Identification of PNRC2 and TLE1 as activation function-1 cofactors of the orphan nuclear receptor ERRγ

BBRC Biochemical and Biophysical Research Communications 312 (2003) 975–982 www.elsevier.com/locate/ybbrc

Identification of PNRC2 and TLE1 as activation function-1 cofactors of the orphan nuclear receptor ERRc Moritz Hentschkea,b and Uwe Borgmeyera,* a

Zentrum f€ ur Molekulare Neurobiologie, Institut f€ur Entwicklungsneurobiologie, Universit€at Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany b Institut f€ur Humangenetik, Universit€at Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany Received 10 October 2003

Abstract Estrogen-related receptor c (ERRc) is an orphan nuclear receptor highly expressed in heart, skeletal muscle, kidney, and brain. To identify activation function-1 (AF-1)-dependent cofactors involved in the transcriptional function of ERRc, we screened for human cDNAs coding for proteins that bind to the bacterial expressed AF-1 by biopanning of a phage display library. Phages displaying fusion proteins with full-length PNRC2 (proline-rich nuclear receptor co-regulatory protein 2), already shown to be a cofactor for other nuclear receptors, and with a polypeptide of the bHLH corepressor TLE1 bound to the AF-1 containing bait. Pull-down analyses demonstrated a direct interaction of the receptor with the newly identified full-length proteins. Surprisingly, not only PNRC2 but also the corepressor TLE1 functioned as ERRc coactivator in a reporter gene analysis. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Orphan nuclear receptor; Transcription factor; ERR3; NR3B3; Transactivation; Phage display; Activation function; PNRC; Groucho

Nuclear receptors (NRs) comprise a large family of metazoan ligand-activated transcription factors that exert both, positive and negative control of gene expression [1]. Generally, NRs display a modular structure composed of an amino-terminal region A/B, followed by a DNA-binding domain (DBD, region C), a hinge region (D), and a ligand-binding domain (LBD, E) [2]. Two transactivation functions, the constitutive activation function-1 (AF-1) originating in the A/B region and the ligand-dependent AF-2 arising in the LBD, mediate the transcriptional activity. The transcriptional control requires the interaction with coregulator proteins (coactivators and corepressors) that act as signaling intermediates with the general transcription machinery [3,4]. Some of these cofactors possess enzyme activities like histone acetyltransferase or deacetylase activities. Local regulation of histone–histone and histone–DNA interactions is mediated in part by three members of a family of 160-kDa proteins that are among the most wellcharacterized coactivators, the steroid hormone receptor *

Corresponding author. Fax: +49-40-42803-5101. E-mail address: [email protected] Borgmeyer).

(U.

0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.025

coactivator (SRC)/p160 family [5]. They contain a cluster of three conserved NR box motifs (LXXLL, where L is leucine and X can be any amino acid) that mediate binding to the AF-2 of essentially all NRs that can function as transcriptional activators. In addition, they recruit a number of secondary coactivator proteins, including histone acetyltransferases such as CREBbinding protein (CBP), the related protein p300, and p300/CBP-associated factor PCAF. Catalyzing the acetylation of histone lysines by the SRC-complex, bound to specific promoters, is thought to initialize chromatin remodeling by counteracting their repressive activity. In addition, CBP/p300 bind subunits of the basal transcription machinery and thus may support the recruitment of the transcription preinitiation complex. Many unliganded NRs actively repress their primary response genes through the recruitment of transcriptional corepressors [6,7]. Corepressors have been found to exist in vivo in multiple, distinct macromolecular complexes that contain histone deacetylase enzymatic activity [8,9]. A large proportion of nuclear receptors are termed orphans, as the natural ligand, if it exists, remains to be identified. ERRa (NR3B1) and ERRb (NR3B2) were

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the first orphans to be isolated by virtue of their homology to the estrogen receptor a (ERa; NR3A1) [10]. While the expression of ERRb is mainly restricted to trophoblast progenitor cells, ERRa shows a widespread expression pattern with high transcript levels in kidney, heart, and brown adipocytes [11,12]. A third member of the family, ERRc (ERR3; NR3B3), recently identified from human and mouse cDNA libraries is most closely related to ERRb (reviewed by Gigu
ere [13]). In the adult mouse, high transcript levels have been detected in heart, skeletal muscle, kidney, and brain [14]. In situ hybridization of the mature mouse brain revealed high transcript levels in the isocortex, the olfactory system, cranial nerve nuclei, and major parts of the coordination centers [15]. In the embryo, transcripts have been detected by means of in situ hybridization, as early as day 10.5 post-coitum (E10.5). During development, expression is most prominent in the nervous system. The gene is preferentially transcribed in already differentiating areas of the nervous system, establishing many features of the adult expression pattern [16]. ERRc binds as a dimer to several variants of a direct repeat of the NR half site resembling the sequence 50 AGGTCA-30 [17]. A fusion protein with the green fluorescent protein revealed constitutive nuclear localization [18]. In cotransfection experiments, ligand-independent reporter gene activation is enhanced by coexpression of p160 family members GRIP1 and pCIP [19,20]. The active conformation of the bacterial expressed ERRc LBD crystallized in the presence of a SRC-1 peptide is in favor of a constitutive active protein [21]. However, interaction with Ca2þ -calmodulin, a Ca2þ -influx-dependent ERRc-mediated transcriptional activation in a reporter gene assay, and cellular factors required for efficient DNA binding suggest additional regulatory mechanisms [17,22]. In addition to p160 cofactors, the peroxisome proliferator-activated receptor c coactivators 1 (PGC-1) a and b (PERC) whose expression pattern widely resembles that of ERRc function as coactivators [18,23–26]. We have recently demonstrated that ERRc contains an AF-1 which mediates activation by PGC-1a but not by PERC and that alternative splicing leads to an isoform lacking the AF-1 [14,18]. Still, much less is still known about the AF-1 which is less well conserved among NRs. In the ER the AF-1 is considered to act synergistically with the AF-2 and to exhibit cell type and promoter context specificity [27,28]. To identify AF-1-specific transcriptional regulators, a human brain cDNA phage display system was employed with the N-terminal domain of ERRc2 as a bait. The interaction of two proteins, PNRC2 and TLE1, with ERRc was confirmed by GST pull-down analysis. The expression patterns are in support of a functional interaction. In addition, cotransfection experiments demonstrate that PNRC2 functions as ERRc-coactivator.

Surprisingly, coexpression of TLE1, previously described as corepressor, results in a robust transcriptional activation.

Materials and methods Plasmid constructs. The plasmid pGEX-ERRc2 (1–124) coding for a GST fusion with the A/B region of mouse ERRc2 (amino acid 1–124) was generated by inserting the respective cDNA fragment into the bacterial expression vector pGEX-KG [29]. The coding sequence of PNRC2 was amplified from human brain cDNA libraries by polymerase chain reaction (PCR) and inserted into pGEM-T easy (Promega) for sequence verification. The insert was then cloned into pGEX-KG and into the eukaryotic expression vector pCMX [30]. Two TLE1 fragments covering the complete open reading frame as well as the open reading frame of an alternatively spliced RNA were amplified by PCR from a cDNA library of the human teratocarcinoma cell line NT2/D1 [31], ligated into pGem-T easy, and then inserted into pCMX. The shorter fragment codes for TLE1DGP with a deletion of amino acids 125–199 with respect to GenBank Accession No. NM_005077. A cDNA with an N-terminal truncation of 115 amino acids (DGP/Q) was generated from the TLE1DGP cDNA by a 50 -deletion up to an endogenous NcoI site. The C-terminal truncation (DWD40) was generated by deletion of a carboxy-terminal SphI fragment. The TLE1 construct WD40 starts with a methionine at an endogenous NcoI site and encodes 296 C-terminal amino acids. Construction of pGSTERRc2, ERRc-218, and pBV-4xSIS-Luc has been described previously [18]. GST fusion proteins were expressed as described [22]. Phage display screen. The fusion protein GST-ERRc2 (1–124), bound to glutathione–Sepharose 4B beads (Amersham Biosciences), was used as affinity matrix. A human brain cDNA library in the T7Select10-3 vector (Novagen) was screened for ERRc-interacting proteins by biopanning. The T7Select vector allows inserts to be expressed as fusions to the carboxy-terminus of the T7 gene 10 major capsid protein with an average of 10 copies per virion. Approximately 1
108 T7 phages were added to 100 ml bacterial culture and incubated at 37 °C under constant shaking until lysis (2.5 h). After diluting 250 ll of this lysate in one volume phosphate-buffered saline (PBS), supplemented with 2
complete protease inhibitor (Roche) and with Tween to achieve a final concentration of 0.1%, it was incubated at room temperature for 60 min with 50 ll immobilized GST-ERRc2 (1–124) under constant rotation. The beads were then precipitated by centrifugation and washed 10 times in 1 ml PBS supplemented with Tween (0.1%) followed by the direct infection of a 100 ml bacterial culture. After six rounds of biopanning, the final lysate was plated at low density. Individual, well-isolated plaques were used for PCR amplification and subjected to dideoxynucleotide sequencing. Inserts derived from the coding region of known cDNAs and in the right orientation with respect to the capsid protein were further analyzed. This led to two clones, PNRC2 [32] and TLE1 [33], previously described as transcriptional cofactors. In vitro-interaction assays. Fusion proteins of full-length ERRc2 and PNRC2 with GST, or GST alone, were used as baits. In vitro, [35 S]methionine-labeled TLE1 and ERRc were produced with the coupled transcription/translation system (Promega). Labeled proteins were incubated with either GST or GST-fusion protein bound to glutathione–Sepharose 4B for 2 h at 4 °C in pull-down buffer (50 mM Tris–HCl, pH 7.5, 120 mM NaCl, 0.1 mM EDTA, 1
complete (Roche), 0.05% Tween, 0.1% BSA, and 1 mM DTT). The beads were washed seven times with 1 ml pull-down buffer. Proteins were eluted by boiling the beads in SDS loading buffer, separated by Tris–glycine SDS–PAGE, and analyzed by autoradiography of the dried gel. Northern blot analysis. Multiple tissue Northern blots (Clontech) were hybridized with cDNA probes of PNRC2 (GenBank Accession No. AF374386: nucleotides 1–375) and of TLE1 (GenBank Accession

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No. NM_005077: nucleotides 451–2763) labeled with [a-32 P]dATP (3000 Ci/mMol) using the Megaprime (Amersham Biosciences) random-prime labeling method. Hybridization was performed at 65 °C in ExpressHyb (Clontech) and the final wash was at 65 °C in 0.1
SSC, 0.1% SDS. Filters were exposed to Kodak MS autoradiograph films. Luciferase expression assays. CV-1 cells were grown in Dulbecco’s modified Eagle’s medium (PAA, Austria) supplemented with 10% newborn calf serum (Biospa, Germany), 200 U/ml penicillin G, and 200 lg/ml streptomycin at 37 °C under a 5% CO2 atmosphere. Cells grown in six-well tissue culture plates were transfected at 50% confluence with FuGENE 6 (Roche), using 2 ll transfection reagent per microgram of DNA. Each well received 1 lg luciferase reporter plasmid (pBV-4xSIS-Luc), 0.25 lg CMX receptor expression plasmid, 0.25 lg CMX cofactor expression plasmid, and 0.1 lg pRAS-bGal [34] as indicated in the figures. Cell lysates were prepared after 24 h to determine the luciferase as well as the b-galactosidase activity using the appropriate kit according to the manufacturer’s instructions (Roche). For both assays, light emission was measured using a MicroLumat LB96P (Berthold Technologies, Germany). The luciferase activity was normalized to the level of b-galactosidase activity derived from the pRAS-bGal control plasmid. Each transfection was performed in triplicate and repeated at least three times.

Results Identification of the NR coactivator PNRC2 and of the bHLH transcriptional corepressor TLE1 as binding partners of ERRc Sequence analysis revealed that the NR coactivator PNRC2 [32] was among the enriched clones that bound the fusion protein coding for the AF-1, suggesting that cofactors specific for NRs can be indeed identified using the phage display system. The chimeric cDNA codes for the complete 16 kDa coactivator protein of 139 amino acids (Fig. 1A). Furthermore, we enriched a chimeric cDNA that encodes 40 amino acids identical with the region encompassing the WD40 domain of TLE1 [33], a corepressor of bHLH proteins (Fig. 1A). In vitro association of cofactors with ERRc To test the validity of the phage display interaction, the full-length PNRC2 cDNA was amplified by PCR and cloned into a pGEX bacterial expression vector. In a GST pull-down assay the fusion protein bound the radioactively labeled ERRc generated by in vitro translation (Fig. 1B). Binding of TLE1 was verified with a pull down of the radioactively labeled full-length protein with GST-ERRc. Efficient capture of TLE1 by the fusion protein, but not by a control GST protein, was observed (Fig. 1C). The GST pull-down assays with GST-PNRC2 and GST-ERRc and as a control with GST alone supported that the phage enrichment of the newly identified partners was based on a direct interaction with the orphan receptor. In addition, the pull down with the full-length proteins suggested that the adhesive epitopes are exposed in the native conformation of both partners.

Fig. 1. Direct interaction of PNRC2 and TLE1 with ERRc. (A) Schematic representation of the captured proteins. The filled bars indicate that full-length PNRC2 and the carboxy-terminal amino acids of TLE1 were isolated as fusion proteins with the phage capsid protein. (B,C) Bacterial expressed GST-PNRC2, GST-ERRc or GST alone, all bound to glutathione–Sepharose beads were incubated with in vitro translated 35 S-labeled ERRc (B) or TLE1 (C) as described in “Materials and methods.” Twenty percent of the in vitro translated proteins used in the binding reaction (20% input) were analyzed for comparison.

Expression of the newly identified putative ERRc cofactors To further substantiate that ERRc and the associating proteins could be in vivo interaction partners, we analyzed their expression by Northern blot analysis. The PNRC2 mRNA exists as two species of 2.9 and 1.9 kb, and both are widely expressed (Fig. 2A). The shorter form is only barely detected. Transcripts are found in all human tissues analyzed with strongest level of expression in kidney, heart, skeletal muscle, and placenta. Hybridization with an ERRc-specific probe (data not shown) revealed that the very same tissues exhibited strong signals. Two TLE1-specific transcripts (4.4 and 2.6 kb) were detected in human tissues (Fig. 2B). High mRNA levels in skeletal muscle and liver and moderate levels in placenta, heart, kidney, and spleen are consistent with a function as a cofactor in ERRc-mediated transcriptional regulation. Transcripts of both cofactors were also detected in human brain. Based on the expression pattern both PNRC2 and TLE1 could be indeed functional in vivo cofactors of ERRc. Functional interaction between ERRc and the newly identified binding partner We next sought to examine whether PNRC2 and TLE1 are limiting transcriptional cofactors of ERRc.

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Fig. 2. Northern blot analysis of PNRC2 and TLE1 mRNA in adult human tissues. A multiple tissue RNA blot (Clontech) was hybridized with cDNA probes specific for PNRC2 (A) and TLE1 (B) as described in “Materials and methods.” RNA size markers (in kb) are aligned at the left of each autoradiograph. The arrows on the right represent the sizes of different cofactor-specific transcripts.

threefold increase in luciferase activity, whereas PNRC2 alone did not stimulate luciferase expression. Surprisingly, coexpression of TLE1, previously described as a corepressor of numerous transcription factors, resulted in an even higher transcriptional activity (Fig. 3B). Together these results strongly suggested that both proteins function as ERRc cofactors, directly interacting with the receptor. AF-1-dependent transcriptional activation

Fig. 3. The effect of PNRC2 and TLE1 overexpression on ERRc transactivation function. CV-1 cells were cotransfected with the reporter plasmid pBV-4xSIS-Luc and either expression plasmids without insert (pCMX), or coding for ERRc, PNRC2 (A), and TLE1 (B) as indicated. Luciferase activities measured 24 h after transfection are presented as fold activation relative to cells which were not transfected with receptor and cofactor expression plasmids.

Therefore, we analyzed their effect on ERRc transcriptional activity in CV-1 cells by transient transfection with a luciferase reporter plasmid consisting of four ERRc-binding sites linked to the basal thymidine kinase promoter [18,35]. As expected, ERRc coexpression leads to an activation of the reporter (Fig. 3A). Coexpression of ERRc and PNRC2 resulted in an additional

The direct in vitro interaction with the 124 aminoterminal amino acids of ERRc2 suggested that the AF-1 is involved in transcriptional activation by PNRC2 and TLE1. To discriminate between AF-1- and AF-2-dependent activation, we compared the transcriptional activity of the amino-terminal truncated protein ERRc124–458 lacking the AF-1 and that of the carboxyterminal truncated protein ERRc-218 lacking the AF-2. Both proteins have the DBD in common. The aminoterminal truncated receptor activated the luciferase promoter by more than 20-fold, demonstrating the importance of the carboxy-terminus for transcriptional activation (Figs. 4A and C). However, coexpression of PNRC2 only weakly affected the transcriptional activity of ERRc-124–458 (Fig. 4A). Expression of the aminoterminal protein ERRc-218 augmented the activation of the reporter gene by a factor of 3 (Figs. 4B and D).

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Fig. 4. Contribution of AF-1 and AF-2 activities to the PNRC2- and TLE1-mediated transactivation. CV-1 cells were cotransfected with the reporter plasmid pBV-4xSIS-Luc and either expression plasmids coding for the C-terminal (A,C) or the N-terminal part of ERRc (B,D), and with PNRC2 (A,B) or TLE1 (C,D) expression plasmids, as indicated. Transcriptional activation was determined as described in the legend to Fig. 3.

In vitro DNA-binding of this small protein has been demonstrated before [17]. The coexpression of PNRC2 led to a further twofold reporter gene activation (Fig. 4B). These experiments indicated that the A/B region, which encodes the AF-1 domain, was necessary for optimal PNRC2-dependent transactivation. Coexpression of TLE1 augmented transcriptional activation of the carboxy-terminal protein by 4 (Fig. 4C) and that of the amino-terminal protein by a factor of 6 (Fig. 4D), suggesting the importance of both AFs for TLE1-mediated activation. Furthermore, and more importantly, these experiments clearly demonstrated that the AF-1 of ERRc operates in an AF2/LBD-independent manner. Domains of TLE1 required for activation of ERRcmediated transactivation Identification of a well-characterized corepressor as a coactivator of ERRc is very unusual. Consequently, deletion constructs of TLE1 were tested for their ability to activate ERRc-mediated transcriptional activation. A newly isolated splice variant encoding a protein without GP domain (TLE1DGP) showed a greatly reduced transcriptional activity (Fig. 5). The amino-terminal

Fig. 5. TLE1 domains involved in the ERRc coactivation function. (A) Schematic view of domain structure of TLE1. Deletion of the GP domain of isoform TLE1DGP is indicated by two arrows. (B) CV-1 cells were transiently transfected with a vector expressing TLE1, TLE1 without GP domain (DGP), without GP and Q domain (DGP/Q), without WD40 domain (DWD40), or the WD40 (WD40) domain alone as indicated, together with the reporter plasmid pBV-4xSIS-Luc and either expression plasmids pCMX or pCMX-ERRc. Transcriptional activation was determined as described in the legend to Fig. 3.

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truncation of this protein (TLE1DGP/Q) abolished the transcriptional activity. The WD40 domain, the only intact domain present in the phage fusion protein, was inactive by itself. However, a TLE1 protein lacking the WD40 domain (TLE1DWD40) also failed in significantly upregulation of the reporter gene activity, supporting an essential function of this domain in ERRc-mediated transcriptional coactivation. Together, these studies suggest that the WD40 domain is important for in vivo recruitment of TLE1 and that the GP and Q domain are important for coactivation.

Discussion Two classes of coactivators, the p160 family proteins on the one hand, and PGC-1 and PERC on the other hand have already been identified as potential coactivators of ERRc [18,19,21,26]. At least two ERRc isoforms that differ by 23 additional amino acids of ERRc2 have been described [14]. In contrast to ERRc1, the A/B domain of ERRc2 has been demonstrated to be actively involved in coactivator recruitment [18]. The AF-1function of nuclear receptors is of primary interest since it is involved in promoter context and tissue specific gene regulation [27,28]. Given the distinct spatial adult expression with high levels in heart, skeletal muscle, kidney, placenta, and brain and the expression in the embryonic central nervous system, it is tempting to speculate that different isoforms are involved in disparate developmental and homeostatic functions. Since distinct coactivator recruitment could be involved to gain specificity, we attempted to identify AF-1 cofactors by a phage display. In contrast to the yeast two-hybrid system, the phage display has only rarely been used to identify new interaction partners for NRs. In the present study, we have characterized two AF-1-binding proteins, previously described as transcriptional coactivator and corepressor, respectively. Importantly, one of them, TLE1, has not been related to NR function, whereas PNRC2 was originally identified by its interaction with the mouse steroidogenic factor 1 (SF1) in the yeast twohybrid system [32]. In addition, the study of Zhou and Chen [32] shows a ligand-dependent PNRC2-interaction with NRs, including estrogen receptor, glucocorticoid receptor, thyroid hormone receptor, progesterone receptor, retinoic acid receptor, and retinoid X receptor. Furthermore, they demonstrate an interaction of the LBD of ERRa and PNRC2 in the yeast two-hybrid system and coactivation in mammalian cells. The mutational analysis revealed the importance of an SH3 domain-binding motif (SDPPSPS) in a proline-rich region of PNRC2 for the interaction with ERRa. It is tempting to speculate that, like ERRa, ERRc recruits PNRC2 via the LBD. However, our studies support an additional function of the A/B domain in PNRC2

recruitment. Previously, PNRC2 has been detected in a series of human cell lines [32]. Our analysis extends this study by detection of a widespread expression of PNRC2 in human tissues. With a DNA probe that is not conserved among PNRC2 and the closely related cofactor PNRC1 [36], a main transcript of 2.8 kb and a minor transcript of 1.9 kb could be detected by Northern blot analysis in various tissues, including the brain. Two transcripts of similar size have previously been detected in a human breast cell line [32]. The high transcript level of both, ERRc and PNRC2 in heart and kidney, suggests a functional interaction in these tissues. In contrast, TLE1 belongs to an important class of corepressors, the TLE (transducin-like enhancer of split) family proteins, mammalian homologues of the Drosophila Groucho, that bind among other transcription factors to Tcf/LEF HMG box transcription factors [33,37]. In Drosophila, Groucho acts together with bHLH proteins of the HES family, both important elements of the Notch cascade, to negatively regulate neuronal differentiation [38]. Several distinct mammalian genes are recognized and are named TLE in human or Groucho-related gene (Grg) in the mouse. In particular, TLE1 is expressed in mitotic neural progenitor cells of the central nervous system, but its expression is down-regulated in new postmitotic neurons [39,40]. The decrease is transient however, as TLE1 expression is reactivated in more mature neurons that have reached their destinations, suggesting that TLE proteins may also be involved in maintenance of the differentiated state. TLE proteins share two highly conserved domains, a carboxy-terminal WD40 repeat domain and an amino-terminal glutamine-rich dimerization domain (Q). These domains separate a variable region including glycine- and proline-rich (GP), casein kinase II and cdc2 phosphorylation sites, nuclear localization sequence (CcN), and serine- and proline-rich (SP) domains [33]. A related protein, designated amino-terminal enhancer of split (AES) encoded by a distinct gene harbors only the Q and GP domain, is a potential dominant negative form of TLE proteins. It has been shown to interact with the amino-terminal fragment of the androgen receptor and to inhibit androgen receptor-mediated transcription in a cell-free transcription system [41]. However, coexpression of TLE1 together with ERRc revealed an activating function of TLE1. The direct in vitro interaction of ERRc and the TLE1 WD40 domain in the phage display assay, as well as the interaction of TLE1 with GST-ERRc in the pull-down experiment, suggested that TLE1 can function as a direct coactivator of ERRc in cell based experiments. It is an interesting hypothesis that expression of a single gene can downregulate one set of genes with bHLH response elements, while up-regulating genes, under the control of ERRc and possibly additional NRs. A function of Groucho as transcriptional activator has previously been discussed

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[42]. In addition, studies in yeast suggest an inherent potential of the Cyc8-Tup1 corepressor for transcriptional activation function [43]. However, we cannot exclude that TLE1 functions by competing an even stronger repressor or by an indirect transcriptional interference through capturing of a corepressor. The Northern blot analysis reveals that TLE1 is widely expressed with high transcript levels in skeletal muscle, liver, and placenta. Therefore, TLE1 may be an important regulator of ERRc in skeletal muscle and placenta where high ERRc transcript levels have previously been reported [44]. However, the spatial distribution of TLE1 and ERRc is best described in the developing and adult rodent brain. Here, regions expressing either high levels of TLE1 or of ERR as well as areas expressing both proteins have been described. In addition to the major 4.4 kb transcript, a shorter TLE1 transcript could be detected. A recent study in mouse suggests that it encodes an alternatively spliced RNA coding for an additional TLE1 isoform. [45]. Indeed, while amplifying the full-length cDNA, we identified an additional human brain cDNA that encodes TLE1DGP lacking the GP-domain but still functioning as a coactivator of ERRc (Fig. 5). The identification of additional proteins as cofactors of ERRc provides a foundation for a better understanding of orphan receptor function. In the future, it will be important to analyze cell specific ERRc target genes and to identify the distinct function of these cofactors for the well-orchestrated gene expression patterns.

Acknowledgments We thank Prof. Schaller for the support of this work. This project was supported by a fellowship to M.H. through the Graduiertenkolleg 255. Special thanks go to Ute S€ usens for technical assistance, to Irm Hermans-Borgmeyer for discussions and comments throughout the course of this project, and to Simon Hempel for help with the figures.

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