Abstract
The expression of estrogen-related receptor-α (ERRα) is stimulated by estrogen in selective tissues. Recently, a correlation between ERRα expression and the induction of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in the liver of fasting animals and in cold-stressed brown-fat tissues and skeletal muscle was shown. To explore the molecular mechanisms of ERRα regulation by diverse signals, the promoter of the human ERRα gene was cloned and characterized. Mutation and deletion analyses revealed that a 53 bp region containing repeated core element AGGTCA motifs of the ERRα gene serves as a multi-hormone response element (MHRE) for several nuclear receptors in transient co-transfection studies of human endometrial carcinoma (HEC-1B) cells. Among the nuclear receptors tested, ERRγ bound to and robustly stimulated the transcription of reporters containing at least two AGGTCA motifs. Ectopic expression of PGC-1α in HEC-1B cells strongly activated the reporter containing the MHRE, presumably via the endogenous nuclear receptor binding to the element. Reducing the endogenous level of ERRγ by small interfering RNA, and increasing the ERRγ level by ectopic expression, substantially decreased and increased respectively the transactivation capability of PGC-1α. The activation function 2 domain of the ERRγ and the L2 and L3 motifs of PGC-1α were essential to transactivate the MHRE. Additionally, PGC-1α increases the amount of endogenous ERRγ bound to the MHRE region as determined by a chromatin immunoprecipitation assay. The present study demonstrates that the MHRE of the ERRα gene is a target for ERRγ transactivation, which is enhanced by PGC-1α.
Introduction
Nuclear receptors constitute an important group of transcription factors that regulate diverse biological functions through multiple signaling pathways (see reviews by Tsai & O’Malley (1994) and Mangelsdorf et al.(1995) and references therein). The activities of many nuclear receptors are controlled by their respective ligands; however, most members have either no or unidentified ligands, and these have been collectively named orphan receptors (see review by Giguere (1999) and references therein). A subfamily close to the estrogen receptor (ER), estrogen-related receptors (ERRα/ NR3B1; ERRβ/NR3B2) was cloned 15 years ago (Giguere et al. 1988) as orphan receptors and a third member (ERRγ/NR3B3) was cloned recently (Hong et al. 1999, Heard et al. 2000). Whether or not a ligand is required for this subfamily remains unclear and controversial. The ERRα has been reported to function in the absence of ligand (Xie et al. 1999, Zhang & Teng 2000) as well as in the presence of a ligand such as serum factors (Vanacker et al. 1999a,b) or protein ligand (Kamei et al. 2003). In contrast, ERRγ consistently functions as a positive activator without exogenous ligand in transient co-transfection experiments (Hong et al. 1999, Coward et al. 2001, Sanyal et al. 2002). Nonetheless, inverse agonists for the ERRs were found. The synthetic estrogen, diethylstilbestrol binds all three ERRs, interrupts the receptor–coactivator interaction and antagonizes the ERRs’ transactivation activities (Tremblay et al. 2001b). In addition, 4-hydroxytamoxifen binds ERRβ and ERRγ (Coward et al. 2001, Tremblay et al. 2001a) whereas micromolar concentrations of some pesticides bind ERRα (Yang & Chen 1999) and inhibit their trans-activation function. Recently, a specific synthetic inverse agonist of ERRα was reported (Mootha et al. 2004).
The expression pattern of ERRα and ERRγ in human and mouse is both overlapping and different (Shi et al. 1997, Shigeta et al. 1997, Hong et al. 1999, Heard et al. 2000). ERRβ is primarily expressed in the embryo and the ERRβ-null mouse is embryonically lethal due to placental defects (Luo et al. 1997). The biological function of ERRγ is not clear, but the roles of ERRα in cellular physiology are emerging. ERRα was found to bind to a TCAAGGTCA element in the human lactoferrin gene promoter and to modulate the estrogen response (Yang et al. 1996). An extensive analysis of ERRα binding preference suggests that this receptor could bind a variety of estrogen-response elements (EREs) (Johnston et al. 1997, Sladek et al. 1997) and regulate similar ER target genes, thus implicating a modulatory role in ER-mediated signaling pathways (Zuo & Mertz 1995, N Yang et al. 1996, C Yang et al. 1998, Giguere 2002, Teng 2002). In addition, ERRα is involved in bone morphogenesis (Vanacker et al. 1998a, Bonnelye et al. 2002) and in energy balance (Sladek et al. 1997, Vega & Kelly 1997, Vanacker et al. 1998b, Vega et al. 2000, Huss et al. 2002, Luo et al. 2003).
Recently, the expression of ERRα was found to be upregulated in mouse uterus and heart by estrogen (Liu et al. 2003) as well as in liver by fasting (Ichida et al. 2002) and in brown fat by exposure to cold (Schreiber et al. 2003). ERRα expression after fasting and cold treatment correlates with the expression of physiological stimuli-inducible peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α (PGC-1α) (Ichida et al. 2002, Schreiber et al. 2003). The mouse PGC-1α was initially identified in brown fat as a coactivator for PPARγ (Puigserver et al. 1998) and the human homologue was subsequently cloned (Knutti et al. 2000). PGC-1α interacts with an array of nuclear receptors by enhancing their transactivation function (Kressler et al. 2002) and importantly, it coordinately regulates genes involved in adaptive thermogenesis and serves as a ‘master’ regulator of cellular energy metabolism (see reviews by Knutti & Kralli (2001) and Puigserver & Spiegelman (2003) and references therein). While searching for proteins that interact with PGC-1α, ERRα was identified as a major interacting partner (Huss et al. 2002, Ichida et al. 2002). More recent evidence demonstrated that PGC-1α and ERRα work in concert to regulate mitochondrial biogenesis (Schreiber et al. 2004) and the oxidative phosphorylation program (Mootha et al. 2004), by directly influencing the expression of subsets of these genes.
The promoter of ERRα gene contains a multi-hormone response element (MHRE) that is a target site for ERα in the estrogen response (Liu et al. 2003). This region also binds ERRα itself and serves as autoregulatory site in the PGC-1α-induced response (Laganiere et al. 2004, Mootha et al. 2004). Since the DNA-binding domain of ERRγ and ERRα are highly conserved (93%) and both receptors are coexpressed in high-energy demanding tissues such as skeletal muscle, heart and kidney, it seems likely that ERRγ would bind and regulate ERRα gene expression. In this study, we demonstrated that ERRγ is a potent activator for ERRα gene expression, which is enhanced by PGC-1α. We show that PGC-1α enhances ERRγ binding to the MHRE, suggesting a potential mechanism for PGC-1α regulation of ERRα gene expression.
Materials and methods
Plasmids
ERRγ, ERRγ449 (deletion of the activation function 2 (AF2) domain, AF2Δ), Myc-ERRγ, and glutathione S-transferase (GST)-ERRγ (Hentschke et al. 2002b) were gifts from U Borgmeyer (University of Hamburg, Germany), ERα and ERβ (Liu et al. 2003), RXRα, RARα and TRα from A Jetten (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA (NIEHS)), PPARα from C Weinberger (NIEHS), RORα, RORβ and RORγ from V Giguere (McGill University, Canada, Montreal, Quebec) and chicken ovalbumin upstream promoter-transcription factor-1 (COUP-TFI) from M Tsai (Baylor College of Medicine, Houston, TX, USA). Coactivator PGC-1α and its mutant version, pcDNA3/HA hPGC-1α, pcDNA3/HA hPGC-1 L2A, pcDNA3/HA hPGC-1 L3A, and pcDNA3/HA hPGC-1 L2A/3A (Huss et al. 2002, Schreiber et al. 2003) were obtained from A Kralli (Scripps Research Institute, La Jolla, CA, USA) and D Kelly (Washington University School of Medicine, St Louis, MO, USA). The human ERRα promoter reporter constructs (0.6-CAT and 0.8-CAT) and the MHREs and its mutant versions (AAB, AB, A, m1, m2, m3, m4 in SV40-CAT) were described before (Liu et al. 2003). The AAB-TATA-Luc was constructed by excising out the AAB element from the AAB-SV40-CAT construct with NheI/XhoI digestion and then cloning into pLuc-MCS (Stratagene, La Jolla, CA, USA) reporter at the XhoI site by blunt-end ligation.
Cell culture and transient transfection
HEC-1B (ATCC# HTB-113, endometrial), HepG2 (ATCC# CRL-8024, liver), PLC/PRF/5 (ATCC# HB-8065, liver) and HeLa (ATCC# CCL-2, cervical) and MCF-7 (ATCC# HTB-22, mammary gland) cells were maintained in Eagle’s MEM. The MCF-7 cells were supplemented with 10 μg/ml insulin. The HEK293 (ATCC# CRL-1573, kidney) cells were cultured in Dulbecco’s MEM medium. All cells were cultured in the presence of 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin at 37 °C under 5% CO2. To investigate the reporter activities in HEC-1B cells, the transfections were carried out with Qiagen Effectene transfection reagent (Qiagen, Valencia, CA, USA) according to the provider’s instruction. The total DNA transfected in each experiment was kept constant with reporter constructs (300 ng/well), internal control PCH 110 plasmid (100 ng/well), expression plasmids (specified in individual experiments) and the carrier DNA to make the total amount of 500 ng. Prior to transfections, cells were plated in six-well plate and grown overnight in medium containing 10% dextran-coated charcoal-stripped serum. Cells were collected 36 h after transfection and the CAT or Luc activities measured (Liu et al. 2003). The reporter activities were normalized by β-galactosidase activities. Qiagen Trans-Messenger Transfection reagent (Qiagen) was used in the experiments intended for quantitative real-time PCR determination, ERRγ mRNA reduction, and the chromatin immunoprecipitation (ChIP) assays.
Transient transfection of small interfering RNA (siRNA)
Synthesized siRNA was ordered from Qiagen-Xeragon (Qiagen, Germantown, MD, USA). The siRNA duplex (500 ng) was mixed with TransMessenger transfection reagent and transfected into the cells for 48 h. The sequence of the control siRNA (non-silencing) is 5′-AAT TCT CCG AAC GTG TCA CGT-3′ and the ERRγ siRNA (specific silencing) is 5′-AAT GGC CAT CAG AAC GGA CTT-3′. The effect of ERRγ mRNA reduction on the ERRα gene activity was determined by first introducing the siRNA (500 ng) to the cells for 24 h and again with the transfection mixture (300 ng reporter plasmid, 100 ng internal control plasmid) for 36 h before the cells were collected and the Luc activity measured.
Quantitative real-time PCR and RT-PCR
The total RNA was extracted with Qiagen RNeasy Mini Kit according to the supplier’s protocol (Qiagen). Quantitative real-time PCR was used to measure the ERRγ and ERRα mRNA levels in HEC-1B cells under various experimental conditions. The primer pair is as follows: for human ERRγ, forward primer 5′-GGC CAT CAG AAC GGA CTT G-3′ and reverse primer 5′-GCC CAC TAC CTC CCA GGA TA-3′ (67 bp amplicon); for human ERRα, forward primer 5′-GGC CCT TGC CAA TTC AGA-3′ and the reverse primer 5′-GGC CTC GTG CAG AGC TTC T-3′ (79 bp amplicon). The 144 bp amplicon of human β-actin was detected with the forward primer 5′-GAC AGG ATG CAG AAG GAG ATC AC-3′ and the reverse primer 5′-GCT TCA TAC TCC AGC AGG-3′. The quantitative real-time PCR method has been previously described in detail (Liu et al. 2003). For standard RT-PCR, 200 ng total RNA were used with the following primers: ERRγ, the forward primer 5′-ATG TCA AAC AAA GAT CGA CAC-3′ and reverse primer 5′-GAC AGG CCC GCT GCC TCC CAG GA-3′ (222 bp); ERRα, the forward primer 5′-AGA TGT CAG TAC TGC AGA GCG T-3′ and reverse primer 5′-CGG CTT CAT ACT CCA GCA-3′ (322 bp). The PCR reaction was run for 25 cycles.
ChIP assay
The ChIP assay was performed according to the instructions of the ChIP Assay Kit (Upstate Biotechnology, Lake Placid, NY, USA) with minor modifications. Twenty-four hours after transfection of either empty vectors or PGC-1α-expressing vectors, protein and DNA were cross-linked with 1% formaldehyde overnight at 4 °C. Cells were washed with cold PBS twice and disrupted in SDS lysis buffer containing a protease inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin and 1 μg/ml pepstatin A). Chromatin was sonicated to an average length of DNA of 200–1000 bp as verified by agarose gel electrophoresis (data not shown). The sheared chromatin was diluted in ChIP dilution buffer and an aliquot of the solution reserved for input control. Fifteen microliters of ERα antibody (mouse monoclonal; NeoMarkers, Fremont, CA, USA) were used as a negative (non-specific antibody) control since HEC-1B cells are ERα-negative (Hopfer et al. 1996). The endogenous ERRγ was detected by polyclonal rabbit ERRγ antiserum (a gift from U Borgmeyer). After addition of the antibodies (15 μl), the chromatin solutions were gently rotated overnight at 4 °C. The Protein A agarose slurry (containing sonicated salmon sperm DNA) was added to the antibody-bound chromatin solution and incubated for 1 h at 4 °C with constant rotation. The agarose beads were collected by centrifugation, washed and the antibody-bound chromatin was released from the agarose beads according to the supplier’s specification. Finally, the DNA was purified by phenol/chloroform extraction and ethanol precipitation. The MHREs region was detected with forward primer 5′-GTC AGT GCA GGA CAG CCC GCG AG-3′ (−758/−734) and the reverse primer 5′-GAT AGG GCC CGG ACG GAG AAA GC-3′ (−649/−627) in PCR reaction. As control, an 8.5 kb region downstream from the MHRE (human genomic sequence AP001453, gi 31790751) was selected and detected with the forward primer 5′-CAG CCC TGG CAG TCT GGA TGG-3′ (at +85 627) and reverse primer 5′-GCC CTC ATC TGC CGA CAT CAA-3′ (at +85 881). The PCR conditions for ChIP assay were 94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s for a total of 35 cycles.
In vitro transcription and translation, and electrophoretic mobility shift assay (EMSA)
PGC-1α and ERRγ were transcribed and translated in vitro with either unlabeled or 35S-labeled L-methionine (Amersham Biosciences, Piscataway, NJ, USA) using the TNT Coupled Reticulocyte Lysate Systems (Promega, Madison, WI, USA). The proteins were used in the EMSAs, the biotin-labeled DNA pull-down and the GST pull-down assays. Double-stranded DNA elements (AAB, AB, A, m1, m2, m3, m4) were cut out from the SV40-CAT reporters by NheI and XhoI, gel purified and used in EMSAs. The AAB and AB double-stranded oligos were labeled with [32P]dGTP by fill-in with Klenow large fragment of DNA polymerase I and the dNTP mixture. The unlabeled AAB, AB, A, m1, m2, m3, m4 elements were used as the competitors. The EMSA has been previously described (Yang et al. 1996, Shigeta et al. 1997).
GST and biotin-DNA pull down assays
The GST- and GST-ERRγ-expressing plasmids were transformed into E. coli BL-21 cells and the expression of GST and GST-ERRγ fusion proteins was induced by isopropyl-1-thio-β-d-galactopyranoside. The bacteria were disrupted by sonication, and the GST and its fusion protein were isolated with a 50% slurry of glutathione-Sepharose beads. Equal amounts of GST, GST-ERRα and GST-ERRγ protein were incubated with the in vitro-translated 35S-labeled PGC-1α for 1 h at 4 °C. Binding of the PGC-1α to the GST-fusion protein was examined with SDS-PAGE and visualized by autoradiography. Two micrograms of biotin-labeled AAB elements (purchased from Sigma Genosys, The Woodlands, TX, USA) from each strand in 200 mM NaCl were heated at 95 °C for 5 min and then slowly cooled down to room temperature. After the double-stranded biotin-labeled AAB elements were bound to 20 μl streptavidin-agarose beads (Sigma, St Louis, MO, USA), the in vitro-transcribed and -translated 35S-labeled ERRγ by itself or in combination with the in vitro-transcribed and -translated unlabeled PGC-1α was added to the biotin-AAB-streptavidin complex beads. The AAB-bound ERRγ was examined with SDS-PAGE and visualized by autoradiography. The intensity of the band was determined by the spot-density analysis program of an Alpha Innotech Chemilmager (San Leandro, CA, USA).
Quantitation of the PCR product or shifted bands in EMSA
Scanning was done in an Innotech ChemiImager 5500 with signal spot densitometry according to the User’s Manual.
Results
MHRE of the ERRα promoter is a pleiotropic nuclear receptor enhancer
We have previously shown that the ERRα gene is estrogen responsive and the MHRE is a major site responsible for the ER-mediated transactivation (Liu et al. 2003). The MHRE does not resemble any typical nuclear receptor response element yet it consists of three TCAAGGTCA (ERRE), an element originally identified to bind ERRα and steroidogenic factor (SF)-1 (Yang et al. 1996, Bonnelye et al. 1997, Johnston et al. 1997), and two AGGTCA motifs of nuclear receptor binding core element. These motifs are arranged in various spacing and orientation within the MHRE that could be recognized by different nuclear receptors. Using a transient transfection approach, we tested a number of nuclear receptors for their ability to activate the ERRα promoter-reporter with or without the MHRE present in the HEC-1B cells (Fig. 1A). Consistent with the earlier findings, the MHRE in the context of GC-rich ERRα promoter is responsive to ligand-bound RXRα and PPARα as either homodimer or heterodimer (Fig. 1B) while RARα and TRα (with or without the presence of their respective ligand) have no effect (data not shown). Interestingly, the ERRα and ERRγ in the absence of exogenous ligand enhanced the reporter activity driven by MHRE 10- and 25-fold respectively (Fig. 1C, 0.8-CAT). We have also tested COUP-TFI, RORα, RORβ and RORγ, and found no significant changes under the current assay conditions (data not shown). Taken together, the ERRα gene is positively regulated by its own gene product and the close family member ERRγ. Since ERRα and ERRγ are coexpressed in many tissues such as skeletal muscle, heart, kidney and pancreas, regulation of ERRα gene expression in those tissues could be greatly influenced by the presence of ERRγ.
Binding properties of ERRγ to the MHRE
The MHRE of the human and mouse ERRα gene is conserved (Fig. 2A) except for a 23 bp region which appears twice in the human gene. The 23 bp region (‘A’), contains one ERRE and one core AGGTCA element and an 11 bp region (‘B’), has a single ERRE (Fig. 2A). Binding of ERRγ to the MHRE of human form (AAB) or the mouse form (AB) was examined by EMSA. The in vitro-transcribed and -translated ERRγ protein binds MHRE of both human (AAB, Fig. 2B, lane 3) and mouse (AB, lane 14), while the reticulocyte lysate protein did not bind (mix, lanes 2 and 13). The specificity of binding was demonstrated by an effective competition with unlabeled wild-type AAB and AB oligos (lanes 4, 5 and 15, 16) and by supershifting the protein-DNA complex with ERRγ antibody (lanes 11 and 20). Preimmune serum (PS, lane 22) and non-relevant antibody (LF, lane 21) had no effect on the mobility of the complexes. To further characterize the binding requirement for ERRγ, several mutant oligos were tested in the competition study. Deletion of the B region from the MHRE (A) or mutation of the A region at the ERRE (CC to AA, m1) reduced but did not abolish the competition (lanes 6, 7, 17 and 18). Interestingly, the same mutation at ERRE of the B region (m3) severely affecting the ERRγ binding (lane 9) and the mutant oligos could only compete at 50% efficiency (scan on top, lane 9). In contrast, mutation of the middle core element (GG to AA, m2) has no obvious effect on ERRγ binding because the mutant oligos (lanes 8 and 19) compete as well as the wild-type oligos (compare to AAB and AB) in the EMSA. As expected, mutations of all three sites (m4) eliminated the ERRγ binding and no competition was found (scan on top, lane 10). These data suggested that ERRγ binds to MHRE specifically and preferably to the two ERREs. Binding of ERRγ as a homodimer complex with multiple synthetic EREs was shown (Hentschke et al. 2002b, Huppunen & Aarnisalo 2004). Whether ERRγ also binds the MHRE of the ERRα gene as homodimer was examined. We transcribed and translated different lengths of ERRγ protein (full length and myc-tagged full length) in vitro and examined their binding patterns in the EMSA (Fig. 2C). Due to the extra myc sequence, the myc-ERRγ-DNA complex moved more slowly than the ERRγ-DNA complex (compare lanes 2 and 7). When different ratios of ERRγ and myc-ERRγ expression plasmids were co-transcribed and translated in vitro, a new protein-DNA complex appeared at the intermediate position formed from the heterodimerization of ERRγ and myc-ERRγ (lanes 3–6). These data are in agreement with the binding study of ERRγ from other laboratories (Hentschke et al. 2002b, Huppunen & Aarnisalo 2004).
Transactivation function of ERRγ and PGC-1α on the MHRE
The above test studies (Fig. 1) demonstrated that ERRγ strongly transactivates MHRE in the context of its natural promoter. ERRγ also transactivated the MHRE in heterologous promoters (SV40-CAT or TATA-Luc) in a dose-dependent manner (data not shown). To identify elements within the MHRE that are required for ERRγ’s function, wild-type or mutant MHRE-reporters were co-transfected with ERRγ expression constructs into HEC-1B cells and the transactivation activities of these reporters were examined (Fig. 3A). ERRγ strongly transactivated the AAB and AB reporters, but weakly with the A reporter. Interestingly, mutation of various elements within the MHRE makes a significant difference in ERRγ transactivation function, such as mutation of the ERRE at m3 position dramatically reduced the transactivation function of ERRγ while a lesser effect as the same ERRE was mutated at the m1 position. The transactivation activity of ERRγ was least affected by the mutation at m2, the ERE core element. When all three sites were mutated (m4), the transactivation capability of ERRγ was blocked. Taken together, the data were consistent with the ERRγ binding characteristics and showed a correlation between the ability of ERRγ to bind and to transactivate the MHRE.
Recently, expression of ERRα in liver and heart was found increased following the induction of PGC-1α (Huss et al. 2002, Ichida et al. 2002, Schreiber et al. 2004). The concerted expression of PGC-1α and ERRα suggests that ERRα is a downstream target of PGC-1α and be may involved in the energy metabolism program. Ectopic expression of the PGC-1α in HEC-1B cells vigorously stimulated the transcriptional activity of the MHRE-reporters (Fig. 3B, AAB and AB) and the required element for this activity mimics the requirement for ERRγ binding and activation of the MHRE (compare Fig. 2B and Fig. 3A to Fig. 3B).
PGC-1α enhances ERRγ transactivation of the MHRE
PGC-1α does not have a typical DNA-binding domain (Puigserver et al. 1998, Knutti et al. 2000); through protein–protein interaction, PGC-1α coactivates a number of nuclear receptors including the ERRα and ERRγ (Huss et al. 2002, Schreiber et al. 2003). Therefore, the strong activation of the MHRE-reporters by PGC-1α expression in HEC-1B cells (Fig. 1B) has to rely on the endogenous nuclear receptors or transcription factors that bind the MHRE. Many nuclear receptors and transcription factors in HEC-1B cell could participate in the PGC-1α-induced transactivation activity of the MHRE-reporter; especially PGC-1α was found to coactivate diverse nuclear receptors and transcription factors (Puigserver & Spiegelman 2003, Schreiber et al. 2003). Our data on the binding and transactivation of ERRα gene by the ERRγ (Figs 1 and 2) suggested that the ERRγ could play a role in PGC-1α-induced reporter activities in HEC-1B cell.
To examine the expression pattern of ERRα and ERRγ in HEC-1B cells as well as several other human cultured cell lines, we performed limited RT-PCR (25 cycles) of total RNA prepared from the HEC-1B (endometrial), HeLa (cervical), MCF-7 (mammary gland), HEK293 (kidney), HepG2 (liver) and PLC/ PRF/5 (liver) cell lines (Fig. 4A). ERRα is ubiquitously expressed and the level of expression is comparable in these cell lines (top panel). In contrast, ERRγ is selectively expressed with a wide range of expression levels (middle panel). For example, a high level of ERRγ was detected in human HEC-1B cells (lane 1) while in the PLC/PRF5 cells (lane 7) it was barely detectable. The ERRγ in MCF-7 (lane 4), HEK293 (lane 5) and HepG2 cells (lane 6) was measurable but at a low level. The high level of ERRγ in the HEC-1B cells may be important in assessing the MHRE-reporter activity by PGC-1α in transient co-transfection experiments. This cell line could also serve as a model to study the molecular mechanism of ERRγ action. To examine whether changing the level of ERRγ in HEC-1B cells affects the transactivation function of PGC-1α, we applied the siRNA technique to reduce the endogenous ERRγ level. After introducing the ERRγ siRNA duplexes into HEC-1B cells for 48 h, the endogenous mRNA levels of both ERRγ and ERRα were significantly reduced as measured by real-time PCR (Fig. 4B, left panel), and the transactivation activity of PGC-1α on MHREs reporter was also reduced (right panel). The control siRNA affected neither the levels of ERRs mRNA nor the transactivation function of PGC-1α. In an opposite experiment, when both PCG-1α and ERRγ were expressed ectopically, the transcriptional activity of the MHRE-reporters was higher than either one alone (Fig. 4C). These experiments demonstrated that ERRγ stimulates the activity of MHRE-reporters and PGC-1α augments its effect. Taken together, PGC-1α enhances but is not required for the ERRγ to transactivate ERRα gene through MHRE.
Mechanism of PGC-1α and ERRγ activation of the MHRE
The AF2 domain of the nuclear receptor and the LXXLL motifs of the coactivator/corepressor are involved in protein–protein interaction and the trans-activation function (Glass et al. 1997, Lanz et al. 1999, McKenna et al. 1999). PGC-1α coactivates the ERRγ on a synthetic ERE and the medium-chain acyl-CoA dehydrogenase response element (Hentschke et al. 2002a, Huss et al. 2002); however, it has not yet been examined with the MHRE of ERRα gene. To investigate the functional relationship of ERRγ AF2 domain and PGC-1α LXXLL motifs on the MHRE, we ectopically expressed the ERRγ AF2 deletion mutant (AF2Δ) (Hentschke et al. 2002a) and PGC-1α L2 and L3 mutant constructs (Huss et al. 2002, Schreiber et al. 2003) in the HEC-1B cells and the MHRE activities were measured (Fig. 5). Expression of ERRγ AF2Δ severely reduced the MHRE-reporter activity and the activity stimulated by PGC-1α suggests that the mutant ERRγ acts as a dominant negative receptor (Fig. 5A). The requirement for the three LXXLL motifs (L1, L2 and L3) of the PCG-1α in nuclear receptor interaction and function has been carefully analyzed (Puigserver et al. 1998, Huss et al. 2002, Schreiber et al. 2003). While the L2 is essential for most of the receptors examined, both the L2 and L3 are needed for ERRs interaction and function (Huss et al. 2002, Kressler et al. 2002, Schreiber et al. 2003). As expected, the coactivation activity of PGC-1α on the MHRE was diminished but not eliminated with either L2 or L3 mutation and completely abolished with double mutations (L2/L3) (Fig. 5B).
PGC-1α could coactivate the ERRγ through multiple ways. In this study, we asked whether the binding of ERRγ to the MHRE is affected by PGC-1α (Fig. 6). The in vitro-labeled 35S-PGC-1α interacted strongly with the GST-ERRγ (Fig. 6A, upper panel, lanes 3 and 4) fusion protein (compare the intensities with the 10% input in lane 1). There was no binding with the GST protein by itself (lane 2), demonstrating the binding specificity and the proper interaction of the in vitro-translated PGC-1α and ERRγ. The result agrees with reports from other laboratories that PGC-1α physically interacts with ERRγ. Interaction of ERRγ and PGC-1α was further investigated by including the MHRE in a DNA pull-down assay. The MHRE was labeled with biotin and immobilized onto streptavidin beads. Binding of 35S-labeled ERRγ to the MHRE was assessed in the presence or absence of in vitro-translated PGC-1α (Fig. 6B). The translation condition that produced similar amount of wild-type and mutant PGC-1α protein was first established and verified by 35S labeling (Fig. 6A, lower panel). An equal amount of the translation reaction mixture (with or without the unlabeled wild-type or mutant PGC-1α) was then mixed with the labeled ERRγ. The total protein in every experiment was kept constant. Interestingly, interaction of 35S-ERRγ with biotin-labeled MHRE increased in the presence of PGC-1α, and the amount of 35S-ERRγ bound to the biotin-MHRE was proportional to the concentration of PGC-1α in protein mixture (Fig. 6B). There was no binding in the absence of biotin-DNA. In addition, mutant PGC-1α did not enhance the interaction between ERRγ and the MHRE (Fig. 6B, bottom, L2 L3). This in vitro study was further investigated by the in vivo ChIP assay (Fig. 6C). PGC-1α or empty vector were transfected into HEC-1B cells for 24 h and the endogenous ERRγ that was cross-linked to the MHRE region by formaldehyde treatment was immunoprecipitated by ERRγ antibody. The amount of ERRγ that binds MHRE region was increased 4-fold in cells expressing PGC-1α (Fig. 6C , top), and with no binding detected in the unrelated region (Fig. 6C, bottom). ERα antibody was used as a negative control, since no ERα was detected in HEC-1B cells by either Western blotting or RT-PCR (C T Teng, unpublished observations). Together, these results demonstrated that the PGC-1α increases ERRγ binding to the MHRE, thus providing a mechanism for PGC-1α amplifying the ERRγ trans-activation function on the MHRE of the ERRα gene.
Discussion
The promoter of the ERRα gene is TATA-less and highly rich in Gs and Cs (Shi et al. 1997). Embedded within the GC-rich region are 11 consensus Sp1-binding sites and this region is estrogen responsive even though there is no typical ERE (Liu et al. 2003). The estrogen response may be mediated through a protein–protein interaction of Sp1 and ERα (Pipaon et al. 1999, Saville et al. 2000). This GC-rich region, however, is not responsive to other ligand-dependent or -independent nuclear receptors examined in this study (Fig. 1). An MHRE functioning as a pleiotropic nuclear receptor response element is present upstream from the GC-rich region. Interestingly, the MHRE is very responsive to ERα but not to ERβ in the presence of ligand (Liu et al. 2003). This observation is similar to the osteopontin gene promoter that is stimulated through an SF response element (sequence identical to ERRE) by ERα and ERRα but not by ERβ (Vanacker et al. 1999a). Based on the composite nature of MHRE, it could be recognized by a variety of nuclear receptors and our initial test results supported the predication. The RXRα and PPARα heterodimer showed strong activation of the MHRE in the presence of their respective ligands. Although the MHRE is pleiotropic, it is also selective in responding to nuclear receptor stimulation. A computer search for motif sequence identity suggested that the MHRE closely resembles the RORE. Several ROR expression vectors (RORα, β,γ) were examined, none of which showed a significant response in the present study. Surprisingly, the MHRE was dramatically stimulated by its own family member ERRα and especially ERRγ.
The protein sequence of ERRα and ERRγ is highly conserved at the DNA-binding domain (93%) and shows moderate homology at the potential ligand-binding domain (53%). The AF2 regions of these receptors are identical with a major difference between these proteins in the N-terminal region (18%) (Shigeta et al. 1997, Hong et al. 1999). The close sequence homology of the ERRα and ERRγ proteins suggests that they could bind and activate similar target genes. However, differential regulation of target genes by these proteins was reported. For example, the small heterodimer partner SHP is constitutively activated by ERRγ and not by ERRα, whereas the thyroid receptor response element is activated by ERRα but not by ERRγ (Vanacker et al. 1999a, Heard et al. 2000, Sanyal et al. 2002). Depending on the response element, ERRγ binds differentially and recruits different cofactors, thus exhibiting different transcriptional activity (Sanyal et al. 2002). These studies demonstrated that the ERRα and ERRγ may recognize similar response elements, but subtle differences in the sequence of a composite response module such as the MHRE could elicit diverse responses by the two proteins and the protein complexes which they assembled. By EMSA, the in vitro-transcribed and -translated ERRα (data not shown) and ERRγ (Fig. 2) bind the MHRE in a similar fashion as a homodimer, in agreement with reports from other laboratories (Hentschke et al. 2002b, Huppunen & Aarnisalo 2004). However, the ERRγ transactivates the MHRE more effectively than the ERRα. In general, ERRα is not a strong activator by itself, it requires PGC-α or another ligand for activity (Vanacker et al. 1999a, Kamei et al. 2003, Mootha et al. 2004). On the other hand, ERRγ is possibly a constitutive activator and could function as a true orphan receptor, which is consistent with the crystal structure of its AF2 domain showing it to be in an active conformation in the absence of ligand (Hong et al. 1999, Greschik et al. 2002). It is reasonable to expect strong transactivation activity by ERRγ when it binds to the response element of the target gene. In a recent study, we showed that the ERRα gene is estrogen responsive in mouse uterus and heart and the ERα-mediated transactivation is via the MHRE (Liu et al. 2003). All three AGGTCA motifs (at m1, m2 and m3 positions) in the mouse MHRE are equally important in estrogen response and mutation at any one site causes similar reduction of estrogen response. In this study, we found that the binding of ERRγ to the MHRE and stimulation of its activity by ERRγ and PGC-1α requires the direct repeat at the m1 and m3 positions because mutations at the m2 had no effect on the binding of ERRγ and only mildly affected the transactivation activity in the transient reporter assay.
The ERRα and ERRγ and the coactivator PGC-1α are coexpressed in adult tissues with a high mitochondrial content which utilize fatty acid oxidation as the primary energy source (Shi et al. 1997, Shigeta et al. 1997, Hong et al. 1999, Knutti et al. 2000, Huss et al. 2002, Ichida et al. 2002, Sanyal et al. 2002, Luo et al. 2003). The PGC-1α and ERRα link in regulation of oxidative phosphorylation (Mootha et al. 2004) and biogenesis of mitochondria (Schreiber et al. 2004) has been recently established. It is not known whether ERRγ is directly involved in the energy balance program or if it functions indirectly by regulating ERRα gene expression. Our present study demonstrated that the ERRγ and PGC-1α indeed cooperate to stimulate the transcriptional activity of the ERRα gene through the MHRE. This finding was further supported by an association of the ERRγ level and the activity of PGC-1α in HEC-1B cells (Fig. 4). PGC-1α coactivates all the nuclear receptors that were examined. Obviously, endogenous nuclear receptors other than ERRγ could influence the function of PGC-1α and the strong activation of MHRE by PGC-1α in HEC-1B cells could be the result of a functional synergy by several nuclear receptors including ERRγ. To understand more of the molecular mechanism of PGC-1α-enhanced transactivation of ERRγ, we performed DNA pull-down assays. This experiment demonstrated that more ERRγ is bound to the MHRE in the presence of wild-type but not mutant PGC-1α, an observation supported by an in vivo ChIP assay (Fig. 6). Whether PGC-1α enhances or stabilizes binding of the ERRγ to MHRE is not clear; it is known that the receptor conformation can be modified and its activity modulated upon binding to the response element and by interaction with the coactivators (Klinge et al. 2001, Loven et al. 2001, Wood et al. 2001, Hall et al. 2002, Sanyal et al. 2002). ERRγ exhibits different coactivator recruitments and transcriptional activity, depending on the response element (Sanyal et al. 2004). Binding of ERRγ to the MHRE could change its conformation and increase interaction with PGC-1α, thus forming a strong activation complex.
In summary, the present work demonstrates that ERRγ and PGC-1α cooperate to stimulate ERRα gene activity. The MHRE of the ERRα gene is the binding site for ERRγ. Furthermore PGC-1α enhances ERRγ binding to the MHRE and increases its transactivational activity. The functional relationship of ERRγ, ERRα and PGC-1α is emerging, and the coexpression pattern of both ERRs and PGC-1α in metabolically active tissues suggests that ERRγ like ERRα could also be involved in adaptive thermogenesis.
The MHRE of the ERRα gene is a pleiotropic response element. (A) Diagrammatic presentation of the ERRα promoter-reporter constructs. Different lengths of ERRα gene 5′-sequences linked to CAT-reporter. The 70% GC region contains 11 Sp1 sites (Liu et al. 2003). The location of the MHRE and the primers used to detect this region in the ChIP assay are marked by arrows. The positions of the 0.6 and 0.8 kb 5′-flanking region of the ERRα gene in relation to the initiation start site (Shi et al. 1997) are indicated. For the transient co-transfection, HEC-1B cells were cultured for 24 h in charcoal-stripped serum before transfection with 300 ng of reporters and 100 ng of expression constructs. Cells were either treated with vehicle or ligand as indicated. (B) Effect of RXRα and PPARα. 9-cis-retinoic acid (9 Cis) at 50 μM and 5,8,11,14-eicosatetraynoic acid (EYTA) at 20 μM. (C) Effect of ERRα and ERRγ. The experiments were repeated three times with duplicated samples. The values are presented as means±S.D.
Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01586
ERRγ binds the MHRE of ERRα gene promoter in an EMSA. (A) Diagrammatic presentation of the wild-type and mutant version of the MHRE. The sequences were linked to either CAT-reporter or Luc-reporter constructs for functional study or made into double-stranded oligos for EMSA. AAB, the human MHRE consists of a 23 bp (‘A’) repeats and an 11 bp (‘B’) sequence; AB, the mouse MHRE; m1, m2 and m3, mutations of GG to AA at the indicated location; m4, all three sites were mutated; A, the 23 bp sequence only. (B) Binding of in vitro-transcribed and -translated ERRγ to AAB or AB probes in an EMSA. Scan of the shifted bands (arrow) is presented on top of the gel. (C) Binding of in vitro-transcribed and -translated ERRγ and myc-ERRγ to the AB probe. 32P-labeled probes are indicated. Arrow indicates the ERRγ-DNA complex. SS, super shifted band;γ, ERRγ antibody (Hentschke et al. 2002b); LF, lactoferrin antibody (Teng et al. 1986); PS, preimmune serum.
Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01586
ERRγ and PGC-1α transactivate the MHRE. (A) Effect of ectopic expression of ERRγ. (B) Effect of ectopic expression PGC-1α. The wild-type or mutant versions of MHRE were linked to the SV40-CAT reporter (300 ng) and the effect of PGC-1α (25 ng) or ERRγ (100 ng) on the reporter activities was measured 48 h after transfection of the HEC-1B cells. Descriptions of the mutant reporters are the same as in Fig. 2. The experiments were repeated three times with duplicated samples. The results are presented as mean±S.D.
Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01586
PGC-1α coactivates the ERRγ transactivation function. (A) ERRα and ERRγ mRNA levels. Total RNA was prepared from the indicated human cultured cell lines. A limited RT-PCR was performed and the PCR product analyzed on the gel. The intensity of each band was determined by image analysis and normalized to the product of HEC-1B cells. Specific primer sets for ERRα, ERRγ and β-actin mRNA detection were described in the Materials and Methods. Molecular markers at lane 1 are included to verify the product size. (B) Endogenous ERRγ mRNA level influences the expression of ERRα and the activation capability of PGC-1α on MHRE. Left panel, the ERRγ siRNA duplex was transfected into HEC-1B cells for 48 h and the endogenous ERRγ and ERRα mRNA levels were determined by the quantitative real-time PCR as described in the Materials and Methods. The experiments were repeated three times and the ERRγ mRNA level from the control siRNA plasmids transfected cells was set as 1. The results are means±S.D. Right panel, the HEC-1B cells were transfected with ERRγ siRNA for 24 h, followed with the transfection of PGC-1α expression constructs (25 ng) and the AAB-TATA-Luc reporters (300 ng). (C) PGC-1α enhances the transactivation activity of ERRγ. PGC-1α (25 ng) and ERRγ (50 ng) expression constructs were co-transfected with the AAB-TATAT-Luc reporters (300 ng) individually or together into the cells for 36 h and the activities measured. The experiments were repeated three times with duplicated samples and results are presented as means±S.D.
Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01586
AF2 of the ERRγ and LXXLL motifs of the PGC-1α are required in functional interaction. (A) Effect of ERRγ AF2 deletion. The 0.8-CAT reporters were co-transfected with expression constructs of the ERRγ wild-type or AF2-deleted (AF2Δ) with or without the PGC-1α. CAT activities were measured 48 h after transfection of the HEC-1B cells. The experiments were repeated three times with duplicated samples and results are presented as means±S.D. The activity from the wild-type PGC-1α was set as 100%. (B) Effect of PGC-1α LXXLL motif(s) mutation(s). The AAB-TATA-Luc reporter were co-transfected with expression constructs of the PGC-1α wild-type or LXXLL motif mutated (L2A, L3A and L2A/L3A) with or without the ERRγ. The Luc activities were measured 36 h after transfection of the HEC-1B cells. The experiments were repeated three times with duplicated samples and results are presented as means±S.D. The activity from the wild-type PGC-1α was set as 100%.
Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01586
PGC-1α enhances ERRγ interaction with the MHRE both in vitro and in vivo. (A) GST pull-down assay. Binding of 35S-PGC-1α to GST-ERRα or GST-ERRγ was analyzed with SDS-PAGE and visualized by X-ray film exposure. (B) Biotin-labeled DNA pull-down assay. Double-stranded biotin-AAB was interacted with 35S-ERRγ alone or in the presence of different concentrations of unlabeled in vitro-translated PGC-1α (5 and 10 μl) and the control samples contain 10 μl of reaction mixture. The amount of 35S-labeled ERRγ pulled down by the beads was resolved in the SDS-PAGE and exposed to X-ray film with an intensifying screen at −70 °C. Top, a representative gel picture. Bottom, relative fold of increase in 35S-ERRγ binding in the presence of wild-type (wt) and mutant (L2/L3) PGC-1α. Results are presented as means±S.D. of three independent experiments. (C) The ChIP assay. PGC-1α or empty vector was ectopically expressed in the HEC-1B cells for 24 h. The ChIP assay was performed with ERRγ or ERα antibodies. Top, detection of the MHRE region by PCR with specific primers (indicated in Fig. 1 and described in Materials and Methods). Bottom, PCR detection of the 8.5 kb region downstream from the MHRE (according to the human chromosome 11 genomic sequence: AP001453; gi 31790751). The intensity of the PCR band detected by the ERα antibody is set to 1 (at bottom of the top graph).
Citation: Journal of Molecular Endocrinology 34, 2; 10.1677/jme.1.01586
(D Liu and Z Zhang contributed equally to this work)
We thank U Borgmeyer, D P Kelly, A Kralli, A Jetten, K Korach, V Giguere, C Weinberger and M Tsai for providing reagents. We appreciate the critical reading and comments of the paper by J Wachsman. L Moore edited the paper. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Bonnelye E, Vanacker JM, Spruyt N, Alric S, Fournier B, Desbiens X & Laudet V 1997 Expression of the estrogen-related receptor 1 (ERR-1) orphan receptor during mouse development. Mechanisms of Development 65 71–85.
Bonnelye E, Kung V, Laplace C, Galson DL & Aubin JE 2002 Estrogen receptor-related receptor alpha impinges on the estrogen axis in bone: potential function in osteoporosis. Endocrinology 143 3658–3670.
Coward P, Lee D, Hull MV & Lehmann JM 2001 4-Hydroxytamoxifen binds to and deactivates the estrogen-related receptor gamma. PNAS 98 8880–8884.
Giguere V 1999 Orphan nuclear receptors: from gene to function. Endocrine Reviews 20 689–725.
Giguere V 2002 To ERR in the estrogen pathway. Trends in Endocrinology and Metabolism 13 220–225.
Giguere V, Yang N, Segui P & Evans RM 1988 Identification of a new class of steroid hormone receptors. Nature 331 91–94.
Glass CK, Rose DW & Rosenfeld MG 1997 Nuclear receptor coactivators. Current Opinion in Cell Biology 9 222–232.
Greschik H, Wurtz JM, Sanglier S, Bourguet W, van Dorsselaer A, Moras D & Renaud JP 2002 Structural and functional evidence for ligand-independent transcriptional activation by the estrogen-related receptor 3. Molecular Cell 9 303–313.
Hall JM, McDonnell DP & Korach KS 2002 Allosteric regulation of estrogen receptor structure, function, and coactivator recruitment by different estrogen response elements. Molecular Endocrinology 16 469–486.
Heard DJ, Norby PL, Holloway J & Vissing H 2000 Human ERR gamma, a third member of the estrogen receptor-related receptor (ERR) subfamily of orphan nuclear receptors: tissue-specific isoforms are expressed during development and in the adult. Molecular Endocrinology 14 382–392.
Hentschke M, Susens U & Borgmeyer U 2002a PGC-1 and PERC, coactivators of the estrogen receptor-related receptor gamma. Biochemical and Biophysical Research Communications 299 872–879.
Hentschke M, Susens U & Borgmeyer U 2002b Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding. European Journal of Biochemistry 269 4086–4097.
Hong H, Yang L & Stallcup MR 1999 Hormone-independent transcriptional activation and coactivator binding by novel orphan nuclear receptor ERR3. Journal of Biological Chemistry 274 22618–22626.
Hopfer H, Rinehart CA Jr, Kaufman DG & Vollmer G 1996 Basement membrane induced differentiation of HEC-1B(L) endometrial adenocarcinoma cells affects both morphology and gene expression. Biochemistry and Cell Biology 74 165–177.
Huppunen J & Aarnisalo P 2004 Dimerization modulates the activity of the orphan nuclear receptor ERRgamma. Biochemical and Biophysical Research Communications 314 964–970.
Huss JM, Kopp RP & Kelly DP 2002 Peroxisome proliferator-activated receptor coactivator-1 alpha (PGC-1 alpha) coactivates the cardiac-enriched nuclear receptors estrogen-related receptor-alpha and -gamma. Identification of novel leucine-rich interaction motif within PGC-1 alpha. Journal of Biological Chemistry 277 40265–40274.
Ichida M, Nemoto S & Finkel T 2002 Identification of a specific molecular repressor of the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha). Journal of Biological Chemistry 277 50991–50995.
Johnston SD, Liu X, Zuo F, Eisenbraun TL, Wiley SR, Kraus RJ & Mertz JE 1997 Estrogen-related receptor alpha 1 functionally binds as a monomer to extended half-site sequences including ones contained within estrogen-response elements. Molecular Endocrinology 11 342–352.
Kamei Y, Ohizumi H, Fujitani Y, Nemoto T, Tanaka T, Takahashi N, Kawada T, Miyoshi M, Ezaki O & Kakizuka A 2003 PPARgamma coactivator 1 beta/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity. PNAS 100 12378–12383.
Klinge CM, Jernigan SC, Smith SL, Tyulmenkov VV & Kulakosky PC 2001 Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor alpha. Molecular and Cellular Endocrinology 174 151–166.
Knutti D & Kralli A 2001 PGC-1, a versatile coactivator. Trends in Endocrinology and Metabolism 12 360–365.
Knutti D, Kaul A & Kralli A 2000 A tissue-specific coactivator of steroid receptors, identified in a functional genetic screen. Molecular and Cellular Biology 20 2411–2422.
Kressler D, Schreiber SN, Knutti D & Kralli A 2002 The PGC-1-related protein PERC is a selective coactivator of estrogen receptor alpha. Journal of Biological Chemistry 277 13918–13925.
Laganiere J, Tremblay GB, Dufour CR, Giroux S, Rousseau F & Giguere V 2004 A polymorphic autoregulatory hormone response element in the human estrogen-related receptor (alpha) (ERR(alpha)) promoter dictates peroxisome proliferator-activated receptor (gamma) coactivator-1(alpha) control of ERR(alpha) expression. Journal of Biological Chemistry 279 18504–18510.
Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai MJ & O’Malley BW 1999 A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97 17–27.
Liu D, Zhang Z, Gladwell W & Teng CT 2003 Estrogen stimulates estrogen-related receptor (alpha) gene expression through conserved hormone response elements. Endocrinology 144 4894–4904.
Loven MA, Likhite VS, Choi I & Nardulli AM 2001 Estrogen response elements alter coactivator recruitment through allosteric modulation of estrogen receptor beta conformation. Journal of Biological Chemistry 276 45282–45288.
Luo J, Sladek R, Bader JA, Matthyssen A, Rossant J & Giguere V 1997 Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 388 778–782.
Luo J, Sladek R, Carrier J, Bader JA, Richard D & Giguere V 2003 Reduced fat mass in mice lacking orphan nuclear receptor estrogen-related receptor alpha. Molecular and Cellular Biology 23 7947–7956.
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P et al.1995 The nuclear receptor superfamily: the second decade. Cell 83 835–839.
McKenna NJ, Lanz RB & O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocrine Reviews 20 321–344.
Mootha VK, Handschin C, Arlow D, Xie X, St Pierre J, Sihag S, Yang W, Altshuler D, Puigserver P, Patterson N et al.2004 ERR(alpha) and Gabpa/b specify PGC-1(alpha)-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. PNAS 101 6570–6575.
Pipaon C, Tsai SY & Tsai MJ 1999 COUP-TF upregulates NGFI-A gene expression through an Sp1 binding site. Molecular and Cellular Biology 19 2734–2745.
Puigserver P & Spiegelman BM 2003 Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocrine Reviews 24 78–90.
Puigserver P, Wu Z, Park CW, Graves R, Wright M & Spiegelman BM 1998 A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92 829–839.
Sanyal S, Kim JY, Kim HJ, Takeda J, Lee YK, Moore DD & Choi HS 2002 Differential regulation of the orphan nuclear receptor small heterodimer partner (SHP) gene promoter by orphan nuclear receptor ERR isoforms. Journal of Biological Chemistry 277 1739–1748.
Sanyal S, Matthews J, Bouton D, Kim HJ, Choi HS, Treuter E & Gustafsson JA 2004 Deoxyribonucleic acid response element-dependent regulation of transcription by orphan nuclear receptor estrogen receptor-related receptor gamma. Molecular Endocrinology 18 312–325.
Saville B, Wormke M, Wang F, Nguyen T, Enmark E, Kuiper G, Gustafsson JA & Safe S 2000 Ligand-, cell-, and estrogen receptor subtype (alpha/beta)-dependent activation at GC-rich (Sp1) promoter elements. Journal of Biological Chemistry 275 5379–5387.
Schreiber SN, Knutti D, Brogli K, Uhlmann T & Kralli A 2003 The transcriptional coactivator PGC-1 regulates the expression and activity of the orphan nuclear receptor ERRalpha. Journal of Biological Chemistry 278 9013–9018.
Schreiber SN, Emter R, Hock MB, Knutti D, Cardenas J, Podvinec M, Oakeley EJ & Kralli A 2004 The estrogen-related receptor (alpha) (ERR(alpha)) functions in PPAR(gamma) coactivator 1(alpha) (PGC-1(alpha))-induced mitochondrial biogenesis. PNAS 101 6472–6477.
Shi H, Shigeta H, Yang N, Fu K, O’Brian G & Teng CT 1997 Human estrogen receptor-like 1 (ESRL1) gene: genomic organization, chromosomal localization, and promoter characterization. Genomics 44 52–60.
Shigeta H, Zuo W, Yang N, DiAugustine R & Teng CT 1997 The mouse estrogen receptor-related orphan receptor alpha 1: molecular cloning and estrogen responsiveness. Journal of Molecular Endocrinology 19 299–309.
Sladek R, Bader JA & Giguere V 1997 The orphan nuclear receptor estrogen-related receptor alpha is a transcriptional regulator of the human medium-chain acyl coenzyme A dehydrogenase gene. Molecular and Cellular Biology 17 5400–5409.
Teng C 2002 Estrogen receptor-related receptor alpha: biological roles. Recent Results in Cancer Research 3 389–400.
Teng CT, Walker MP, Bhattacharyya SN, Klapper DG, DiAugustine RP & McLachlan JA 1986 Purification and properties of an oestrogen-stimulated mouse uterine glycoprotein (approx. 70 kDa). Biochemical Journal 240 413–422.
Tremblay GB, Bergeron D & Giguere V 2001a 4-Hydroxytamoxifen is an isoform-specific inhibitor of orphan estrogen-receptor-related (ERR) nuclear receptors beta and gamma. Endocrinology 142 4572–4575.
Tremblay GB, Kunath T, Bergeron D, Lapointe L, Champigny C, Bader JA, Rossant J & Giguere V 2001b Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR beta. Genes and Development 15 833–838.
Tsai MJ & O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annual Review of Biochemistry 63 451–486.
Vanacker JM, Delmarre C, Guo X & Laudet V 1998a Activation of the osteopontin promoter by the orphan nuclear receptor estrogen receptor related alpha. Cell Growth and Differentiation 9 1007–1014.
Vanacker JM, Bonnelye E, Delmarre C & Laudet V 1998b Activation of the thyroid hormone receptor alpha gene promoter by the orphan nuclear receptor ERR alpha. Oncogene 17 2429–2435.
Vanacker JM, Pettersson K, Gustafsson JA & Laudet V 1999a Transcriptional targets shared by estrogen receptor-related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta. EMBO Journal 18 4270–4279.
Vanacker JM, Bonnelye E, Chopin-Delannoy S, Delmarre C, Cavailles V & Laudet V 1999b Transcriptional activities of the orphan nuclear receptor ERR alpha (estrogen receptor-related receptor-alpha). Molecular Endocrinology 13 764–773.
Vega RB & Kelly DP 1997 A role for estrogen-related receptor alpha in the control of mitochondrial fatty acid beta-oxidation during brown adipocyte differentiation. Journal of Biological Chemistry 272 31693–31699.
Vega RB, Huss JM & Kelly DP 2000 The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor alpha in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Molecular and Cellular Biology 20 1868–1876.
Wood JR, Likhite VS, Loven MA & Nardulli AM 2001 Allosteric modulation of estrogen receptor conformation by different estrogen response elements. Molecular Endocrinology 15 1114–1126.
Xie W, Hong H, Yang NN, Lin RJ, Simon CM, Stallcup MR & Evans RM 1999 Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2. Molecular Endocrinology 13 2151–2162.
Yang C & Chen S 1999 Two organochlorine pesticides, toxaphene and chlordane, are antagonists for estrogen-related receptor alpha-1 orphan receptor. Cancer Research 59 4519–4524.
Yang C, Zhou D & Chen S 1998 Modulation of aromatase expression in the breast tissue by ERR alpha-1 orphan receptor. Cancer Research 58 5695–5700.
Yang N, Shigeta H, Shi H & Teng CT 1996 Estrogen-related receptor, hERR1, modulates estrogen receptor-mediated response of human lactoferrin gene promoter. Journal of Biological Chemistry 271 5795–5804.
Zhang Z & Teng CT 2000 Estrogen receptor-related receptor alpha 1 interacts with coactivator and constitutively activates the estrogen response elements of the human lactoferrin gene. Journal of Biological Chemistry 275 20837–20846.
Zuo F & Mertz JE 1995 Simian virus 40 late gene expression is regulated by members of the steroid/thyroid hormone receptor superfamily. PNAS 92 8586–8590.
