role of in light signaling: the number of known substrates has increased significantly; the biological processes regulated by has expanded; and activity can be directly modulated by two types of photoreceptor. Studies have also identified the functional unit of and DET1 as two independent multimeric ligases SPA and CDD. DET1 was also found to be a transcriptional corepressor, recruited by specific transcription factors to their target genes. Many interesting questions on the structure, function and regulation of SPA and CDD remain to be addressed. First, why does require SPA proteins to function in vivo and how is the complex able to bind as a multimer? Also, how do 0 and DET1, two non-dwds, mediate their interaction with? Structural and in vitro studies on these complexes will be valuable for addressing these questions. Second, as discussed above, the function of CDD remains obscure. Further experiments will be needed to clarify its relationship with SPA and its potential role in DET1-mediated gene repression (Figure 4). Finally, it remains unknown how phytochromes inhibit and what mediates the nuclear export of (Figure 2b). Whether the CDD complex is regulated by light or other signals also remains to be explored. With all these challenging questions, the future of COP/DET/FUS research will require carefully designed experiments that integrate biochemical, structural and genetic approaches to uncover the remaining mysteries. Acknowledgments We thank Graham Dow for critical reading of the manuscript. Relevant research in our laboratory is supported by a National Institutes of Health (NIH) grant (GM-47850) and a National Science Foundation (NSF) 2010 grant (MCB ) to X.W.D. 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