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Molecular Endocrinology 20 (2): 302-310
Copyright © 2006 by The Endocrine Society

A Single Nucleotide in an Estrogen-Related Receptor {alpha} Site Can Dictate Mode of Binding and Peroxisome Proliferator-Activated Receptor {gamma} Coactivator 1{alpha} Activation of Target Promoters

Janelle B. Barry, Josée Laganière and Vincent Giguère

Molecular Oncology Group (J.B.B., J.L., V.G.), McGill University Health Centre and Departments of Biochemistry (J.L., V.G.), Medicine and Oncology, McGill University, Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: Dr. Vincent Giguère, Molecular Oncology Group, Room H5-21, McGill University Health Centre, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1. E-mail: vincent.giguere{at}mcgill.ca.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The orphan nuclear receptor estrogen-related receptor {alpha} (ERR{alpha}, NR3B1) is a constitutively active transcription factor that controls multiple processes, most notably mitochondrial function. ERR{alpha} preferentially binds to a nine-nucleotide extended half-site sequence TNAAGGTCA, referred to as the ERRE, as either a monomer or a dimer, although how the mode of DNA binding is dictated remains to be determined. Here, we used variants of the extended half-site sequence and selective DNA binding domain mutants of ERR{alpha} to investigate the effects of ERRE sequence specificity on ERR{alpha} DNA binding mode, transactivation and interaction with the coactivator protein peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}). We found that the base at the N position of the TNAAGGTCA sequence dictated ERR{alpha} binding preference as a monomer or dimer. In addition, we demonstrated that the threonine residue at position 124 (Thr124) was a determinant of ERR{alpha} DNA-dependent dimerization. Transfection experiments also indicated that substituting a thymidine for a cytosine at the N position in the ERRE of the native ERR{alpha} target promoter trefoil factor 1 (TFF1) considerably diminished the transcriptional response of the ERR{alpha}/PGC-1{alpha} complex. These results suggest that a single nucleotide in an ERR{alpha} binding site can determine specific configuration to the receptor and productive interaction with the coactivator PGC-1{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ESTROGEN-RELATED receptor {alpha} (ERR{alpha}, NR3B1) was the first orphan member of the nuclear receptor superfamily identified, on the basis of its sequence similarity with the estrogen receptor {alpha} (ER{alpha}, NR3A1) (1). ERR{alpha} is constitutively active and does not respond to estradiol or other natural estrogens (1), although its transcriptional activity can be inhibited by synthetic drugs such as diethylstilbestrol (2) and XCT790 (3). ERR{alpha} may play a role in classical estrogen-responsive tissues (4) because it is a prognostic marker of breast cancer (5, 6), controls the expression of genes in breast cancer cells (7, 8), and is involved in bone remodeling (9, 10, 11, 12). However, the main physiological role for ERR{alpha} may be that of an essential regulator of energy metabolism. ERR{alpha} is expressed in tissues with high ß-oxidation activity including brown fat, kidney, heart, and muscle (1, 13), and its expression is induced in response to stimuli that increase energy demand, including fasting and exposure to cold (14, 15). ERR{alpha} controls the expression of genes involved in mitochondrial biogenesis and other aspects of energy metabolism (13, 16, 17, 18, 19), and ERR{alpha} null mice are resistant to high-fat diet-induced obesity (20). The action of ERR{alpha} in the control of energy metabolism has been shown to be closely linked with that of the coactivator coactivator protein peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}), another key regulator of energy homeostasis (reviewed in Refs.21 and 22). PGC-1{alpha} induces the expression of ERR{alpha} and enhances ERR{alpha} transcriptional activity on both synthetic and natural reporters (15, 18, 23, 24, 25) and has also been described as a repressor of PGC-1{alpha} activity on the phosphoenolpyruvate carboxykinase 1 (PEPCK) promoter (14). Recently, ERR{alpha} and PGC-1{alpha} were also shown to function interdependently on the ESRRA promoter to regulate the level of ERR{alpha} expression (24).

ERR{alpha} is known to recognize DNA as both a dimer and as a monomer. The initial, nonbiased approach used to define the binding site for ERR{alpha} showed that the orphan receptor recognized a nine-nucleotide extended half-site sequence with the consensus TNAAGGTCA, referred to as the ERRE (13). ERR{alpha} can also bind the inverted repeat estrogen response element (ERE) sequence as a dimer (26), although it is not clear whether the ERE is a physiologically relevant binding site for ERR{alpha} in vivo. There is evidence to suggest that ERR{alpha} can bind the ERRE as either a monomer or a dimer. Solution structure studies indicate that ERRß binds the consensus ERRE as a monomer, with the carboxy-terminal extension of the DNA binding domain (DBD) forming a pseudo-dimer interface to stabilize binding by interacting with both the DNA and core DBD residues (27). In addition, EMSA experiments indicate that ERR{alpha} binds a consensus ERRE and the lactoferrin promoter ERRE as a monomer (28, 29). In contrast, it has been shown that ERR{alpha} binds preferentially as a homodimer to the multiple ERREs present in its own promoter (24). ERR{alpha} also dimerizes on the SFRE element (TCAAGGTCG), presumably with one ERR{alpha} molecule tightly contacting the core half-site and the second binding loosely to surrounding flanking sequences (30, 31, 32). The mode of binding may have an important transcriptional outcome because recent studies have shown that DNA-bound ERR{alpha} dimer but not monomer interacts with the coactivator PGC-1{alpha} on DNA (8), and that amphioxus ERR{alpha} homodimer binding to the SFRE is needed to activate transcription through this element (33). Despite this evidence, little is known about how the sequence of the 5' half-site extension may affect the mode of ERR{alpha} DNA binding, nor about which specific residues of the ERR{alpha} DBD directly mediate the DNA binding effects.

In this study, we investigated the effects of ERRE sequence specificity on the mode of ERR{alpha} DNA binding because evidence suggests it can bind to distinct ERRE elements as a monomer or a dimer. Because of the paucity of information about which residues of the ERR{alpha} DBD mediate its DNA binding, we also mutated selected residues and assessed their effects on ERR{alpha} DNA binding, transactivation and PGC-1{alpha} interaction. We found that the base at the N (–2) position of the TNAAGGTCA extended half-site sequence dictated ERR{alpha} binding preference as a monomer or dimer. In addition, the threonine residue at position 124 (Thr 124) was found to be important for ERR{alpha} DNA-dependent dimerization. Transfection experiments indicated that one nucleotide in synthetic or natural ERREs and Thr124 dictated ERR{alpha}/PGC-1{alpha} transactivation potential. This study thus demonstrates that the sequence of the ERRE determines how ERR{alpha} recognizes DNA and whether it will form a productive interaction with its main coactivator partner, PGC-1{alpha}, on an ERR{alpha} target promoter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ERRE Sequence and DBD Residue Thr124 Dictate ERR{alpha} Mode of DNA Binding
Our initial unbiased analysis of the ERR{alpha} monomeric binding site showed it to be in the form of the consensus extended half-site sequence TNAAGGTCA. In this site was a preference for C, G, or A at the N' or –2 position, whereas T was observed with a very low frequency (4%) (13). Furthermore, structural studies have shown that, in contrast to other monomeric DNA binding receptors such as NGFI-B (NR4A1), ERR{alpha} can accommodate a C or G nucleotide at the –2 position (27). However, the functional consequence of ERR{alpha} being able to recognize both C/G and A/T pairs at this position has not been investigated. Therefore, to further explore the effects of ERRE sequence specificity on the mode of ERR{alpha} DNA binding and transactivation, consensus ERRE probes containing either a C, G, T, or A at this position were generated. Structural studies have also shown that the threonine residue present in the D-box of ER{alpha}, conserved in ERRß and ERR{gamma}, and substituted for a serine residue (Ser118) in ERR{alpha} (Fig. 1AGo), participates actively in forming the dimer interface of the ER{alpha} homodimer when bound to an ERE (34). Residue Thr124 was also chosen because its corresponding amino acid in ERRß (Thr148) has been shown using solution structure studies to move considerably after DNA binding (27). This study suggested that the observed change in chemical shift for Thr148 in ERRß reflects DNA-induced structure formation in the DBD, which may have an impact on the function of the receptor. The residues Ser118 and Thr124 were thus mutated individually to alanine, and their effects on DNA binding to the above-mentioned ERREC, -G, -T or -A probes were assessed using EMSA experiments. ERR{alpha} expressed in COS-1 cells bound to the ERREC probe as both a monomer and dimer (Fig. 1CGo), with a greater intensity of monomeric binding. Both species were supershifted with an antibody directed against ERR{alpha}. The ERR{alpha}S118A mutant behaved the same as ERR{alpha}, with monomer and dimer bands of equal intensity to ERR{alpha} being shifted by the antibody. The ERR{alpha}T124A mutant behaved differently, binding the ERREC element predominantly as a monomer. In addition, ERR{alpha}T124A was not able to produce the same dimer species as ERR{alpha} on ERREC. Equivalent expression of ERR{alpha} and the mutant proteins was confirmed by Western blotting (Fig. 1BGo). A similar result was obtained using the ERREG probe (Fig. 1DGo), with a higher monomer to dimer ratio for ERR{alpha} and ERR{alpha}S118A but greatly reduced binding for ERR{alpha}T124A. In EMSA using the ERRET probe (Fig. 1EGo), the ratio of monomer to dimer binding was reversed for ERR{alpha} and the ERR{alpha}S118A mutant. Moreover, the ERR{alpha}T124A mutant was now unable to bind to the ERRET as a monomer, although its dimeric binding was similar in intensity to ERR{alpha}. A similar pattern was observed on the ERREA element (Fig. 1FGo). These data suggest that the sequence of the ERRE element determines ERR{alpha} preference for dimeric or monomeric binding, with more monomer binding to ERREC or -G and preferential dimeric binding to ERRET or -A. These results also indicate that, although Thr124 does not contact DNA, the residue plays a role in determining ERR{alpha} dimerization that is dependent on the primary sequence of ERRE.


Figure 1
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  		Fig. 1. ERRE Sequence and DBD Residue Thr124 Regulate ERR{alpha} DNA Binding

A, Schematic representation of the ERR{alpha} DBD highlighting the two zinc fingers, the P-box, the D-box, and the carboxy-terminal extension (CTE). Residues Ser118 and Thr124 (denoted by arrows) were mutated to alanine. B, Western blot (WB) of nuclear extracts with the ERR{alpha} antibody. C, EMSA of nuclear extracts from COS-1 cells transiently transfected with ERR{alpha}, ERR{alpha}S118A, or ERR{alpha}T124A constructs. The ERREC probe was used, which contains a C at the N position of the TNAAGGTCA extended half-site sequence. Extracts were supershifted with the ERR{alpha} antibody (Ab). M and D denote monomer and dimer bands, respectively. D–F, EMSA as in C, but with ERREG, -T or -A probes, respectively.

 
ERR{alpha}T124A Has Defective Dimerization Activity that Is DNA Dependent
As mentioned above, the residue equivalent to Ser118 in ER{alpha} is involved in the formation of the dimer interface when ER{alpha} is bound to the ERE, whereas Thr124 clearly participate in dictating the dimerization potential of ERR{alpha} on the ERRE. To further investigate the role of these residues in ERR{alpha} recognition of specific response elements, we next performed EMSA using an ERE element, to which ERR{alpha} binds exclusively as a dimer. As expected, ERR{alpha} bound to the ERE as a dimer (Fig. 2AGo), whereas, in contrast to its role in ER{alpha} dimer formation, mutation of Ser118 did not affect ERR{alpha} binding to the ERE. However, binding to the ERE was completely abolished for the ERR{alpha}T124A mutant, indicating an unsuspected and important role for this residue in dimerization.


Figure 2
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  		Fig. 2. ERR{alpha}T124A Has Defective Dimerization Activity that Is DNA Dependent

A, EMSA of nuclear extracts expressing ERR{alpha}, ERR{alpha}S118A, or ERR{alpha}T124A on the ERE probe. Extracts were supershifted with the ERR{alpha} antibody (Ab). B, Coimmunoprecipitation of COS-1 extracts expressing HA-ERR{alpha} or HA-ERR{alpha}T124A without or with ECFP-ERR{alpha}. Lysates were immunoprecipitated (IP) with the anti-HA polyclonal antibody. Electrophoresed products were blotted with the anti-GFP (green fluorescent protein) antibody and then with the anti-HA monoclonal antibody. WB, Western blot.

 
To examine whether the defective dimerization function of ERR{alpha}T124A is dependent on DNA binding, coimmunoprecipitation was performed using transfected HA-ERR{alpha} or HA-ERR{alpha}T124A and ECFP-ERR{alpha} (Fig. 2BGo). ERR{alpha} and ERR{alpha}T124A were immunoprecipitated with the HA antibody, and ECFP-ERR{alpha} was coimmunoprecipitated equivalently with ERR{alpha} and ERR{alpha}T124A. These data indicate that ERR{alpha} and ERR{alpha}T124A dimerize equivalently in solution, and confirm that the role played by Thr124 in dimerization is DNA dependent.

Dimeric DNA Binding Dictates ERR{alpha}/PGC-1{alpha} Transactivation and Interaction
The data obtained with the different ERREs and ERR{alpha} DBD mutants suggest that the DBD and potentially the receptor as a whole undergoes a significant ERRE-dependent change in conformation that could influence the interaction with coactivators and the transactivation potential of the receptor. We therefore performed transient transfections with ERR{alpha} or the DBD mutants, together with the coactivator PGC-1{alpha} and reporter constructs driven by ERREC or ERRET previously used in EMSA. PGC-1{alpha} displayed high activity on the ERREC TkLUC reporter even in the absence of ERR{alpha} (Fig. 3AGo). We and others (15, 24) have previously shown that this activity is transduced via endogenous ERRs. Surprisingly, cotransfection of ERR{alpha} on the ERREC (Fig. 3AGo) reporter inhibited the activity of PGC-1{alpha}. The ERR{alpha}S118A mutant produced the same inhibitory effect as ERR{alpha}. In sharp contrast, ERR{alpha}T124A was unable to inhibit PGC-1{alpha} activity on this reporter. ERR{alpha} and ERR{alpha}S118A, but not ERR{alpha}T124A, can dimerize effectively on this ERRE (Fig. 1Go). These results suggest that the inhibitory effect observed with ERR{alpha} and ERR{alpha}S118A on PGC-1{alpha}-dependent transactivation is due to squelching of the endogenous active receptor species by ectopically expressed ERR{alpha}. However, cotransfection of ERR{alpha} or ERR{alpha}S118A on the ERRET reporter (Fig. 3BGo) did not significantly affect the constitutive activity of PGC-1{alpha}, which is lower on this element. In addition, introduction of PGC-1{alpha} led to a superactivation of the ERRET-based reporter in the presence of ERR{alpha}T124A. The enhanced ERR{alpha}T124A/PGC-1{alpha} transactivation compared with ERR{alpha} suggests that ERR{alpha}T124A, which can dimerize effectively on ERRET, may assume a conformation on this element that selectively enhances its interaction with PGC-1{alpha}.


Figure 3
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  		Fig. 3. ERR{alpha}T124A Regulates ERR{alpha}/PGC-1{alpha} Transactivation

A, Transient transfection of COS-1 cells with constructs expressing ERR{alpha}, ERR{alpha}S118A, or ERR{alpha}T124A alone or with PGC-1{alpha}. The reporter construct ERRECtkLUC, which contains three copies of the TCAAGGTCA extended half-site sequence, was cotransfected. B, COS-1 cells were transfected as in A except with the reporter ERRETtkLUC. In all transfections, the measured luciferase activity was corrected using the ß-galactosidase transfection efficiency control. All results are representative of three independent transfections performed in duplicate. RLU, Relative light units.

 
To further test the dependence of receptor dimerization on ERR{alpha}/PGC-1{alpha} transcriptional activity, EMSA experiments were performed using a PGC-1{alpha} supershift. As in Fig. 1Go, both ERR{alpha} and ERR{alpha}T124A bound to ERREC (Fig. 4AGo) more as monomers than dimers. ERR{alpha}T124 dimerization was again limited, with no appearance of the fastest migrating dimer species as seen with ERR{alpha}. In the case of both ERR{alpha} and ERR{alpha}T124, dimer bands were supershifted with PGC-1{alpha}, whereas monomer bands remained unchanged. On the ERRET probe (Fig. 4BGo), both ERR{alpha} and ERR{alpha}T124 bound strongly as dimers. PGC-1{alpha} bound to and supershifted both ERR{alpha} and ERR{alpha}T124A dimers but did not alter the limited ERR{alpha} monomer migration. These data clearly indicate that receptor dimerization on DNA is required for PGC-1{alpha} interaction. They also provide a structural basis for the transfection data that suggest ERR{alpha} dimerization is needed for transactivation involving PGC-1{alpha}.


Figure 4
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  		Fig. 4. Receptor Dimerization Is Necessary for ERR{alpha}/PGC-1{alpha} Binding

EMSA of nuclear extracts from COS-1 cells transiently transfected with ERR{alpha} or ERR{alpha}T124A constructs on ERREC (A) or ERRET (B) probes. Extracts were supershifted with the ERR{alpha} antibody (Ab) to confirm specificity of the shifted bands. Extracts were also incubated with purified GST-PGC-1{alpha} (P). M, D, and S denote monomer, dimer, and PGC-1{alpha} supershifted bands, respectively.

 
One Nucleotide in the ERRE Can Dictate Target Promoter Activation by ERR{alpha}
Having found that a single nucleotide can change how ERR{alpha} activates transcription on synthetic reporter elements, we investigated whether a similar change could influence ERR{alpha} function on a native promoter. Transient transfections were performed using the TFF1 (trefoil factor 1, also known as pS2) promoter reporter construct previously shown to be responsive to ERR{alpha} (7). On the TFF1 promoter (Fig. 5AGo), PGC-1{alpha} strongly enhanced the transactivation activity of ERR{alpha}. The TFF1 promoter contains both an ERRE and an ERE (7), so to confirm that the effects of PGC-1{alpha} were through the ERRE, this element was deleted. The effect of PGC-1{alpha} on ERR{alpha} transactivation was almost completely abolished, indicating that the response is indeed dependent on ERR{alpha} binding to the ERRE (Fig. 5AGo). The TFF1 promoter ERRE contains a T at the N base of the TNAAGGTCA extended half-site sequence. To further understand the importance of ERR{alpha} target gene ERRE sequence on ERR{alpha}/PGC-1{alpha} transactivation, the N base of TNAAGGTCA was mutated in the context of the native TFF1 promoter, with the sequence being changed from TTAAGGTCA to TCAAGGTCA. The transactivation activity of ERR{alpha} either alone or in combination with PGC-1{alpha} was significantly inhibited on the TFF1ERREC promoter (Fig. 5BGo). This result confirms the suggestion that ERR{alpha} dimer is the active form of the receptor, known because changing the ERRE from an element on which ERR{alpha} preferentially dimerizes (T) to one with monomer preference (C) significantly reduces transactivation on this target promoter. These data also indicate that the ERRE sequence TTAAGGTCA within the native TFF1 promoter context is highly important for efficient ERR{alpha}/PGC-1{alpha} activation.


Figure 5
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  		Fig. 5. ERRE Sequence Determines ERR{alpha}/PGC-1{alpha} Response on the TFF1 Promoter

A, COS-1 cells were transfected with constructs expressing ERR{alpha} alone or with PGC-1{alpha}. The TFF1LUC or TFF1LUC{Delta}ERRE reporter was cotransfected. B, COS-1 cells were transfected with the reporters TFF1LUC or TFF1LUC-ERRE-C, the latter of which contains a single point mutation changing the ERRE from TTAAGGTCA (TFF1) to TCAAGGTCA (T->C) in the context of the normal TFF1 promoter. Constructs expressing ERR{alpha} either alone or with PGC-1{alpha} were cotransfected. In all transfections, the measured luciferase activity was corrected using the ß-galactosidase transfection efficiency control. All results are representative of three independent transfections performed in duplicate. RLU, Relative light units.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Previous studies indicate that ERR{alpha} can bind as a monomer or dimer to EREs and distinct ERREs, either synthetic or those contained in a variety of ERR{alpha} target promoters. In addition, these reports have described ERR{alpha} as having a potent constitutive transcriptional activity, showing no activity when transfected in the absence of coactivators or even behaving as a transcriptional repressor (see introductory text). Several factors may contribute to explaining these findings. These include the presence or absence of endogenous ligand or cell-specific coregulatory proteins, and the particular ERR{alpha} binding site used in the study. In this work, we examined the role of particular sequences associated with distinct ERR{alpha} binding sites. Our results showed that the sequence of the ERR{alpha} site is responsible for determining the mode of ERR{alpha} binding, and that dimerization is required for a productive interaction with the coactivator PGC-1{alpha}.

We found that the nucleotide at the N' position (–2) of the TNAAGGTCA ERRE extended half-site sequence influenced ERR{alpha} preference for monomeric or dimeric DNA binding. We also found that residue Thr124, an amino acid that undergoes significant chemical shift upon DNA binding (27), is an element-specific determinant of ERR{alpha} dimeric DNA binding and is important in the PGC-1{alpha} activation of sequence-specific ERRE containing reporters. In particular, we showed that ERR{alpha} has a preference for monomeric DNA binding on a C- or G-containing TNAAGGTCA element, and a preference for dimer on ERREA or -T (Fig. 1Go). These data indicate that the mode of ERR{alpha} ERRE binding is dependent on the extended half-site sequence and may help to explain the varying reports of monomeric vs. dimeric binding on assorted ERREs. In addition, the ERR{alpha}T124A mutant has attenuated dimeric DNA binding and normal monomer binding on ERREC and -G, whereas it dimerizes but cannot bind as a monomer on ERREA or -T (Fig. 1Go). Both the ERR{alpha} and ERR{alpha}T124A results indicate that the receptor assumes distinct conformations upon binding to the different elements. Taken together, these results demonstrate that the sequence of the ERR{alpha} site carries information that is conveyed to the bound ERR{alpha}, changing its configuration, and determining whether PGC-1{alpha} will form a productive interaction with ERR{alpha}.

There is considerable evidence that nuclear receptor conformational changes occur after DNA binding, for example with the retinoid X receptor {alpha} (NR2B1), changes occur within the D-box and the second zinc finger, as well as at more carboxy-terminal sites (35, 36). For the thyroid hormone receptor/retinoid X receptor heterodimer, it has been shown that varying the sequence of the binding element changes the conformation and transcriptional activity of the heterodimer (37). Similarly, specific sequences within EREs modulate the conformation of bound ER{alpha} and ERß, resulting in differential coactivator recruitment to the bound receptors (38, 39, 40). ERR{gamma} (NR2B3) assumes distinct conformations on the stf4 and SF-1RE ERRE consensus elements, which vary in sequence in the conserved 6-bp part of the half-site. This change also alters the receptor affinity for coactivator (41). In addition, solution structure studies with ERRß indicate that DNA binding induces the carboxy-terminal extension to form a pseudo-dimer interface by interacting with both the DNA and core DBD residues (27). Our study suggests that the conformation of ERR{alpha} bound on ERREC or -G presumably makes the major dimerization interface less exposed, resulting in preferential monomer binding. In contrast, DNA binding on ERRET or -A would induce a receptor conformation such that the dimerization interface is more accessible. In the case of ERR{alpha}T124A, mutation of the threonine residue must disrupt the DNA-induced movement that is normally seen in this region of the DBD. This disruption presumably results in a less exposed dimerization interface on ERREC and -G, whereas on ERREA and -T it directly interferes with monomeric DNA binding.

In addition to showing that the extended half-site sequence dictates ERR{alpha}/PGC-1{alpha} activation of synthetic ERRE reporters, we have found that a single base change within the ERRE dictates activation of a native ERR{alpha} target promoter (Fig. 5Go). Mutation of the TFF1 promoter ERRE from TTAAGGTCA to TCAAGGTCA significantly inhibits ERR{alpha} and ERR{alpha}/PGC-1{alpha} activation. The TFF1 results indicate that a T rather than a C in the ERRE is required for full ERR{alpha} activation of this promoter. The TFF1 promoter contains a single ERRE; however, some target promoters such as ESRRA possess multiple ERREs (24). It is currently not known at the moment whether the presence of multiple ERREs may promote ERR{alpha} dimer binding or oligomerization and thus diminish or totally alleviate the need for sequence specificity. Finally, in the case of the TFF1 promoter, the more efficient activation observed with the T-containing half-site likely relates to the finding that ERR{alpha} binds preferentially to ERRET than ERREC as a dimer, this being the functional form of the receptor.

We have identified a residue within the ERR{alpha} DBD that is an important determinant of receptor dimerization. This is an effect dependent on DNA binding because ERR{alpha}T124A dimerization in solution is equivalent to the wild-type receptor (Fig. 2Go). Residue Thr124 is also an important determinant of ERR{alpha}/PGC-1{alpha} transactivation activity on sequence-specific ERRE reporters (Fig. 3Go). The inability of ERR{alpha}T124A to inhibit PGC-1{alpha} activity on ERREC likely relates to its defective dimerization function because dimeric DNA binding is needed for PGC-1{alpha} binding (Fig. 4Go). Conversely, the increased transactivation potential of ERR{alpha}T124A/PGC-1{alpha} on ERRET element, on which ERR{alpha}T124A dimerizes efficiently, suggests that this mutant has a conformation here with higher coactivator affinity than ERR{alpha}. Thr124 has the potential to be an important residue in the regulation of ERR{alpha} target gene expression. The residue is exposed and moves significantly after DNA binding (27). It is also a potential candidate residue for posttranslational modification because the DBD of ERR{alpha} is phosphorylated by protein kinase C {delta} (8), and Thr124 lies within a consensus protein kinase C {delta} phosphorylation site. Posttranslational modification of orphan nuclear receptors in the DBD is known to change their DNA binding and receptor conformation (42, 43, 44). In addition, EGF-induced phosphorylation of ERR{alpha} in the DBD induces selective activation of ERR{alpha} target genes (8). It would be of interest to investigate whether posttranslational modification of Thr124 could influence DNA-induced conformational changes in the DBD and thus be involved in controlling activation of ERR{alpha} target genes.

In conclusion, we have found that a single nucleotide in the extended ERRE half-site can determine the mode of DNA binding by ERR{alpha}. Significantly, the half-site sequence is also important for efficient activation of the TFF1 promoter by ERR{alpha} and PGC-1{alpha}. In addition, we have shown that residue Thr124 is an important regulator of ERR{alpha} dimeric DNA binding and PGC-1{alpha}-mediated activation of selected ERR{alpha} target sequences. Our study thus highlights the functional importance of the sequence of the ERRE in determining how ERR{alpha} can regulate gene expression and achieve its developmental and physiological functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mutagenesis and Cloning
The constructs pCMXERR{alpha}S118A and pCMXERR{alpha}T124A were prepared by PCR-based mutation of pCMXERR{alpha}, introducing a single residue change to alanine at Ser118 or Thr124. PCR products were digested with Kpn1 and Sph1 and cloned into the Kpn1 and Sph1 sites of pCMXERR{alpha} that had been digested to remove the wild-type sequence. The constructs pCMXHA-ERR{alpha} and pCMXHA-ERR{alpha}T124A were generated by PCR cloning using pCMXERR{alpha} and pCMXERR{alpha}T124A as templates. Primers were generated to incorporate the HA tag sequence at the 5' end and products were cloned into the Kpn1 and EcoRI sites of pCMX. The pECFP-ERR{alpha} fusion construct was made by cloning the ERR{alpha} cDNA as a BamH1/EcoRI fragment from glutathione-S-transferase (GST)-human ERR{alpha} (hERR{alpha}) into the BglII/EcoRI sites of pECFP-C1 (Promega, Madison, WI). The reporter construct ERRETTkLUC was generated using an oligonucleotide containing three copies of the sequence 5'-TCGACGCTTTTAAGGTCATATGCG-3', which has a T at the N position of the extended half-site sequence. Oligonucleotides were cloned into the Sal1 and BamH1 sites of pTkLUC. The reporter TFF1LUC-ERRE-C was generated by PCR-based mutation of TFF1LUC to change the underlined T of the TTAAGGTCA ERRE sequence to C. Products were digested with Kpn1 and HindIII and cloned into the Kpn1 and HindIII sites of pGL3 (Promega).

EMSA
EMSAs were performed as described (45) using the consensus ERRE probe 5'-TCGACGCTTTNAAGGTCATATGCG-3' with the nucleotides C, T, G or A at the N position of the underlined extended half-site sequence. The sequence of the ERE probe was 5'-TCGACAAAGTCAGGTCA-CAGTGACCTGATCAAG-3'. Nuclear extracts were prepared from COS-1 cells that had been transiently transfected with 5 µg pCMXhERR{alpha}, Ser118A or Thr124A for 16 h. Two micrograms of extract were used per binding reaction. Four microliters of the ERR{alpha} polyclonal antibody (24) was used for supershift reactions. For PGC-1{alpha} supershift experiments, extract was combined with 2 µg of purified GST-PGC-1{alpha} (8).

Western Blot Analysis
Nuclear extracts prepared for EMSA were electrophoresed, transferred to polyvinylidene difluoride membranes, and blotted in 5% skim milk powder/PBST with the hERR{alpha} antibody (24).

Coimmunoprecipitation
COS-1 cells were plated in 10-cm dishes and cotransfected with 2.5 µg pCMXHA-ERR{alpha} or Thr124A and 2.5 µg pECFP-ERR{alpha} for 40 h. Whole cell extracts were prepared using modified RIPA buffer, and 200 µg lysate was immunoprecipitated with the anti-HA polyclonal antibody (Upstate Biotechnology, Lake Placid, NY). Immunoprecipitated proteins were electrophoresed, transferred to polyvinylidene difluoride membranes, and blotted with the anti-GFP polyclonal antibody (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) in 20% horse serum/PBST. Membranes were stripped in 10 mM Tris/150 mM NaCl (pH 2.3) and blotted with the anti-HA monoclonal antibody (Covance Research Products, Denver, PA) in 3% skim milk powder/PBST.

Cell Culture and Transient Transfection
COS-1 cells were maintained in DMEM with 10% fetal bovine serum. Cells were plated in 12-well plates in DMEM with 10% charcoal-stripped fetal bovine serum, and transfections were performed using FuGene 6 (Roche Diagnostics, Indianapolis, IN). Cells were transfected with 100 ng pCMXERR{alpha} construct, 200 ng PGC-1{alpha} expression construct, 400 ng Luciferase reporter, and 100 ng pCMVßgal transfection efficiency control plasmid. The synthetic ERRE reporter constructs were ERRECTkLUC and ERRETTkLUC. The promoter reporter constructs were TFF1LUC, TFF1LUC{Delta}ERRE, (7), and TFF1LUC-ERREC. Cells were transfected for 40 h and harvested for Luciferase and ß-galactosidase activity.


   FOOTNOTES
 
Financial support was provided by the Canadian Institutes for Health Research (CIHR). J.L. is a recipient of a U.S. Department of Defense Breast Cancer Research Program Predoctoral Traineeship Award (No. W8IWXH-04-1-0399). J.B. was supported by an internal fellowship of the McGill University Health Centre.

First Published Online September 8, 2005

Abbreviations: DBD, DNA binding domain; ER{alpha} or NR3A1, estrogen receptor {alpha}; ERE, estrogen response element; ERR{alpha} or NR3B1, orphan nuclear receptor estrogen-related receptor {alpha}; ERRE, nine-nucleotide extended half-site sequence TNAAGGTCA; GST, glutathione-S-transferase; hERR{alpha}, human ERR{alpha}; PGC-1{alpha}, peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}); TFF1, trefoil factor 1; Thr124, threonine residue at position 124.

Received for publication August 1, 2005. Accepted for publication September 1, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERRα
Coregulators:   PGC-1



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