Nucleic Acids Research, 2001, Vol. 29, No. 24 4901-4908
© 2001 Oxford University Press
Retinoic acid receptor 
1 variants, RAR1
B and RAR1BC, define a new class of nuclear receptor isoforms
1Laboratoire de Biologie Cellulaire Hématopoïétique, INSERM U00-03, Université D. Diderot–Paris VII, Institut d’Hématologie, Hôpital Saint-Louis, Paris, France, 2Department of Haematology, University of Wales College of Medicine, Cardiff, UK, 3Sección de Inmunología, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain, 4Fondation Jean Dausset–CEPH, Hôpital Saint-Louis, Paris, France and 5Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France
Received October 1, 2001; Accepted October 22, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retinoic acid (RA) binds and activates retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimers, which regulate the transcription of genes that have retinoic acid response elements (RARE). The RAR isotypes (
, ß and 
) are comprised of six regions designated A–F. Two isoforms of RAR, 1 and 2, have been identified in humans, which have different A regions generated by differential promoter usage and alternative splicing. We have isolated two new splice variants of RAR1 from human B lymphocytes. In one of these variants, exon 2 is juxtaposed to exon 5, resulting in an altered reading frame and a stop codon. This variant, designated RAR1
B, does not code for a functional receptor. In the second variant, exon 2 is juxtaposed to exon 6, maintaining the reading frame. This isoform, designated RAR1BC, retains most of the functional domains of RAR1, but omits the transactivation domain AF-1 and the DNA-binding domain. Consequently, it does not bind nor transactivate RARE on its own. Nevertheless, RAR1BC interacts with RXR and, as an RXR/RAR1BC heterodimer, transactivates the DR5 RARE upon all-trans-RA binding. The use of RAR- and RXR-specific ligands shows that, whereas transactivation of the DR5 RARE through the RXR/RAR1 heterodimer is mediated only by RAR ligands, transactivation through the RXR/RAR1BC heterodimer is mediated by RAR and RXR ligands. Whilst RAR1 has a broad tissue distribution, RAR1BC has a more heterogeneous distribution, but with significant expression in myeloid cells. RAR1BC is an infrequent example of a functional nuclear receptor which deletes the DNA-binding domain. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Retinoic acid (RA) regulates the growth and differentiation of a wide variety of embryonic and adult cell types (1, and references therein). Two classes of receptors bind RA, the retinoic acid receptors (RAR) and the retinoic X receptors (RXR). They belong to the superfamily of steroid-thyroid nuclear hormone receptors (2). The known ligands for the RARs are all-trans-RA (ATRA) and 9-cis-RA and for the RXRs 9-cis-RA only (3–5). Each class of receptor is composed of three genes, named
, ß and (6–8). Based on sequence homology, the nuclear receptors are structured in modules. The RARs are composed of six regions, A–F (Fig. 1A and C), and the RXRs of five regions, A–E. The A and B regions possess a promoter-specific, ligand-independent transcription activating function (AF-1) (9). The C region constitutes the DNA-binding domain, through which the RARs bind to retinoic acid response elements (RARE), which are specific DNA sequences generally located in the vicinity of target genes. RAREs consist of direct repeats of the consensus sequence (A,G)G(T,G)TCA separated by 1–5 nt (DR1-5) (10–13). RARs bind to RAREs as heterodimers with RXRs. The E region contains the ligand-binding domain, a dimerization interface, the ligand-dependent transcription activating function (AF-2) and the corepressor binding and the coactivator association domains (9,14,15).
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The physiological effects of RA are mediated through activated RXR/RAR heterodimers that stimulate gene transcription (16). In the absence of RA, RXR/RAR binds the nuclear receptor corepressor (N-CoR) or its homolog SMRT, which recruit histone deacetylase leading to transcriptional repression of target genes (reviewed in 15,17). RA binding to RXR/RAR induces dissociation of corepressors, enabling the heterodimer to associate with nuclear receptor coactivator complexes, which include various histone acetyltransferases (15,17,18). These data indicate that RARE-bound, RA-activated RXR/RAR heterodimers recruit the transcriptional apparatus to RARE-containing genes.
Seven isoforms of RAR (RAR1–7), four of RARß (RARß1–4) and seven of RAR (RAR1–7) have been identified in mice (19–23). The isoforms differ in their 5'-untranslated region (5'-UTR) and their A region, which is either encoded by different exons or deleted. The most abundant isoforms of RAR in mice, RAR1 and RAR2, have also been cloned in human. The human RAR gene, located on chromosome 17q21, consists of 10 exons (Fig. 1A; adapted from refs 24–26 and our unpublished observations). Two promoters, located in front of exons 1 and 3, control expression of RAR1 and RAR2. Start codons of RAR1 and RAR2 lie in exons 2 and 3, respectively. RAR1 is expressed in a wide variety of tissues at similar levels (27). In contrast, RAR2 is expressed in a tissue-specific manner (21) and is up-regulated upon RA- or granulocyte colony-stimulating factor-induced differentiation (28,29). The specific function of each isoform is unknown. Targeted disruptions of single RAR isotypes show normal embryonic development and adult phenotypes (reviewed in 30). However, compound null mutants for RAR1 and total RARß, for RAR1 and total RAR or for other RAR and/or RXR isoforms exhibit malformations and are short lived (30). These data suggest that a degree of functional redundancy exists, but certain combinations of isoforms are irreplaceable, underlining the complexity of RA signaling.
In this paper, we report the identification of two new RAR isoforms which result from usage of the A1 region and alternative splicing of other regions. One of these isoforms, designated RAR1B, splices out the B region and, as a consequence of an altered reading frame and a premature termination codon, lacks the rest of the functional domains. The second isoform, designated RAR1BC, splices out the B and C regions, which comprise the AF-1- and DNA-binding domains, while the remaining functional domains are intact. RAR1BC represents an infrequent example of a functional nuclear receptor that deletes the DNA-binding domain.
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MATERIALS AND METHODS |
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Fresh cells and cell lines
Peripheral blood (PB) or bone marrow (BM) mononuclear cells (PBMC and BMMC, respectively) of normal donors and B chronic lymphocytic leukemia (B-CLL) patients were isolated by gradient centrifugation. PB CD3+ T cells, CD19+ B cells and CD56+ natural killer (NK) cells from normal donors were separated using immunomagnetic particles and processed for RNA purification as previously described (31). CD34+ hematopoietic progenitor cells from the BM of normal individuals were purified using the Ceprate LC kit (CellPro, Bothell, WA). Myeloid cell lines NB4 (PML-RAR
-expressing acute promyelocytic leukemia), HL60 and U937, T cell acute leukemia cell line Jurkat, Epstein–Barr virus (EBV)-negative Burkitt lymphoma (BL) cell line DG75 and an EBV-transformed B lymphoblastoid cell line derived from normal B cells (B-LCL) were cultured in RPMI-1640 medium, supplemented with 10% fetal calf serum (FCS). Epithelial cell line MCF-7 and COS monkey fibroblasts were grown in DMEM with 10% FCS.
Cloning and sequencing
A 
DR2 cDNA expression library (Clontech, Palo Alto, CA) made from total RNA obtained from malignant cells of a B-CLL patient (1) was screened by hybridization as previously described (32), using an RAR probe (1.9 kb EcoRI fragment containing the entire coding region of human RAR1 cDNA; 6). A positive clone was plaque purified, excised and circularized into the recombinant plasmid pDR2 as described by the manufacturer. The cloned insert was sequenced using an ABI automatic sequencer (Perkin Elmer, Branchburg, NJ), appearing to be a splice variant of RAR1. The plasmid was called pDR2-RAR1BC (see Results). RAR cosmids 121 and 124 were gifts of E. Solomon (London, UK). Amplified RT–PCR products (described below) from two B-CLL patients (1 and 2) and from a normal individual (CD19+ 1) were cloned, using the pCR-Trap cloning vector primer kit (Genehunter, Nashville, TN), and sequenced. The DNAstar software was used for database searches and molecular biology programs (Madison, WI).
Semi-quantitative competitive RT–PCR
Total RNA was obtained from human or mouse tissues or obtained from the fresh cells and cell lines described above. Part of the RNA panel of human tissues was purchased (Clontech). RNA (1, 0.3 or 0.1 µg) was reverse transcribed using random primers for 15 min at 42°C, followed by 25 cycles of PCR consisting of 25 s at 95°C, 1 min at 59°C and 3 min at 72°C, in a Perkin Elmer thermal cycler. The upstream primer R6 (5'-GGTGCCTCCCTACGCCTTCT-3') was located within exon 2 of the RAR gene. The downstream primer RARD/E (5'-AGAGGGCAGGGAAGGTTTCC-3') was located within exon 6. PCR products were electrophoresed and blotted onto Hybond N+ nylon membranes (Amersham, Little Chalfont, UK). Filters were hybridized with [-32P]ATP-labeled oligonucleotide probes RAR21 (5'-GAGCTCCCCCACCTCCGGCGT-3'), upstream to RARD/E within exon 6, or RAR22 (5'-TCCCCAGCCACTGTGAGAAAC-3'), comprising the junction between exons 2 and 6, and autoradiographed. Mouse primers and probes were homologs of the human set: R6M (5'-AGTACCCCCCTACGCCTTCT-3'); RARD/EM (5'-AGAGGGCCGGGAAGGTCTCC-3'); RAR21M (5'-GAGCTCGCCCACCTCAGGAGT-3'); RAR22M (5'-TCCCCAGCCACGGTGCGAAAC-3').
Constructions
For transient transfections, a pSG5-RAR1BC construct was generated by excising a 2.2 kb BamHI fragment from pDR2-RAR1BC and subcloning into the BamHI site of pSG5. The pSG5-hRAR1 (6) and pSG5-mRXR (4) expression vectors and the RARE3-tk-luc luciferase reporter (33) were also used. The RSV-ß plasmid (Promega, Madison, WI) was used as a control for transfection efficiency. To produce GST fusion recombinant protein, a pGEX-RAR1BC construct was generated by PCR (20 cycles under the conditions described above), using pDR2-RAR1BC as PCR template and primers carrying EcoRI and XhoI restriction sites at the 5'-end (DBC-Eco, 5'-TCTGAATTCATGGCCAGCAACAGCAGCTC-3'; DBC-Xho, 5'-AATCTCGAGTGTGTCCATGTGGCGTGGGC-3'). The PCR product was digested with EcoRI and XhoI and cloned into the EcoRI and XhoI sites of pGEX-4T-1 (Pharmacia, Uppsala, Sweden). The pGEX-RAR1 and pGEX-RXR (34) constructs were also used.
Antibodies
A rabbit polyclonal antibody directed against the F region of RAR [RP(F)] (35) was used in western blot analysis. A rabbit antiserum directed against the predicted A1–D domain junction of RAR1BC was made by sequential intradermal injections using the synthetic peptide NH2-YSTPSPATVRNDRNKC-CONH2 (Syntem, Nîmes, France). This antibody, designated RP1, was used in immunofluorescence studies. Mouse monoclonal antibodies (mAb) directed against the F region of RAR [Ab9(F)] (35) and against the D–E region of RXR (4RX3A2) (36) were also used in immunofluorescence as well as in electrophoretic mobility shift assay (EMSA) experiments.
Western blot analysis
The immunoblotting procedures have been previously described (31,37). Briefly, 10 µg whole cell protein extracts or 1 µg purified recombinant protein was fractionated on 10% Tris–glycine/SDS/polyacrylamide gels and electrotransferred onto Hybond-ECL nitrocellulose membranes (Amersham). Filters were blocked for 3 h in 5% non-fat milk in PBS. After overnight incubation in PBS with RP(F) diluted at 1:1000 or RP1 diluted at 1:100, the filters were washed five times for 10 min, blocked for 10 min in 2.5% non-fat milk and incubated for 30 min with protein A linked to horseradish peroxidase (Amersham) diluted at 1:10 000. After five additional washes, the proteins were visualized using ECL chemiluminescent reagents.
Immunofluorescence
In order to localize RAR1BC in the cell, immunofluorescence was undertaken on transiently transfected COS cells as previously described (37). Briefly, cells were fixed in 4% formaldehyde and incubated overnight at 4°C with RP1 or Ab9 antibodies at 1:100 dilution. Cells were visualized using fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody at 1:100 dilution or cyanin 5 (cy5)-conjugated anti-mouse antibody at 1:50 dilution (Caltag, San Francisco, CA). Nuclei were stained with Hoechst 33258. Cells were analyzed by confocal fluorescence microscopy. As negative controls, non-transfected cells were stained in parallel using the antibodies.
Electrophoretic mobility shift assays
The procedures were similar to those previously described (37). RAR1, RXR and RAR1BC recombinant proteins were produced and purified from bacterial lysates and incubated, alone or in combinations, with double-stranded [-32P]ATP-labeled probes made using the following oligonucleotides and their complements: DR5 RARE (5'-GATCAGGGTTCACCGAAAGTTCACTCGCATATATTA-G-3') and DR1 RXRE (5'-GATCAGGTCACAGGTCACAGGTCACAGTTCA-3'). For supershifts, 1 µg Ab9(F) or 4RX3A2 mAb was added 10 min before the recombinant protein. Binding reactions were electrophoresed in 10% polyacrylamide gels for 1 h. Gels were dried and autoradiographed.
In vitro interaction assay
GST ‘pull-down’ assays were performed as previously described (37). Briefly, bacterial lysates containing GST–RXR or GST protein were bound for 2 h on glutathione–Sepharose beads. After four washes, beads were incubated for 1 h at 4°C with whole cell protein extracts of COS cells transfected with pSG5-RAR1BC or pSG5-RAR. After four washes, SDS loading buffer was added. Proteins were denatured for 10 min at 100°C, loaded onto SDS–PAGE gels and processed for immunoblotting using the RP(F) antibody as described above.
Transfections and transactivation assays
COS cells were transfected by the calcium phosphate precipitation method and B-LCL cells by electroporation, as described previously (31). Briefly, 3 x 105 COS cells/35 mm dish were plated the day before transfection, then transfected with 0.1 µg each receptor plasmid following different combinations and 1 µg reporter constructs. B-LCL cells (20 x 106) were electroporated in the presence of 5 µg each expression construct, 7.5 µg RARE3-tk-luc and 2.5 µg RSV-ß. The quantities of DNA in each experiment were equalized with the pSG5 vector. Cells were grown in carbon-treated FCS (Gemini, Calabasas, CA) in the presence or absence of 10–6 M ATRA (Hoffman-La-Roche, Basel, Switzerland) or synthetic RAR agonist CD336 or RXR agonist CD2809 (CIRD-Galderma, Sophia Antipolis, France). A reporter lysis buffer was added to cells 24 h after transfection and protein was extracted according to the manufacturer’s instructions (Promega). Standard assays were performed to measure luciferase (Promega) and ß-galactosidase activities (Boehringer-Mannheim, Mannheim, Germany) using a Berthold luminometer. Luciferase activity was normalized to ß-galactosidase activity. Each experiment was done in triplicate at least twice. Results are expressed as fold induction of luciferase activity induced by transfected receptors relative to the pSG5 vector.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of novel RAR
splice variantsExons 1 and 2 of the RAR
gene encode the 5'-UTR and A1 region of the RAR1 isoform, exon 3 encodes the 5'-UTR and A2 region of the RAR2 isoform and exons 4 and 5 encompass regions B, C and the first 3 amino acids of D. The B–F regions are common to both isoforms (Fig. 1A and C; 21,26). Screening a DR2 cDNA library from a B-CLL patient (1) with a full-length RAR1 cDNA probe revealed a clone which, upon sequencing, lacked exons 3–5 of the RAR gene. As the A1 region is retained in this clone and the major deleted regions are B and C, this variant receptor was designated RAR1BC. The junction between exons 2 and 6 maintains the reading frame of the D region (Fig. 1B). As a consequence, RAR1BC represents a short form of the RAR1 isoform where the A1 region is juxtaposed to the D–E–F regions (Fig. 1C and D). Using primers flanking the A1–D junction (R6 and RARD/E, Fig. 1C), an RT–PCR technique was set up to detect RAR1BC in total RNA samples. By the use of such primers the RAR1 isoform was co-amplified in the same tube. Southern blot analysis and hybridization with the RAR21 oligonucleotide probe, which comprises sequences of exon 6 upstream of RARD/E (Fig. 1C), allowed specific and simultaneous detection of both RAR1 and RAR1BC amplified fragments (Fig. 2A, left). Hybridization with the RAR22 oligonucleotide probe, which comprises the A1–D junction (Fig. 1C), allowed specific detection of the RAR1BC fragment (Fig. 2A, right). The RAR1BC fragment was subsequently cloned from three independent RT–PCR products, including one from the original patient (1), one from another B-CLL patient (2) and one from a normal individual (CD19+ 1). Sequencing of these clones confirmed the A1–D junction observed in the original RAR1BC cDNA clone. The splice donor and acceptor sites were sequenced using RAR cosmids and primers R6 and RARD/E (data not shown). Sequences previously reported of the exon 2/intron 2 and intron 5/exon 6 boundaries were confirmed (24,26).
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The RT–PCR method generated, in addition to the fragments corresponding to the RAR
1 and RAR1BC mRNAs, another product of intermediate size detected with the RAR21 probe (Fig. 2A, left). Cloning and sequencing of this product from the B-CLL patient 1 and normal CD19+ 1 samples showed that it consisted of sequences of exon 2 juxtaposed to exon 5. Therefore, exon 4, which encodes the B and part of the C regions of RAR, was deleted. Thus, this fragment may represent a novel RAR isoform, which was designated RAR1B (Fig. 1C). The junction between exons 2 and 5 resulted in an altered reading frame and a stop codon in the C region at position 100 (Fig. 1D). Therefore, RAR1B represents a truncated form of RAR1 where the A region is fused to a short amino acid stretch derived from exon 5 (C region) and which does not code for a functional receptor. In conclusion, we have isolated two new RAR isoforms, RAR1BC and RAR1B, with the exceptional feature that other functional domains apart from the A region are spliced out.
Transcript and protein tissue expression and nuclear localization of RAR1BC
The competitive RT–PCR technique described above was used to detect expression of RAR1BC and RAR1B mRNAs in different tissues and cell lines. As the RAR1 mRNA is expressed at similar levels in a broad spectrum of tissues, including hematopoietic cells (27), it provides a competitor template which is appropriate as an RT–PCR internal control. The RT–PCR conditions were set up to end the reaction in the exponential phase of amplification of RAR1, RAR1B and RAR1BC (data not shown). Therefore, Southern blot hybridization of the RT–PCRs with the RAR21 probe provided a semi-quantitative analysis of the expression of the novel isoforms. From a panel of human tissues, RAR1BC mRNA was expressed in mononuclear cells from bone marrow (Fig. 2B, lane 1) and from peripheral blood (not shown). As previously shown (Fig. 2A), RAR1BC mRNA was also expressed in normal CD19+ B lymphocytes and malignant B-CLL cells. In addition, it was expressed in CD3+ T lymphocytes and CD56+ NK cells (not shown). RAR1BC mRNA was also detected as a less intense band in spleen, colon, small intestine, testis, trachea and lung (Fig. 2B, lanes 2, 4, 5 and 7–9, respectively). Lower levels or absence of RAR1BC mRNA expression was observed in tissues such as thymus, prostate, liver, kidney, heart, brain, skin, breast, tonsil and thyroid (Fig. 2B, lanes 3, 6 and 10–17, respectively). RAR1BC mRNA was not detected in a panel of mouse tissues which included brain, heart, kidney, liver, lung, skeletal muscles, spleen, lymph node and bone marrow in the presence of a positive signal for RAR1 (data not shown).
Western blot analysis of transiently transfected COS cells with a polyclonal antibody against the F region of RAR [RP(F)] showed that, as expected from the nucleotide sequence of its cDNA, RAR1BC is expressed as a 40 kDa protein (Fig. 3A, lane 2), smaller than wild-type RAR1 (50 kDa) (Fig. 3A, lane 1). Native RAR1BC protein of the same size was detected in nuclear extracts of HL60 and NB4 myeloid cell lines and total extracts of BMMC (Fig. 3A, lanes 4, 6 and 8, respectively). However, it was not detected in total extracts of BM CD34+ hematopoietic progenitor cells (lane 7), PBMC (not shown) or in nuclear extracts of monocytic U937, B lymphoid DG75, T lymphoid Jurkat or epithelial MCF-7 cell lines (not shown), in the presence of the signal for the RAR1 isoform, which served as a control. Thus, from the panel of tissues and cell lines analyzed it can be concluded that RAR1BC has a more restricted and weaker expression than RAR1. Furthermore, as it is not expressed in mouse, RAR1BC may be a human-specific RAR isoform.
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The presence of RAR
1BC in nuclear extracts is consistent with retention of the nuclear localization signal (NLS) of the RAR main isoforms, located in the D region (38). Nevertheless, experiments were performed to determine whether absence of the C domain, which is involved in DNA binding, affected subcellular localization of the receptor. A polyclonal antibody generated against the A1–D junction (RP1), which recognized RAR1BC efficiently but RAR1 only marginally, as shown by immunoblotting using recombinant proteins (Fig. 3B, lane 6), was used. Immunofluorescence analysis of RAR1BC-transfected COS cells labeled with the RP1 antibody showed that the RAR1BC protein was located in the nucleus (Fig. 3D), with a diffuse pattern similar to that of RAR1, as shown in RAR1-transfected COS cells labeled with a mAb against the F region [Ab9(F)] (Fig. 3C). Staining of non-transfected control cells using the antibodies did not produce significant background (data not shown). Thus, absence of the DNA-binding domain did not affect the subcellular distribution of RAR1BC.
RAR1BC is found in a complex which binds the RARE and RXRE, through interaction with RXR
In RAR, the B region contains the AF-1 activity and the C region contains the DNA-binding domain. To determine whether absence of the B and C regions might affect the DNA-binding capacity of RAR1BC, EMSA analysis was performed using a DR5 RARE and the recombinant receptors. It was first observed that, on its own, RAR1BC did not bind to a DR5 response element, unlike RAR1 or RXR (Fig. 4A, compare lane 2 to lanes 3 and 4). However, when RAR1BC was tested in the presence of RXR, a retarded complex was observed (compare lanes 4 and 5). This complex was shifted with antibodies against either RAR (lane 6) or RXR (lane 7). The intensity of the complexes was less than that observed with the complex between RAR1 and RXR (lanes 8–10), suggesting that RXR/RAR1 dimers bind the RARE more efficiently than RXR/RAR1BC dimers. Similar results were obtained with a DR1 RXRE (data not shown). Altogether, these results indicate that, though unable to bind DNA due to absence of the C region, RAR1BC protein is still found in the complex which binds the RARE, probably through heterodimerization with RXR.
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To obtain additional evidence that RAR
1BC may interact with RXR, GST ‘pull down’ experiments were performed using a GST–RXR fusion protein bound to glutathione–Sepharose beads. After immunoblotting, RAR1BC was found to directly interact with RXR in the absence of RA (Fig. 4B, lane 6), as was RAR1 (Fig. 4B, lane 3).
RAR1BC and RXR contribute to the transactivation mediated by the RAR1BC/RXR heterodimer on DR5 response elements
The ability of RAR1BC to bind RXR raises the question of a potential dominant negative function of RAR1BC on RAR1. To test this hypothesis, the transactivation capacities of RAR1BC were analyzed by luciferase reporter assays after transient co-transfection. Experiments performed in the presence of ATRA in COS cells (Fig. 5, white bars) or in B-LCL cells (not shown) showed that, as expected, RAR1BC alone could not significantly induce the activity of a luciferase reporter under the control of DR5 response elements (compare white bars 1 and 4), whereas expression of RAR or RXR induced 3- and 2.3-fold increases above empty vector (white bars 2 and 3, respectively). However, when co-expressed with RXR, RAR1BC significantly increased transactivation of the promoter, as did RAR1, though to a lesser extent (3.2- and 4.6-fold, white bars 7 and 5, respectively), showing that in vitro RAR1BC is a functional receptor when heterodimerized with RXR and does not exert a dominant negative effect. These conclusions were further supported when RAR1, RXR and RAR1BC were co-transfected (4.7-fold, white bar 8).
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As ATRA isomerizes in vivo into 9-cis-RA, the ligand of RXR, specific retinoids were used to dissect the contribution of the receptors to the transactivating capacity of the RXR
/RAR1BC heterodimers. As shown in Figure 5 (red bars), transactivation of the DR5 RARE induced by the RAR agonist CD336 through the RXR/RAR1BC heterodimer was dependent on binding of the ligand to RAR1BC [compare red bars 7 and 3, representing 2.1- and 1.2-fold increases over empty vector (red bar 1), respectively]. When the RXR agonist CD2809 was used (Fig. 5, green bars), transactivation mediated through RXR (green bar 3) was inhibited by RAR1 fixation (green bar 5). Interestingly, CD2809 induced transactivation through the RXR/RAR1BC heterodimer (green bar 7). These findings suggest that transactivation of the DR5 RARE by the RXR/RAR1 heterodimer is mediated only by RAR ligands, whilst both RAR and RXR ligands transactivate the DR5 RARE through binding to the RXR/RAR1BC heterodimer. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A novel isoform of the RAR
gene derived from usage of promoter P1 and alternative splicing of the B and C regions was identified. This isoform, designated RAR1BC, lacks the DNA-binding domain and part of the AF-1 domain, whereas the ligand-binding domain, AF-2 domain and other functional domains remain intact. A second isoform was detected, derived from usage of promoter P1 and alternative splicing of the B region. This isoform, designated RAR1B, generates a premature termination codon and lacks most of the RAR functional domains, making it unlikely that it codes for a functional receptor. In addition, its mRNA expression appears to be minor, and we have not studied its significance further. In contrast, the RAR1BC protein is expressed in hematopoietic cells, particularly in primary myeloid cells and cell lines. Whilst it was originally cloned from B-CLL cells, RAR1BC mRNA was expressed at apparently equal levels in normal B lymphocytes and B-CLL cells, as observed by a sensitive RT–PCR assay, ruling out the hypothesis of an association of RAR1BC expression with malignant B lymphocytes.
The different spatial and temporal expression of the RAR, ß and isoforms suggests that they have specific activities. RAR1BC tissue expression was found to be more restricted and weaker than that of RAR1, but with significant expression in myeloid cells. This suggests that this variant receptor may have distinct functions from RAR1. Both receptors were localized in the nucleus, as expected from retention of the nuclear translocation signal in the D domain (38), and were found in the same nuclear compartment. As for the rest of the RAR isoforms, the exact role of RAR1BC remains to be elucidated. The RARs are phosphoproteins and phosphorylation of specific serine residues influence their functional properties. Two Ser-Pro motifs within the B region of RAR, S74 and S77, which are targets for CDK7 within TFIIH, are critical for AF-1 activity and transcription of RA-inducible genes (39–41). Another serine of RAR1, S157, can be phosphorylated in vitro by protein kinase C (42). Although the exact role of these post-translational modifications has not yet been defined, the absence of these residues in RAR1BC further suggests a divergent role from RAR1.
The transcriptional properties of RAR1BC and RAR1 were compared. Absence of the DNA-binding domain would explain why RAR1BC bound to neither a DR5 RARE nor to a DR1 RXRE. Nevertheless, our results show that it can interact with RXR in the absence of ligand, consistent with presence of the heterodimerization interface. Moreover, these heterodimers bind to RARE and RXRE, likely through the RXR half-site, although apparently with less affinity than RXR/RAR1 heterodimers. Despite this, the RXR/RAR1BC heterodimers are functional in transactivating a DR5 RARE in transfected COS cells in the presence of ATRA or an RAR-specific retinoid. Thus, our results rule out that RAR1BC could exert a dominant negative effect and suggest that binding of the RXR/RAR1BC heterodimer through the RXR half-site of the RARE would be sufficient to transduce the activating signal triggered by RA. Furthermore, the results of our transactivation experiments using RAR- and RXR-specific retinoids allow us to conclude that whereas transactivation through RXR/RAR1 is mediated preferentially or exclusively by RAR ligands (reviewed in 17), transactivation through RXR/RAR1BC may be mediated by both RAR and RXR ligands. An interesting question which remains to be elucidated is whether the response element repertoire of the RXR/RAR1BC heterodimer differs from that of the regular RXR/RAR heterodimer in a target gene promoter context. Although further studies are needed to investigate their specific function, RAR1B and RAR1BC represent a new class of receptor variants which may provide another tier of control in RA signaling.
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ACKNOWLEDGEMENTS |
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We thank P. Chambon for the RAR
1 and RXR plasmids and antibodies, H. de Thé for the RAR, PML-RAR and RARE3-tk-luc plasmids and useful discussions, E. Solomon for the RAR cosmids and Fabien Zassadowski, Nicole Balitrand, Michel Schmid and Christelle Dolliger for technical assistance. This work was supported by grants from the Leukaemia Research Fund of Great Britain, the Welsh Bone Marrow Transplant Research Fund, the Ligue Nationale Contre le Cancer of France, the Association pour la Recherche contre le Cancer and Subprograma para el Perfeccionamiento de Doctores y Tecnólogos del Ministerio de Educación y Ciencia of Spain.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Sección de Inmunología, Hospital Universitario Virgen de la Arrixaca, El Palmar 30120, Murcia, Spain. Tel: +34 968 36 95 99; Fax: +34 968 36 96 78; Email: aparrado{at}ono.com
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REFERENCES |
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