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

David J. Heard1, Peder L. Norby, Jim Holloway and Henrik Vissing

Department of Molecular Genetics (D.J.H., P.L.N., H.V.) Novo Nordisk A/S Novo Allè, DK-2880 Bagsvaerd, Denmark
ZymoGenetics Inc. (J.H.) Seattle, Washington 98102


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear receptor protein superfamily is a large group of transcription factors involved in many aspects of animal development, tissue differentiation, and homeostasis in the higher eukaryotes. A subfamily of receptors, ERR{alpha} and ß (estrogen receptor-related receptor {alpha} and ß), closely related to the ER, were among the first orphan nuclear receptors identified. These receptors can bind DNA as monomers and are thought to activate transcription constitutively, unaffected by ß-estradiol. Studies of the expression patterns of ERR{alpha} and gene disruption experiments of ERRß indicate that they play an important role in the development and differentiation of specific tissues in the mouse. In this work we demonstrate the existence in humans of a third member of this subfamily of receptors, termed ERR{gamma}, which is highly expressed in a number of diverse fetal and adult tissues including brain, kidney, pancreas, and placenta. The ERR{gamma} mRNA is highly alternatively spliced at the 5'-end, giving rise to a number of tissue-specific RNA species, some of which code for protein isoforms differing in the N-terminal region. Like ERR{alpha} and ß, ERR{gamma} binds as a monomer to an ERRE. A GAL4-ERR{gamma} fusion protein activates transcription in a ligand-independent manner in transfected HEK293 cells to a greater degree than either the GAL4-ERR{alpha} or -ß fusion proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear receptor (NR) superfamily is comprised of both ligand-regulated receptors and a large group of receptors for which no ligands have been found, the orphan receptors. The functions of many of the ligand-regulated receptors are well understood from the molecular level to their roles in development and homeostasis of the adult organism (for reviews see Refs. 1, 2, 3, 4, 5). In contrast to the liganded NRs, the biological functions of the orphan receptors are only beginning to be understood. Knockout experiments have demonstrated their biological relevance as the orphans COUP-TFI (chicken ovalbumin upstream promoter-transcription factor I), COUP-TFII, and ERRß (estrogen receptor-related receptor-ß) are essential genes, and SF-1/Ftz-F1 and DAX1 are required for the differentiation of the adrenal glands and gonads (reviewed in Refs. 1, 2, 6). Orphan NRs can be separated into four groups, based on the way they interact with DNA (Ref. 5 and references therein): Group 1 receptors require heterodimerization with retinoid X receptor to bind to their response elements [i.e. nuclear growth factor I-B (NGFI-B)]. Receptors in group 2 bind DNA as homodimers (i.e. COUP-TFI and related receptors). Group 3 receptors bind as monomers to an extended NR half-site (i.e. SF-1/Ftz-F1, NGFI-B, ROR{alpha} and -ß, ERR{alpha} and -ß). The group 4 receptors are those with an unusual structure in that they lack either a DNA-binding domain (DAX1) or a ligand-binding domain (Drosophila Knirps).

The estrogen receptor-related receptors, ERR{alpha} and -ß, are two group 3 orphan receptors, which were initially identified in human testis, kidney, and cardiac cDNA libraries by screening with probes derived from the DNA-binding domain (DBD) of human ER{alpha} (7). Although these receptors are related to ER at the amino acid level, functionally they are quite distinct. ER{alpha} and ß bind as homodimers to estrogen response elements (ERE), consisting of an inverted repeat of the common NR hexameric half-sites separated by three nucleotides, AGGTCANNNTGACCT (where N is any nucleotide) (2, 5). In contrast to the ERs, ERR{alpha} and -ß can bind to DNA as monomers to an extended NR half-site, TCAAGGTCA (half-site in bold) (8, 9, 10). This sequence is also the response element for group 3 orphan receptors of the SF-1/Ftz-F1 subfamily (2, 5).

Although discovered 10 yr ago, investigation of the transcriptional activity of the ERR family of receptors is still ongoing. Until recently, ERR{alpha} and -ß were thought to activate transcription constitutively (in the absence of ligand). However, two recent reports suggest that ERR{alpha} may indeed be a ligand-activated receptor. Yang and Chen (11) have shown that two organochlorine pesticides, toxaphene and chlordane, appear to act as antagonists for ERR{alpha} activation in HepG2 cells, whereas these compounds are weak ER{alpha} agonists. Also, activation by both ERR{alpha} and -ß may be regulated by an agonist found in the serum which is removable by treatment with charcoal (12, 13). In certain instances ERR{alpha} may also act as a repressor of transcription as it inhibits expression from the SV40 major late promoter during the switch from early to late gene expression (14). ERRß has been shown to specifically repress transcription induced by the glucocorticoid receptor (GR) in CV-1 but not HeLa cells perhaps via titration of cofactors required for activation by GR (15).

The expression patterns of ERR{alpha} and ß are quite different. Mouse ERR{alpha} is highly expressed in adult heart, brain, skeletal muscle, kidney, and testis and more weakly expressed in liver, lung, and vagina (7, 16, 17, 18). ERR{alpha} is also known to be expressed in osteoblasts during bone development (16) and in the uterus (endometrium carcinoma cell lines) (8). During mouse development, ERR{alpha} mRNA can be detected in embryonic stem (ES) cells, and by its expression pattern it has been implicated in the development of heart and skeletal muscle, central and peripheral nervous system, the epidermis, and the epithelium of the intestine and urogenital tract (17). In contrast to ERR{alpha}, ERRß expression is barely detectable in a few tissues of the adult rat including kidney, heart, testis, brain, and prostate (7), whereas in the mouse it is weakly expressed in adult kidney and heart (19). In the mouse, ERRß has been shown to be involved in early placental development and its expression appears to be restricted to a subset of extraembryonic tissues in a small window between 5.5 days post coitum (d.p.c.) and 8.5 d.p.c. (19, 20). In agreement with this observation, homozygous ERRß-/- knockout mouse embryos die by 9.5 d.p.c. due to abnormal chorion formation (20). Therefore it appears that both members of the ERR subfamily of receptors play important roles during development and that ERR{alpha} is also involved in gene expression in a number of adult tissues.

In this work we describe the molecular cloning and characterization of a human orphan receptor, ERR{gamma}, which has 77% overall sequence identity to its closest relative, ERRß. In contrast to ERRß, ERR{gamma} is highly expressed in a number of adult human tissues including brain, skeletal muscle, heart, kidney, and retina. This receptor is also expressed in several human fetal tissues including placenta, brain, heart, skeletal muscle, kidney, and lung. These findings suggest a possible role for ERR{gamma} in the differentiation and maintenance of these tissues. Interestingly, there are multiple tissue-specific alternatively spliced forms of the ERR{gamma} mRNA, some of which give rise to different protein isoforms. Full-length ERR{gamma} binds as a monomer to an extended NR hexamer half-site (TCAAGGTCA) in vitro, suggesting potential redundancy of function with ERR{alpha} in tissues where both receptors are expressed. The ligand-binding domain of ERR{gamma}, when fused to the GAL4 DBD, activates transcription in a constitutive manner, and this activity is not affected by estradiol. Furthermore the activation potential of the GAL4-ERR{gamma} is much greater than GAL4-ERR{alpha} or -ß in transfected HEK293 cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of ERR{gamma} and Sequence Analysis
To identify further members of the NR family the dbEST database was mined for sequences similar to known NRs. An EST from a human retina library (GenEMBL Accession No. W26275) was identified which had significant homology to ERRß. On the basis of the EST sequence, oligonucleotide primers were designed and human cDNA libraries were screened for the presence of this EST by PCR. 5'- and 3'-RACE (rapid amplification of cDNA ends)-PCR was then performed on human placenta, skeletal muscle, kidney, and pancreas cDNA libraries (CLONTECH Laboratories, Inc., Palo Alto, CA) using oligonucleotide primers derived from the EST sequence and primers for the Marathon Ready cDNA ends (CLONTECH Laboratories, Inc.). Several clones were obtained by 5'-RACE-PCR (Fig. 1AGo) from different cDNA libraries. When these sequences were compared by multiple alignment, it was obvious that while all clones start with a common sequence, they diverge at an AGGT motif, a sequence commonly found at splice junctions, indicating that these clones arise as a result of alternative splicing. Most of the alternative splicing appears to take place in the 5'-untranslated region (UTR) and does not affect the open reading frame (ORF). However, up to three different protein isoforms are predicted by the 5'-RACE-PCR clones (Fig. 1AGo). In all clones homology is restored 15 nucleotides upstream of an AUG, which is the putative translation start site for one of the isoforms (ERR{gamma}1; see below and Fig. 1Go). A clone obtained from placenta cDNA libraries contains an AUG codon in a good Kozak sequence environment 207 nucleotides upstream of the initiation codon described above. The 5'-end of this clone is identical to the ERR{gamma}2 cDNA described by Chen et al. (21). The sequence of this RACE-PCR clone is 99% identical to the initial retina EST sequence identified, W26275 (corresponds to nucleotides 71–561 of ERR{gamma}2; see Fig. 1BGo). All four alternatively spliced ERR{gamma} transcripts derived from kidney cDNA libraries have the same ORF starting at the next AUG downstream of the putative start site in ERR{gamma}2 (see Fig. 1AGo) and would produce an isoform similar to a ERR{gamma} sequence reported recently by Eudy et al. (22). Here we refer to these cDNAs as ERR{gamma}1.1 to 1.4 (Fig. 1AGo). A third ERR{gamma} cDNA found in muscle libraries contains a potential non-AUG translation start codon as it contains an in-frame AGAGUGG sequence (putative translation start site in bold) 34 codons upstream of the predicted translation start site for the ERR{gamma}1 protein (Fig. 1AGo). This mRNA could potentially give rise to a third isoform, ERR{gamma}3, with a different N-terminal domain than predicted for either ERR{gamma}1 or -2. Use of alternative translation start sites has been observed previously for a number of NRs (2, 23, 24, 25).



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Figure 1. Alignment of ERR{gamma} 5'-RACE-PCR Clones and Full-Length ERR{gamma}2 Sequence

A, Multiple alignment of clones derived from kidney (ERR{gamma} clones 1.1–1.4), placenta (2 ), and skeletal muscle (3 ) cDNA libraries. Boxes indicate sequences common to all clones. The sequence of the 5'-linker/primer from the Marathon Race cDNA library (CLONTECH Laboratories, Inc.) is in italics. Positions of PCR primers used for amplification of specific transcripts are indicated in bold. Putative translation start signals are underlined. Gaps in the alignment are indicated by dots. B, Nucleotide and predicted amino acid sequence of the full-length ERR{gamma}2 clone. Positions of oligonucleotides used for PCR are indicated by arrows above the sequence. The arrowhead in panels A and B indicates the position at which homology in all clones is restored. C, Schematic comparison of the predicted ERR{gamma}-1 and {gamma}-2 protein sequences with other closely related human nuclear receptors. Domains A–F are indicated above. The arrow indicates the position of the methionine where translation begins for the ERR{gamma}-2 isoform. Numbers above the boxes refer to amino acid sequence positions. Numbers within the boxes indicate percent identity to ERR{gamma}, and no number indicates no significant homology.

 
When 3' RACE-PCR was performed, only one product was obtained from placenta cDNA libraries. The sequence of this fragment, which ends in a stretch of 26 adenines, is indicated in Fig. 1BGo. The 3'-UTR sequence reported here is 100% identical to the consensus sequence predicted by five ESTs (accession nos. Z42949, R12217, R19987, H06208, and H08990); further sequence analysis of the 3'-UTR was not performed. Our 3'-UTR sequence is also nearly identical to the one described by Eudy et al. (22) up to the poly-A stretch, but the Eudy sequence continues for another 3.2 kb. Given that no consensus polyadenylation signal could be found near the poly-A terminus of the 3'-sequence (Fig. 1BGo) and that the sequence described by Eudy et al. (22) is closer to the expected size of the RNA from Northern blots (see below), it is likely that the 3'-RACE-PCR clone obtained by us is derived from a false priming by the oligo-dT primer at the stretch of adenines during cDNA library construction. Alternatively, our 3'-RACE-PCR clone may represent a nonabundant message arising from alternative polyadenylation.

To confirm the sequence of the existence of the various ERR{gamma} transcripts, oligonucleotides specific for the three different 5'-RACE-PCR clones (Fig. 1AGo) and an oligonucleotide derived from the 3'-RACE-PCR clone (primer 5, see Fig. 1BGo) were used to amplify the full-length coding sequence from the appropriate tissues: ERR{gamma}2 was amplified from a placenta library; ERR{gamma}1 from kidney cDNA, and ERR{gamma}3 from a skeletal muscle library. The PCR products were cloned into an expression vector giving rise to constructs pERR{gamma}1, -2, and -3. From these sequences, predictions of the amino acid sequence and size of the various protein isoforms were made. The ERR{gamma}2 isoform is 458 amino acids long and has a predicted molecular mass of 51.3 kDa. The ORF in the ERR{gamma}1 clone gives rise to a 435-amino acid protein with a predicted molecular mass of 48.7 kDa. If the putative GUG start codon present in the muscle-derived ERR{gamma}3 isoform is used, the resulting protein would be 469 amino acids in length and have a predicted molecular mass of 52.6 kDa. If this alternative start site is not recognized, translation of this mRNA should start at the first downstream AUG codon, which is the same as that in the pERR{gamma}1 clone (see below).

Comparison of the predicted amino acid sequence of ERR{gamma} with related NRs revealed highest homology with the human ERRß receptor (Fig. 1CGo). The DNA binding domain (domain C) of ERR{gamma} is 99% identical to that of ERRß and 93% identical to that of ERR{alpha}. The ligand-binding domain (domain E/F) is less conserved between ERRß and {gamma}, only 77% identical, and the hinge region (domain D) is 70% identical. The N-terminal A/B domain is 59% identical between ERR{gamma}1 and ERRß. There is no significant homology between the putative A/B domains present in ERR{gamma} clones and other NRs. A number of differences exist between the ERR{gamma} sequence of Eudy et al. (22) and the one reported here. These include 10 base changes within the coding sequence, 6 of which alter the amino acid sequence of ERR{gamma}1. Sequencing and comparison of all independently derived RACE-PCR and full-length clones (PCR amplified by Pfu polymerase) have confirmed the validity of the coding region sequence presented here. The sequence of the ERR{gamma}2 clone presented here is also 100% identical to the amino acid sequence for ERR{gamma}2 described recently by Chen et al. (21).

ERR{gamma} Isoforms Are Expressed in a Tissue-Specific Manner in Adult Tissues and during Development
Northern blot analysis of the expression pattern of ERR{gamma} in adult human tissues (MTN blots, CLONTECH Laboratories, Inc.) was performed using a probe derived from the N-terminal region of ERR{gamma}2 but extending to the beginning of the DNA binding domain (nucleotides 166–569), therefore containing sequences common to all spliced forms so far identified. Autoradiograms revealed that ERR{gamma} is encoded by an mRNA of approximately 5.5–6.5 kb. ERR{gamma} mRNA is highly expressed in heart and skeletal muscle, kidney, pancreas, placenta, and brain. Weak expression is also seen in prostate, spleen, testis, and small intestine (Fig. 2Go). This Northern blotting experiment was repeated with a second batch of MTN blots with identical results (data not shown). To further confirm our Northern results and analyze the expression patterns of the different ERR{gamma} mRNAs, panels of human cDNA libraries (CLONTECH Laboratories, Inc.), corresponding to the Northern blots, were screened by PCR using primers specific for the ligand-binding domain of ERR{gamma} and primers designed to specifically amplify the ERR{gamma}1, -2, or -3 cDNA (see Fig. 1Go). A retina cDNA library (CLONTECH Laboratories, Inc.) was also included due to the fact that EST W26275 originated from this tissue. Amplification of the ligand-binding domain (LBD) of ERR{gamma} from multitissue cDNA panels using primers 2 and 4 (Fig. 1BGo) indicates a pattern of expression very similar to that observed on the Northern blots (compare Fig. 2Go and Fig. 3AGo). Also there appears to be strong ERR{gamma} expression in the retina (lane 17). However, the signal observed in muscle by PCR is rather weak compared with the Northern blot. This may reflect the amount of muscle mRNA loaded on the Northern blot (Fig. 2Go, compare the ß-actin signal in lane 7 to other lanes). Also, in comparison to the Northern blot, there is only a weak amplification of the ERR{gamma} LBD from small intestine cDNA, which may indicate a further spliced form in this tissue (see Discussion). Interestingly, this method indicates weak ERR{gamma} expression in lung, thymus, ovary, and colon where no expression was observed in Northern blots.



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Figure 2. ERR{gamma} Expression in Adult Tissues

MTN blot probed with a 32P-labeled ERR{gamma}2 probe (top panel, probe corresponds to nucleotides 143–546 see Fig. 1BGo) or ß-actin control probe (bottom panel). Positions of RNA markers and size in kilobases (Kb) are indicated on the left. Sk. Muscle, Skeletal muscle; Sm. Intestine, small intestine; P. Blood Leuk., peripheral blood leukocytes.

 


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Figure 3. Tissue Distribution of ERR{gamma} Isoforms in Adult and Fetal Human Tissues

A and C, Expression of ERR{gamma} mRNA as determined by RT-PCR from adult (B) or fetal (C) human multitissue cDNA library panels. Panels represent amplification products of (from top to bottom): a fragment of the LBD common to all mRNAs (primers p2 and p4 see Fig. 1BGo); ERR{gamma}2 mRNA-specific 5'-primer and p3; ERR{gamma}1 mRNA-specific primer and p3; ERR{gamma}3 mRNA-specific primer and p5; amplification of control glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA. Size of mol wt markers in kilobases (1 kb ladder) are indicated. Control reactions in which no cDNA was added are indicated (Control, lane 18). Note that in panel B the control lane for ERR{gamma}1 contains 1 µg of human genomic DNA. Abbreviations are the same as for Fig. 2Go.

 
PCR with primers specific for the different ERR{gamma} cDNAs (Fig. 1AGo) and a downstream primer (primer 3 for {gamma}1 and 2 and primer 5 for {gamma}3, indicated in Fig. 1BGo) demonstrates that the different transcripts are expressed in a tissue-specific manner (Fig. 3AGo). The ERR{gamma}2 mRNA is most abundant in placenta and retina but is also present in heart, brain, and perhaps weakly in colon. As expected, amplification of using ERR{gamma}1-specific oligonucleotides gives rise to multiple bands due to the presence of several differentially spliced transcripts (see Fig. 1AGo). ERR{gamma}1 is expressed most highly in adult kidney and pancreas but is also seen in heart, brain, prostate, and retina and weakly in spleen and ovary. The expression of ERR{gamma}3 mRNA appears to be restricted to skeletal muscle. Control reactions using primers supplied by the manufacturer indicate a similar amount of mRNA in all reactions (Fig. 3AGo, G3PDH). Taken together the results of Northern blotting and PCR experiments essentially substantiate each other, although PCR also indicates weak ERR{gamma} expression in adult lung, thymus, ovary, and colon. These weak PCR signals are further corroborated by amplification of the specific ERR{gamma}1 and -2 cDNAs from adult colon and ovary libraries, respectively (Fig. 3AGo).

Analysis of ERR{gamma} expression in human fetal tissues by PCR of the LBD revealed that, reflecting the pattern seen in adult tissues, ERR{gamma} is highly expressed in the fetal brain, kidney, heart, and skeletal muscle (Fig. 3BGo). Weak ERR{gamma} expression also seems to be present in spleen, thymus, and lung whereas there is no detectable expression in these tissues in the adult. Interestingly, the three ERR{gamma} isoform mRNAs are also restricted to expression in specific tissues during development. The ERR{gamma}2 signal is strong in heart but is also detectable in a number of other fetal tissues including brain, kidney, lung, skeletal muscle, and liver. ERR{gamma}1 appears to be most highly expressed in fetal kidney and is also weakly expressed in skeletal muscle. As in the adult, ERR{gamma}3 expression appears to be completely restricted to the skeletal muscle (Fig. 3BGo). Therefore, ERR{gamma} is highly expressed in some tissues during development and as in the adult the three different ERR{gamma} mRNAs demonstrate a tissue- restricted expression pattern somewhat different from that seen in adult tissues.

In Vitro Translation of the ERR{gamma} Transcripts
To determine whether the various ERR{gamma} clones code for proteins of the expected size, the cDNAs were translated in vitro in the presence of 35S-labeled methionine and products separated on a denaturing 4–12% gradient gel. The most abundant protein species produced from the pERR{gamma}2 clone is migrating at approximately 51 kDa, indicating that the first AUG in the ORF is used preferentially as the translation start site in this mRNA (Fig. 4AGo, band 1). The protein arising from the ERR{gamma}1 clone is migrating just above the 46 kDa marker, suggesting that the predicted molecular mass of 48.7 kDa is correct for this isoform (band 2). The ERR{gamma}2 clone gives rise to a similar protein band, suggesting some read-through of the upstream AUG in the reticulocyte lysate. The majority of protein produced from the pERR{gamma}3 construct also migrates at approximately 48 kDa (band 2), suggesting that the translation start site is not at the putative alternative start codon located upstream of this GUG. Another labeled polypeptide arising from the ERR{gamma}3 clone runs at a molecular weight higher than expected if the GUG codon had been used (band 3); therefore the identity of this polypeptide is unclear. Interestingly, in comparison to the 48 kDa band produced by the ERR{gamma}1 clone, the polypeptide migrating at 48 kDa arising from ERR{gamma}3 appears more abundant, giving a thicker more diffuse band. It seems for the ERR{gamma}3 mRNA that the majority of translation initiates at the first AUG but that the sequence upstream of this site has an influence on the efficiency of translation. Finally, a polypeptide of approximately 44 kDa is produced by all clones (band 4). This corresponds to the polypeptide expected if translation began at the next downstream AUG, 72 residues downstream of the ERR{gamma}2 start site (Fig. 1BGo), which is in a better Kozak sequence environment than the AUG at codon position 24 in the ERR{gamma}2 sequence (see Fig. 1BGo).



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Figure 4. In Vitro Translation of ERR{gamma} Gene Products and DNA Binding

A, Rabbit reticulocyte lysates were programmed with empty vector (Ctrl) or specific ERR{gamma} clones (pERR{gamma}1 to 3) and 35S-labeled proteins were separated on denaturing polyacrylamide gels. Positions and size of protein mol wt markers indicated to the right (in kDa). The various polypeptides observed (1 2 3 4 ) are discussed in the text. B, EMSA with 32P-labeled WT or mutant (Mut) double stranded (ds) ERRE probes (lower panel) incubated with rabbit reticulocyte lysates programmed with pERR{gamma}2 or empty vector (Ctrl). Binding was competed with 0,5 or 25 molar excess of unlabeled ds WT or Mut probes.

 
ERR{gamma} Binds to an ERRE Element as a Monomer
As the DBDs of all the ERR receptors are more than 90% conserved, it seemed likely that similarly to ERR{alpha} and ß, ERR{gamma} should bind as a monomer to an extended NR half-site. To test this notion, an electrophoretic mobility shift assay (EMSA) was performed with unlabeled in vitro translated ERR{gamma}2 (Fig. 4BGo). The results demonstrate that, as expected, this protein binds to a 32P-labeled, double-stranded oligonucleotide probe containing a single wild-type (WT) ERRE (TCAAGGTCA) (lane 2) and that this interaction can be efficiently competed with excess cold WT ERRE probe, but not by a probe containing a mutant (Mut) ERRE (TCAAccTCA) (compare lanes 3–6). Consequently, no binding of ERR{gamma}2 to the labeled Mut ERRE probe was observed (lane 7). Proteins in control lysates programmed with empty vector do not bind the probes (lanes 1 and 8). It is possible that the appearance of two bands in the EMSA experiment is due to the multiple translation start sites observed for this mRNA in vitro (see Fig. 4AGo). An alternative explanation may be that two ERR{gamma}2 proteins bound to two different probe molecules can interact forming a higher order structure. Regardless, these data strongly support the conclusion that ERR{gamma} can bind to DNA as a monomer.

The ERR{gamma} Ligand-Binding Domain Has a Transcription Activation Function
Both ERR{alpha} and -ß have been found to activate transcription constitutively, in the absence of ligand, and their activity is unmodified by estrogen. To test whether this is also true for ERR{gamma}, the LBD (amino acids 189–458, Fig. 1BGo) was fused to the DBD of GAL4 (amino acids 1–147). The ability of this fusion protein to activate transcription of a reporter construct containing GAL4 binding sites was determined in transiently transfected HEK293 cells (Fig. 5AGo). The results indicate that, as expected, the GAL4-ERR{gamma} fusion protein activates transcription more than 50-fold over the signal with the GAL4 DBD alone. This activity is not significantly altered in the presence of 5 x 10-7 M ß-estradiol (Fig. 5AGo). A control construct in which the GAL4 DBD is fused to the ER{alpha} LBD demonstrates the activity of ß-estradiol in this system as transcription activation by this fusion protein was strongly induced upon addition of ß-estradiol (Fig. 5AGo). Some squelching of transcription is observed when 1 µg of the GAL4-ER{alpha} construct is used. Note that in the presence of ligand, activation by the GAL4-ER{alpha} construct is more than 5-fold greater than that seen with the GAL4-ERR{gamma} construct.



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Figure 5. GAL4-ERR{gamma} Fusion Proteins Activate Transcription in Transient Transfection Assays

A, Comparison of GAL4-ERR{gamma} with GAL4-ER{alpha} in the presence or absence of ß-estradiol. B, The activation potential of the GAL4-ERR{gamma} construct as compared with GAL4-ERR{alpha} and GAL4-ERRß. The graphs represents the means of at least three independent experiments where each transfection was done in quadruplicate. Numbers below the x axis represent amounts of plasmid expressing the indicated fusion proteins used in each transfection (in micrograms). Error bars indicate SEM. Fold activation is increase in the normalized luciferase signal over the signal obtained when 1 µg of the GAL4 DBD plasmid is transfected. Ethanol indicates ethanol vehicle control; ß-estradiol concentration was 5 x 10-7 M.

 
The activation potential of the LBD of ERR{gamma} was compared with those of human ERR{alpha} and -ß LBDs using GAL4 fusion proteins (Fig. 5BGo). Similar to the findings above, transfection of the GAL4-ERR{gamma} construct resulted in a 65-fold stimulation of transcription in this set of experiments. In contrast, the highest activation observed with the ERR{alpha} construct is only 10 fold above the background observed with the GAL4 DBD alone. This level of stimulation is in agreement with previous findings using the full-length ERR{alpha} (12, 13). Surprisingly, cotransfection of the GAL4-ERRß construct did not significantly alter the transcription of the reporter gene (see Discussion). Therefore it seems that the LBD of ERR{gamma} contains the most potent activation domain of the ERR subfamily of receptors described to date.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ERR{gamma} pre-mRNA is extensively alternatively spliced at the 5'-end giving rise to at least six different transcripts that appear to code for two different ERR{gamma} protein isoforms. The results also suggest that other tissue-specific ERR{gamma} transcripts and perhaps protein isoforms may be found in tissues such as muscle, testis, small intestine, and lung. This is evident from the fact that while the ERR{gamma} mRNA is apparent in these tissues by Northern blotting and/or RT-PCR screening when screened with a portion of the message common to all six ERR{gamma} clones identified, oligonucleotides specific for the 5'-ends of the different mRNAs fail to amplify ERR{gamma} fragments in these tissues. Apart from the tissue-specific expression of the ERR{gamma}1 and {gamma}2 protein isoforms, the explanation for the extensive alternative splicing of the ERR{gamma} mRNA is unclear. An analysis of the 5'-UTRs of the six different ERR{gamma} transcripts has not revealed the presence of sequences or structures known to be involved in the control of translation initiation (data not shown, see Ref. 26); however, this possibility cannot be ruled out at present. Also further experiments with anti-ERR{gamma} antibodies must be performed to determine which protein isoforms are expressed in vivo.

Northern blots indicate that ERR{gamma} is highly expressed in several adult human tissues including heart, brain, placenta, skeletal muscle, kidney, pancreas, and retina. ERR{gamma} expression is also observed in prostate, testis, spleen, and small intestine. RT-PCR with ERR{gamma} LBD-specific primers also indicates weak ERR{gamma} expression in lung, thymus, ovary, and colon, which were all negative in Northern blot experiments even after long exposure (data not shown). PCR with primers specific for the ERR{gamma}1 and 2 mRNAs supports the observation of weak expression of ERR{gamma} in colon and ovary but not in lung and thymus, perhaps suggesting the existence of further mRNAs coding for ERR{gamma} in these tissues. Our Northern blot results differ from those published by Eudy et al. (22), who reported high expression of human (h)ERR{gamma} in lung where we see nothing on the Northern and only a trace signal by RT-PCR with the LBD-specific primers. Further, these authors found no expression of hERR{gamma} in skeletal muscle or kidney, whereas our Northern and RT-PCR data indicate relatively high expression in these tissues. The pattern of expression of hERR{gamma} also differs somewhat from that of the mouse (m)ERR{gamma} as seen in a paper that appeared while this manuscript was being revised (27). While these authors also detected high mERR{gamma} expression in the mouse kidney using a probe for the N-terminal region of the mRNA corresponding to the hERR{gamma}2 transcript, it appears that, in contrast to humans, ERR{gamma} mRNA is not present in mouse skeletal muscle but is present in liver. These differences may be explained in part by the large number of tissue-specific alternative splicing events observed for the 5'-end of the ERR{gamma} mRNA in human tissues. If the same is true in mouse, the probe used by Hong et al. (27) may not detect other spliced forms of the mRNA.

The ERR family of orphan NRs appear to be important transcription factors involved in the differentiation of specific tissues during mouse development. In the case of ERRß, this has been demonstrated by production of a knockout mouse, which in homozygous mutants results in lethality at 9.5 d.p.c. due to a defect in the development of extraembryonic tissues (20). This phenotype correlates exactly with the restricted expression pattern of ERRß in the developing embryo (19, 20). For ERR{alpha}, a role in development is suggested by its highly restricted expression pattern during mouse embryogenesis (16, 17). We report here that ERR{gamma} is also expressed in a tissue-restricted manner during development in humans, suggesting that this receptor may be an important factor in the differentiation of tissues such as cardiac and skeletal muscle, brain, liver, and kidney. Intriguingly, the different ERR{gamma} mRNA species are also expressed in a tissue-specific manner during development. As in the adult, ERR{gamma}2 is expressed in the fetal heart and brain but is also expressed in kidney, muscle, lung, and liver. ERR{gamma}1 is most highly expressed in the fetal kidney but is also present in brain, muscle, lung, and spleen cDNA libraries. Like in the adult, the ERR{gamma}3 mRNA is restricted to the developing skeletal muscle. Extrapolating from studies of ERR{alpha} expression in the rat and mouse (7, 17, 18), it appears that high expression of human ERR{alpha} and -{gamma} could overlap in several tissues including heart, brain, skeletal muscle, and kidney. Since results presented here demonstrate that, similarly to ERR{alpha} and ß, ERR{gamma} binds as a monomer to an extended hormone response element half-site of the ERRE type (TCAAGGTCA), this suggests a possible functional redundancy of ERR{alpha} and {gamma} in these tissues, and future gene knockout experiments should take this into account.

A GAL4-ERR{gamma} fusion protein increases reporter gene transcription up to 60-fold over GAL4 alone, and this activity is unaffected by ß-estradiol, indicating that the LBD of ERR{gamma} contains a transcriptional activation domain that is unaffected by estrogen, as reported previously for ERR{alpha} and -ß (13). The level of activation by the GAL4-ER{alpha} construct in the presence of ß- estradiol is more than 5-fold greater than that of ERR{gamma}. While ERR{gamma} may simply have a weaker activation potential as compared with ER{alpha}, this result may also indicate either that the full-length ERR{gamma} receptor is required or that binding to a ligand is necessary to achieve full activation. However, in comparison to other members of the ERR subfamily of receptors, the LBD of ERR{gamma} appears to have the strongest activation potential. Activation by the ERR{alpha} LBD is 10-fold over background, which corresponds well to previous observations (12, 13), but is 5- to 6-fold lower than the ERR{gamma} signal. In contrast to ERR{alpha} and -{gamma}, no detectable activation was observed with GAL4-ERRß construct. Although full-length ERRß receptor has been shown to result in activation levels similar to full-length ERR{alpha} in CV-1 cells (13), transcription activation by GAL4-ERRß fusion proteins has not been previously reported. The results presented here may indicate that the ERRß LBD alone is insufficient for activation.

A 10- to 12-fold activation by the full-length mouse ERR{gamma} receptor was reported recently using a reporter construct containing two estrogen response elements (ERE) in CV-1 cells (27). In our hands full-length hERR{gamma}1 and {gamma}2 receptors showed only a 2-fold increase over background expression in cotransfection with a reporter construct containing a single WT ERRE (results not shown). In these experiments background luciferase expression was 6-fold higher for reporter constructs containing a single WT ERRE compared with those containing a mutant ERRE (data not shown). Therefore, further work is necessary to elucidate the functional properties of the ERR{gamma}1 and {gamma}2 isoforms and their ability to activate transcription from an ERRE in vivo.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
All routine RNA and DNA manipulations were performed as described by Sambrook et al. (28) unless otherwise indicated. When kits were used, the manufacturers instructions were followed unless otherwise stated. All constructs were confirmed by DNA sequencing. Sequence analysis, amino acid, and mol wt prediction were performed using the Wisconsin Package version 9.1 (Genetics Computer Group, Madison WI).

Database Mining and Molecular Cloning of ERR{gamma} cDNAs
The EST databases were screened by BLAST searches (29) for sequences similar to the known NRs. An EST (GenEMBL Accession no. W26275) was identified and RACE-PCR was used to amplify the 5'- and 3'-ends of the corresponding cDNA using Marathon-Ready cDNA libraries (CLONTECH Laboratories, Inc.) following the manufacturers recommended protocol. Fragments were cloned into the vector pCRII by Topo-TA cloning (Invitrogen, San Diego, CA) and analyzed by DNA sequencing. Sequences of RACE-PCR fragments containing the original EST sequence were used to design oligonucleotides for PCR amplification of the full- length sequence from appropriate cDNA libraries by Pfu DNA polymerase (Stratagene, La Jolla, CA). Full-length ERR{gamma} PCR products were cloned into the mammalian expression vector pcDNA3.1/V5/His by TA cloning (Invitrogen).

Analysis of Gene Expression Patterns by Northern Blotting and RT-PCR
Northern blots of Poly-A+ mRNA derived from a variety of human tissues (MTN blots, CLONTECH Laboratories, Inc.) were probed according to the manufacturers instructions. A PCR-amplified fragment derived from the putative N-terminal A/B domain of ERR{gamma}2 (corresponding to nucleotides 143–546 of the full-length sequence; see Fig. 1AGo) was labeled with [{alpha}-32P]-dCTP by random priming using the Prime-a-Gene kit (Promega Corp., Madison, WI). Briefly, the blots were prehybridized for 30 min at 68 C and then hybridized to the radioactive probe for 2 h at 68 C in Expresshyb hybridization solution (CLONTECH Laboratories, Inc.). Stringent washes were performed at 50 C in 0.1x SSC, 0.1% SDS. Blots were exposed either overnight to a PhosphorImager screen (Molecular Dynamics, Inc.) or to x-ray film at -80 C using an intensifying screen for the appropriate length of time.

PCR was performed on multitissue cDNA panels (CLONTECH Laboratories, Inc.) according to the manufacturers instructions. PCR conditions were 94 C, 30 sec; 55 C, 30 sec; 72 C, 2 min for 35 cycles. For analysis of tissue-specific expression of the different ERR{gamma} isoforms, a downstream primer localized in the ligand binding domain (primer 3 for ERR{gamma}1 and 2 and primer 5 for ERR{gamma}-3; see Fig. 1BGo) was combined with primers specific for each of the different 5'-sequences identified (Fig. 1AGo). The ligand-binding domain was also PCR amplified (primers 2 and 4; Fig. 1BGo) as this portion of the molecule should be present in all mRNAs. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA was amplified as a control using primers supplied by the manufacturer (CLONTECH Laboratories, Inc.).

In Vitro Translation and Electrophoretic Mobility Shift Assays
ERR{gamma} isoforms were synthesized in vitro in rabbit reticulocyte lysates using the T7 transcription-coupled translation system (Promega Corp.) in the presence of 35S-methionine (Amersham Pharmacia Biotech) following the manufacturers instructions. Proteins were separated by denaturing 4–12% NuPAGE gradient gels using the MOPS (3-[N-morpho- lino]propanesulfonic acid) buffer system (Novex, San Diego, CA) and the gel was dried and exposed to a PhosphorImager screen or x-ray film.

For the EMSA assays, ERR{gamma}2 protein was synthesized as above in the absence of 35S-methionine. Double-stranded (ds) oligonucleotides containing an extended half-site sequence or ERRE (5'-CCGGGGCTTTCAAGGTCATATGCA-3') or a mutation within this sequence (5'-CCGGGGCTTTCAAccTCATATGCA-3') (8) were labeled with [{alpha}-32P]-dCTP by filling in the XmaI and BglII sites at the 5'- and 3'-ends of the ds oligos using the Klenow fragment of DNA polymerase I. DNA binding was performed as described by Kliewer et al. (30). Protein-DNA complexes were separated by electrophoresis through a 6% DNA retardation gel (Novex) at 4 C using 0.5xTBE as a buffer. The gel was dried and exposed to a PhosphorImager screen (Molecular Dynamics, Inc., Sunnyvale, CA) for 30 min.

GAL4 Fusions and Transient Transfection Assays
The DEF regions of ERR{gamma} (amino acids 189–458 of ERR{gamma}2; see Fig. 1BGo), human ER{alpha} [amino acids 263–595 (31)], human ERR{alpha} [amino acids 237–521 (7)], and human ERRß [amino acids 164–438 (21)] were PCR amplified using Pfu DNA polymerase such that a BamHI site was engineered into the 5'-end of the PCR products. PCR products were digested with BamHI and fused in-frame downstream of the DBD of the yeast GAL4 transcription factor [amino acids 1–147 (32)] which was cloned previously into the HindIII and BamHI sites of the vector pcDNA3.1(+) (Invitrogen). These constructs, along with a reporter construct containing five GAL4 binding sites upstream of the firefly luciferase gene in the pGL2-Promoter vector (Promega Corp.), were cotransfected into HEK293 cells plated in 96-well plates using FuGene transfection reagent (Roche Molecular Biochemicals). After 24 h the transfected cells were treated with 5 x 10x7 M 17ß-estradiol or ethanol vehicle and the cells incubated for a further 16–24 h. Luciferase activity was measured in a luminometer (Packard Instruments, Meriden, CT) using the FireLite detection system (Packard). Results were normalized by comparison to the RenLite signal from the cotransfected pRL-CMV construct (Promega Corp.). The amount of DNA was kept constant in each transfection by the addition of empty vector (pcDNA3.1).


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Koen Doechering of Organon bv. Netherlands for supplying the ERRß clone before publication. Thanks also to Lissi Aagaard, Iben Bredmose, and Vibeke Pedersen for excellent technical assistance and members of the group for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Henrik Vissing, Department of Molecular Genetics, Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark.

1 Current Address: Exonhit Therapeutics SA, 65 Boulevard Masséna, F-75013 Paris, France. Back

P.L.N. was supported by the European Commission DG XII Biomed 2 Grant PL962433.

Received for publication April 12, 1999. Revision received November 8, 1999. Accepted for publication December 1, 1999.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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