Molecular Plant Advance Access originally published online on October 11, 2007
Molecular Plant 2008 1(1):58-67; doi:10.1093/mp/ssm005
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© The Author 2007. Published by Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.
The Clock Protein CCA1 and the bZIP Transcription Factor HY5 Physically Interact to Regulate Gene Expression in Arabidopsis
Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095–1606, USA
1 To whom correspondence should be addressed. E-mail etobin{at}ucla.edu, tel. 1–310–825–7700, fax 1–310–206–3987.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODS |
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The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes—Lhcb1*1 and Lhcb1*3—and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCA1-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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Circadian rhythms enable organisms to anticipate and respond to diurnal changes in the environment. These endogenous rhythms with a period of about 24 h were first recorded in plants, and they occur in organisms ranging from cyanobacteria to humans (Barak et al., 2000). Studies of these daily rhythms have led to a simple conceptual model consisting of a central oscillator that generates the rhythms, input pathways that transduce time-keeping signals from the environment to the central oscillator, and output pathways, by which the rhythms are made manifest. Understanding the molecular mechanisms involved in linking the central oscillator to the various output pathways is important for a full understanding of these rhythms and is currently under investigation in a number of different model systems (Harmer et al., 2001; Yakir et al., 2007). The fact that the rhythms can occur with different phases and amplitudes in a single organism suggests that there are multiple factors that affect the output pathways.
In plants, the circadian clock regulates a plethora of biological processes ranging from rhythmic leaf movements to the rhythmic transcription of many genes (reviewed in Barak et al., 2000). Circadian rhythms are also involved in the regulation of developmental processes such as flowering in many different plants species (Searle and Coupland, 2004). In the model plant Arabidopsis, two Myb-related transcription factors—CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL)—as well as the pseudo-response regulator TOC1 (TIMING OF CAB EXPRESSION 1) are proposed to be components of the core oscillator (Wang and Tobin, 1998; Schaffer et al., 1998; Alabadi et al., 2001). Each of these putative oscillator components shows a circadian rhythm of expression of both its RNA and protein (Wang and Tobin, 1998; Kim et al., 2003; Más et al., 2003). Evidence supports the position that CCA1 and LHY act to negatively regulate TOC1 expression by binding to its promoter and that TOC1 participates in the positive regulation of CCA1 and LHY expression by an unknown mechanism (Alabadi et al., 2001).
Expression profiling studies indicate that up to 36% of the Arabidopsis genome exhibits circadian rhythms, with different genes showing rhythms that peak at different times throughout the day and night, thus having different phases of expression (Harmer et al., 2000; Schaffer et al., 2001; Michael and McClung, 2003; Edwards et al., 2006). Two similar cis-elements—a CCA1-binding site (CBS: AAA/CAATCT) (Wang et al., 1997) and an evening element (EE: AAAATATCT)—are necessary to confer circadian-regulated transcription to a set of Arabidopsis genes (Harmer et al., 2000). Recombinant CCA1 and LHY proteins specifically interact with both the CBS and EE (Wang et al., 1997; Alabadi et al., 2001). However, the CBS and EE cis-elements in the TOC1 and CATALASE3 (CAT3) promoters do not by themselves confer robust mRNA oscillations, suggesting that additional cis-elements and transcription factors are required for normal circadian control of gene expression (Michael and McClung, 2002).
Examination of the promoters of two nuclear genes—Lhcb1*1 and Lhcb1*3 (also called CAB2 and CAB1)—which encode Arabidopsis light-harvesting chlorophyll a/b proteins, has identified several cis-acting sequence elements important for light and circadian control of these genes (Anderson et al., 1994; Anderson and Kay, 1995; Carré and Kay, 1995; Kenigsbuch and Tobin, 1995; Millar and Kay, 1996; Maxwell et al., 2003; Terzaghi and Cashmore, 1995) and transcription factors binding these elements have been characterized (Giuliano et al., 1988; Sun et al., 1993; Menkens et al., 1995; Degenhardt and Tobin, 1996; Wang et al., 1997). Figure 1A shows the DNA sequence of fragments that contain the CBS for each of these promoters. The CBS is present in a region of the Lhcb1*1 promoter that is critical for circadian control, phytochrome regulation and high expression (Sun et al., 1993; Carré and Kay, 1995; Kenigsbuch and Tobin, 1995). Close to the CBS in both the Lhcb1*1 and Lhcb1*3 promoters is a G-box element—a motif commonly found in the promoters of light-regulated plant genes (Terzaghi and Cashmore, 1995). It is also an essential promoter element regulating circadian rhythms in other model organisms, including mice and Drosophila, where it is known as an E-box (Wang et al., 2001; Munoz et al., 2002). Several basic leucine zipper (bZIP) DNA-binding proteins that are able to bind G-box elements have been identified in plants (Menkens et al., 1995; Chattopadhyay et al., 1998).
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One of these proteins—HY5—is a positive regulator of photomorphogenesis (the control of plant development by light), and HY5 can bind G-box elements in a number of different light-regulated promoters in Arabidopsis (Oyama et al., 1997; Chattopadhyay et al., 1998; Gao et al., 2004; Lee et al., 2007). Phytochrome is a plant photoreceptor that regulates the expression of many genes and, in hy5-null Arabidopsis mutants, the phytochrome-mediated induction of G-box-containing promoters is reduced (Chattopadhyay et al., 1998). HY5 can act either as a homodimer or as a heterodimer with a related bZIP transcription factor to control gene expression (Holm et al., 2002). Studies in two different plant species have shown that the functional interaction of several cis-acting elements (and the transcription factors binding them) is required for integrating the response of a particular gene to different signals (Puente et al., 1996; Chattopadhyay et al., 1998; Martinez-Hernandez et al., 2002). Interaction of the CBS and G-box and its importance for circadian regulation has not previously been demonstrated. We show here that HY5 binds the G-box element of Arabidopsis Lhcb promoters and provide evidence that suggests that CCA1 can alter HY5-binding to the G-box through a direct protein–protein interaction. Furthermore, the absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression without affecting the period of CAT3, which is another circadian-regulated gene that lacks a G-box element in its promoter. Our results demonstrate that HY5 and CCA1 act in concert to regulate the circadian expression of Lhcb genes.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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HY5 Specifically Binds the G-box Element of the Lhcb1*3 Promoter
We tested whether recombinant HY5 could bind to a fragment of the Lhcb1*3 promoter that contains a G-box and whether the binding requires the G-box. Figure 1A shows that the core ACGT sequence characteristic of G-box elements is present in both the Lhcb1*1 and Lhcb1*3 promoters in the vicinity of the CBS. HY5 has been shown to bind to G-box elements in DNA fragments and in the context of the Arabidopsis RBCS–1A gene (Chattopadhyay et al., 1998). Figure 1B shows that recombinant HY5 binds the A2 (Figure 1A and Sun et al., 1993) fragment of Lhcb1*3. When the core ACGT element of the G-box was mutated to TATT (Figure 1A, MutG fragment), HY5 did not bind, showing that HY5 specifically binds to the G-box element. To confirm the binding of HY5 to the A2 fragment of the Lhcb1*3 promoter, we used nuclear extracts from HY5-overexpressing (HY5-OX) plants in an electrophoretic mobility shift assay (EMSA) with the A2 fragment. HY5-OX plants were used because HY5 protein levels are low in wild-type seedlings over 5 d old (Hardtke et al., 2000). Addition of nuclear extracts from HY5-OX plants to the A2 fragment led to the appearance of a binding activity with retarded mobility that was not detected in wild-type nuclear extracts (Figure 2, cf. lanes 2 and 3). Including HY5 antibodies in the binding assay led to the replacement of this band with a band of highly reduced mobility (Figure 2, lane 4), demonstrating that HY5 was part of the HY5-OX-binding activity seen in lane 3. When nuclear extracts of HY5-OX plants were incubated with the MutG fragment, no HY5-OX-binding activity could be detected (Figure 2, lane 7), demonstrating that the HY5-OX-binding activity is specific to the G-box element in the Lhcb1*3 promoter.
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CCA1 Can Physically Interact with HY5
Because the G-box element in both Lhcb1*1 and Lhcb1*3 promoters is present upstream of, and in close proximity to, CBSs (Figure 1A), we tested whether CCA1 and HY5 might physically interact with each other to regulate Lhcb gene expression. Figure 3A (left side) shows that HY5 was able to bind GST–CCA1 in vitro but not GST alone, demonstrating that CCA1 and HY5 are able to physically interact. As a control, GBF4 (another bZIP transcription factor) was tested for interaction with GST–CCA1. No interaction between GBF4 and GST–CCA1 was detected (Figure 3A, right side), suggesting that CCA1 interacts specifically with HY5 rather than non-specifically with bZIP transcription factors. To further confirm the in vitro interaction between CCA1 and HY5, we fused CCA1 to the GAL4 DNA-binding domain (CCA1-BD) and HY5 to the GAL4 transcription–activation domain (HY5-AD) and co-expressed them in a yeast two-hybrid system. Figure 3B demonstrates that CCA1 and HY5 were able to specifically interact in yeast cells, as shown by the production of blue yeast colonies due to the expression of the β-galactosidase reporter. No such interaction was observed when CCA1-BD was co-expressed with the AD alone or when HY5-AD was co-expressed with an unrelated yeast protein (SNF1-BD) (Celenza and Carlson, 1986).
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CCA1 and CKB3 Alter the Binding Activity of HY5 on the Lhcb1*3 Promoter
We examined whether recombinant CCA1 and HY5 can affect each other's binding activity on the A2 fragment in an EMSA. HY5-binding activity on the A2 fragment was detected as a low mobility band (Figure 4A, lane 2) similar to that obtained in Figure 1B. Figure 4A also shows the CCA1-binding activity was observed as a relatively higher mobility band (lane 3 and cf. Wang et al., 1997 and Sugano et al., 1998). When both CCA1 and HY5 were incubated with the A2 fragment, a new, very low mobility binding activity was observed (Figure 4A, lane 4), suggesting that CCA1 and HY5 can simultaneously bind the Lhcb1*3 promoter possibly as a complex. When CCA1 and HY5 were incubated with the MutG fragment, only a barely visible amount of HY5-binding activity was evident and the very low mobility band representing the putative CCA1–HY5–DNA complex was not detected (Figure 4A, lane 8). In order to test whether the binding of CCA1 to the A2 fragment was required for the appearance of the very low mobility band or whether the physical interaction between CCA1 and HY5 was sufficient, we carried out an EMSA with the m1 fragment (Figure 1A) as a probe. This DNA fragment is identical to the A2 fragment, except that bases essential for CCA1-binding have been mutated so that CCA1 can no longer bind (Wang et al., 1997). Figure 4A shows that CCA1 was unable to bind the m1 fragment, while HY5 was still able to bind due to the intact G-box element (lanes 9 and 10). However, when CCA1 and HY5 were co-incubated with the m1 fragment, no very low mobility band was observed (lane 11), indicating that binding of CCA1 is necessary for the formation of the very low mobility binding activity with HY5 on the Lhcb1*3 promoter.
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CCA1-binding to the Lhcb1*3 promoter can be increased either by phosphorylation by the protein kinase CK2 or by incubation with CK2 regulatory β-subunits which, alone, cannot phosphorylate CCA1 (Sugano et al., 1998; Daniel et al., 2004). However, phosphorylation of HY5 decreases its binding to G-box-containing promoters (Hardtke et al., 2000). It is not known whether interaction with CK2 β-subunits can alter the binding of HY5 to DNA. Figure 4B shows that incubation of HY5 with the β3-subunit of CK2 (CKB3) led to a large increase in HY5 DNA-binding activity to the A2 fragment (Figure 4B, lane 4), which could not be mimicked by replacing CKB3 with the non-specific protein, BSA (Figure 4B, lane 6). This observation suggests that interaction with CK2 can enhance binding of both CCA1 and HY5 to the Lhcb1*3 promoter. The fact that no shift of the HY5-binding activity occurs upon the addition of CKB3 suggests that any interaction between HY5 and CKB3 is transient and/or unstable under the conditons of the EMSA. This is similar to the interaction between CCA1 and CKB3, which increases CCA1-binding on the Lhcb1*3 promoter without a shift in the mobility of the CCA1-binding activity (Sugano et al., 1998).
CCA1 Increases the Binding of HY5 to the Lhcb1*1 Promoter
We undertook a second approach to understand how CCA1 affects HY5-binding activity. We tested whether CCA1 and HY5 can similarly alter each other's activity in the context of the Lhcb1*1 promoter and whether a physical interaction between the two proteins may contribute to any alteration in their binding activities. The binding of CCA1-AD and HY5-AD to the Lhcb1*1 promoter (–221 to +7 relative to transcription start; Figure 1A), was tested in a yeast one-hybrid assay. Figure 5A shows that expression of CCA1-AD led to a small increase in β-galactosidase activity compared with background activity, demonstrating that CCA1 is able to bind to the Lhcb1*1 promoter in yeast, albeit at low efficiency. Expression of HY5-AD induced a large increase in β-galactosidase activity, showing that HY5 is also able to bind the Lhcb1*1 promoter in yeast (Figure 5B). Co-expression of CCA1-AD and HY5-AD led to an increase in β-galactosidase activity greater than that of HY5-AD alone. The differences observed in β-galactosidase activity were not due to differences in expression levels of CCA1-AD or HY5-AD (data not shown). We surmised that this increased activation of the Lhcb1*1 promoter could be due to at least two reasons: (i) CCA1-AD and HY5-AD could both be binding to the Lhcb1*1 promoter and thus the addition of an extra AD could lead to increased transcription of the LacZ gene, or (ii) CCA1-AD might physically interact with HY5-AD and increase its binding to the Lhcb1*1 promoter. In order to test these possibilities, we removed the AD from CCA1 and co-expressed it with HY5-AD. We observed the same increase in β-galactosidase activity greater than that of HY5-AD alone, even though CCA1 was no longer fused to the AD (Figure 5B). Overall, these results suggest that CCA1 is able to affect the binding of HY5 to the Lhcb1*1 promoter and that this may be due to a physical interaction between the two proteins.
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Absence of HY5 Alters the Period of Lhcb1*1 mRNA Oscillations
In order to test whether binding of HY5 to the Lhcb1*1 promoter has any functional significance for gene expression, we examined circadian expression of the Lhcb1*1 gene in hy5-null seedlings (Koornneef et al., 1980; Oyama et al., 1997). Wild-type and hy5 seedlings were entrained in a photoperiod of 12 h light:12 h dark and then transferred to continuous light. Figure 6A shows that the absence of HY5 led to a shorter period of Lhcb1*1 circadian oscillations compared with wild-type, suggesting that HY5 is involved in regulating Lhcb1*1 expression. However, it is possible that the altered circadian rhythm of Lhcb1*1 is due to an overall effect of the hy5 mutation on the circadian clock. Therefore, we examined the expression of another circadian output gene—CAT3—whose promoter architecture differs from that of Lhcb1*1. The CAT3 promoter contains an EE at –191 relative to transcription start but is devoid of consensus G-box elements. The overall level of CAT3 mRNA was significantly higher in the hy5 mutant than in wild-type (data not shown). However, circadian oscillations of CAT3 were similar to wild-type (Figure 6B), suggesting that the absence of functional HY5 has no effect on the circadian oscillator. Rather, in the context of the CAT3 promoter, HY5 appears to be involved in reducing overall CAT3 mRNA levels. These results support the idea that HY5 is involved in the combinatorial control of Lhcb1*1 expression.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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Clock mechanisms of model organisms comprise transcription–translation negative feedback loops that involve a group of clock genes, many of which encode transcriptional regulators (Young, 2000; Harmer et al., 2001). Although binding sites of these clock proteins in the promoter region of clock-controlled genes are necessary for rhythmic expression, they are alone insufficient to confer fully functional circadian transcription, suggesting that additional cis-elements are required for proper control of circadian gene expression (Lyons et al., 2000; Michael and McClung, 2002; Munoz et al., 2002). However, little is known regarding the identity of such cis-elements in any organism.
We have presented several lines of evidence showing that the bZIP transcription factor HY5 interacts with CCA1 to regulate the expression of Lhcb genes. We have shown that HY5 is able to specifically bind the G-box element on the Lhcb1*3 promoter (Figures 1B, 2 and 4A
). HY5 was recently shown to bind the Lhcb1*3 promoter in vivo (Lee et al., 2007). We have also demonstrated that not only are CCA1 and HY5 able to interact, but that they can simultaneously bind DNA (Figures 3A, 3B and 4A). This finding is supported by our previous data showing that CCA1-containing Lhcb1*3-binding activity from whole plant extracts has a lower mobility on an EMSA than recombinant CCA1 (Sugano et al., 1998). The fact that DNA-bound HY5 does not interact with unbound CCA1 (Figure 4A, lane 8), nor DNA-bound CCA1 with unbound HY5 (Figure 4A, lane 11) suggests that they interact when bound to their respective cognate sequences. Furthermore, the yeast one-hybrid data show that co-expression of CCA1-AD and HY5-AD in a yeast one-hybrid assay increases the transcriptional activity of the Lhcb1*1 promoter above the activity observed when HY5-AD is expressed alone (Figure 5B). Moreover, this increased Lhcb1*1 promoter activity is independent of whether CCA1 is fused to the AD. It is unclear why CCA1-AD binds only weakly to the Lhcb1*1 promoter in the yeast one-hybrid assay. It is possible that the CCA1-AD protein is post-transcriptionally modified in yeast in such a way as to cause weak binding. Nevertheless, the fact that mutation of the CBS (such that CCA1 can no longer bind) abolishes the putative CCA1-HY5-binding activity (Figure 4A), combined with the yeast one-hybrid results, support a model whereby binding of CCA1 to its recognition site either recruits or stabilizes HY5-binding to Lhcb promoters.
Combinatorial control of transcription by multiple transcription factors has been reported from both animal and plant systems (Peers et al., 1995; Chen et al., 1996; Molkentin and Olson, 1996; Giangrande et al., 2003, 2004; Narusaka et al., 2003; Lara et al., 2003; Hartmann et al., 2005). For example, the homeodomain protein STF1, which is required for mammalian pancreatic development, interacts cooperatively with and targets the homeobox protein Pbx to a subset of STF1-binding site-containing promoters (Peers et al., 1995). Examples of combinatorial control have also been described in plants (Chen et al., 1996; Lara et al., 2003; Narusaka et al., 2003; Weltmeier et al., 2006). Indeed, HY5 itself was recently shown to act cooperatively with the bHLH transcription factor, PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), to regulate transcription of anthocyanin biosynthesis genes (Shin et al., 2007). Plant bZIP transcription factors other than HY5 have been implicated in combinatorial control of gene expression (Lara et al., 2003; Weltmeier et al., 2006). However, only one other report has demonstrated recruitment of a plant bZIP transcription factor by another DNA-binding protein (Chen et al., 1996). By contrast, in other organisms, it is well established that DNA-binding affinity, specificity and enhancement of transcriptional activation by bZIP transcription factors usually require accessory protein factors (Tjian and Maniatis, 1994; Baranger, 1998). Interestingly, in Drosophila, one bZIP transcription factor—PDP1—which appears to function in a complex with the MADS box transcription factor MEF2 to regulate transcription of muscle genes (Lin et al., 1997), also functions within the Drosophila circadian oscillator (Cyran et al., 2003).
We have demonstrated here that HY5 is necessary for proper control of Lhcb1*1 circadian gene expression in Arabidopsis. A shorter period of Lhcb1*1 circadian mRNA oscillations was observed in hy5-null seedlings (Figure 6A). Moreover, HY5 appears to act at the level of the Lhcb1*1 promoter rather than affecting the circadian oscillator, since CAT3 circadian oscillations were not affected in hy5 (Figure 6B). Thus, the hy5 northern data in the present study support the in vitro data and the contention that CCA1 and HY5 interact to control Lhcb gene expression. The idea that HY5 controls circadian expression of Lhcb genes is also supported by a report showing that in etiolated seedlings irradiated with a pulse of red light, the circadian peak of Lhcb1*1 expression is attenuated by 50% in hy5 seedlings compared with wild-type, whereas the acute response (the light-dependent, clock-independent activation of Lhcb1*1 expression) is unaffected (Anderson et al., 1997). Our Lhcb1*1 expression results differ from other studies showing that 5'-deletion or mutation of the G-box element leads to a reduction in overall Lhcb1*1 mRNA levels but has no effect on circadian rhythms (Anderson et al., 1994; Anderson and Kay, 1995). However, deletion or mutation of the G-box element would preclude binding of any transcription factors to this sequence, not just HY5. Other bZIP transcription factors can selectively bind the G-box of the A2 fragment (C. Andronis and E.M. Tobin, unpublished results). In addition, there exists a HY5-like protein—HYH—which has distinct and overlapping functions with HY5, can physically interact with HY5, and can bind as a HY5–HYH complex to the G-box of the RBCS–1A promoter (Holm et al., 2002). Any of these bZIP transcription factors might also play a role in regulating the expression of Lhcb1*1 through its upstream G-box element.
It is tempting to speculate how HY5 and CCA1 might function in controlling circadian expression of Lhcb1*1. The rhythmically oscillating level of CCA1 protein peaks just after dawn (Wang and Tobin, 1998). HY5 protein levels are controlled by the photomorphogenic repressor COP1, which targets HY5 for degradation in the dark (Osterlund et al., 2000). Upon exposure to light, HY5 no longer interacts with COP1 and HY5 protein levels accumulate while COP1 subsequently leaves the nucleus (von Arnim and Deng, 1994). Thus, peak levels of CCA1 might coincide with rising HY5 levels and CCA1 may aid in the recruitment of HY5 to the Lhcb1*1 promoter, leading to induction of Lhcb1*1 expression.
An additional kind of regulation may involve CK2. CK2 has been shown to play a role in regulating the circadian clock of Arabidopsis, Neurospora and Drosophila (Sugano et al., 1999; Akten et al., 2003; Blau, 2003; Daniel et al., 2004). Furthermore, CK2 phosphorylation of CCA1 or interaction of CCA1 with CK2 β-subunits can enhance the DNA-binding activity of CCA1 (Sugano et al., 1998). CK2 can also phosphorylate HY5, leading to reduced interaction with COP1 (Hardtke et al., 2000). We show that incubation of HY5 with the β3-subunit of CK2 enhances HY5 DNA-binding activity (Figure 4B). Thus, HY5 interaction with CK2 regulatory subunits, together with its interaction with CCA1, may lead to strong binding of HY5 to the G-box element and strong induction of Lhcb1*1 expression in the morning.
Taken together, our results suggest that the G-box element and the CBS define part of a circadian-responsive module and that physical interaction of CCA1 and HY5 bound to these elements provides a basis for circadian regulation of Lhcb1*1 by these proteins.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODS |
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Plant Material and Growth
Wild-type, HY5 overexpressor (HY5-OX) (Ang et al., 1998) and hy5 (Koornneef et al., 1980) Arabidopsis seedlings (all in the Landsberg erecta background) were germinated and grown on MS2S medium (Green and Tobin, 1999) at 23°C under a photoperiod of 12 h light (150 µE m–2s–1):12 h dark. For circadian rhythm experiments, wild-type and hy5 seedlings were grown for 12 d and then transferred to continuous light.
Northern Blot Analysis
RNA was extracted from liquid-N2-frozen tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Synthesis of RNA probes was carried out as described (Wang and Tobin, 1998). Hybridization of probes with RNA gel blots was performed as described (Wang et al., 1997).
Expression of Recombinant Proteins
Plasmids pHY5-FULL (Oyama et al., 1997) and pGBF4 (Menkens and Cashmore, 1994) were used to synthesize their respective proteins in vitro in 50 µl of rabbit reticulocyte lysate using a coupled transcription/translation system (Promega TNT system). Glutathione–S-transferase (GST), GST–CCA1 and GST–HY5 were expressed in E. coli strain BL21 (DE3) and purified as described (Frangioni and Neel, 1993; Sugano et al., 1998). Removal of GST from purified GST–HY5 and GST–CCA1 was performed using thrombin and factor Xa, respectively, following the manufacturer's protocols (Amersham Biosciences, Buckinghamshire, UK). CKB3 was produced as described (Sugano et al., 1998).
Preparation of Whole Plant Nuclear Extracts
Nuclear extracts were purified from 2-week-old wild-type, hy5 and HY5-OX seedlings as described (Maxwell et al., 2003).
Electrophoretic Mobility-Shift Assays (EMSAs)
The Lhcb1*3 promoter fragments A2 (Sun et al., 1993), m1 (Wang et al., 1997) and MutG were end-labeled as described (Sugano et al., 1998). MutG is identical to the A2 fragment, except that the ACGT motif was changed to TATT. Nuclear extracts were pre-incubated with 50 mM Tris-HCl (pH 7.5), 0.1 mM Na2EDTA, 5 mM DTT and 2 mM MnCl2 for 5 min on ice. EMSAs were performed as described (Maxwell et al., 2003) except that a 6% bis-acrylamide gel was used for electrophoresis. Specificity of the HY5 antibodies was described previously (Osterlund et al., 2000).
Yeast Two-Hybrid Assay
Yeast expression plasmids and strains were as described (Sugano et al., 1998). The entire coding sequence of HY5 was fused to the GAL4 transcription–activation domain (AD) in pACT (Arabidopsis Biological Resource Center). β-galactosidase assays were carried out by colony filter assay as described (Sugano et al., 1998).
In Vitro Binding Assay
GST pulldown assays were performed as described (Sugano et al., 1998) except that proteins were resolved on a 7.5% bis-acrylamide gel.
Yeast One-Hybrid Assay
A fragment of the Lhcb1*1 promoter spanning –221 to +7 bp relative to transcription start was amplified by PCR using the following primers: Lhcb1*1F, 5'-CGTACCGTTGAAGTATTCAG-3’ and Lhcb1*1R, 5'-CTCGAGAGTGATTAAAACTG-3'. The promoter fragment was ligated into the vector pLacZi (URA marker, Clontech, Mountain View, CA, USA) and sequenced. Full-length HY5 cDNA was subcloned into pGAD424 (LEU marker, Clontech). The AD coding sequence from pGAD424 was subcloned into p10CLA13
400 (TRP marker) to form p10CLA13400–AD and full-length CCA1 cDNA was subcloned into p10CLA13400–AD. All yeast transformations were performed following the manufacturer's protocols (Clontech). β-galactosidase activity was measured using the substrate Chlorophenolred–β-D-galactopyranoside (Roche Molecular Biochemicals, IN, USA) following the manufacturer's protocols (Clontech).
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Acknowledgements |
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We would like to thank Dr T. Oyama (Nagoya University, Japan) for HY5-OX seeds and HY5 antibody and for providing the pHY5-FULL and pGST–HY5 clones. We are grateful to Dr May Ong for providing the MutG DNA probe and to Dr R. Green (The Hebrew University of Jerusalem, Israel) for valuable comments on the manuscript. Funding was provided by the National Institutes of Health (GM–23167 to E.M.T.).
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Notes |
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a These authors contributed equally to this work.
b Present address: Biovista, 34 Rodopoleos St, 16777, Athens, Greece.
c Present address: Albert Katz Dept. Drylands Biotechnology, Jacob Blaustein Insts. for Desert Research, Ben–Gurion University of the Negev, Midreshet Ben–Gurion, 84990, Israel.
d Present address: Plant Disease Resistance Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
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