Synovial Sarcoma Translocation (SYT) Encodes a Nuclear Receptor Coactivator
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《内分泌学杂志》
Discovery Biology Research and Clinical Investigation, Lilly Research Laboratories, Eli Lilly & Co. (T.I., W.W.C.), Indianapolis, Indiana 46285
Department of Integrative Physiology, Gunma University Graduate School of Medicine (T.I., N.K.), Maebashi, Gunma 371-8511, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corp. (T.I., N.K.), Kawaguchi, Saitama 332-0012, Japan
Abstract
We previously cloned and characterized a novel RNA-binding motif-containing coactivator, named coactivator activator (CoAA), as a thyroid hormone receptor-binding protein-interacting protein using a Sos-Ras yeast two-hybrid screening system. A database search revealed that CoAA is identical with synovial sarcoma translocation (SYT)-interacting protein. Thus, we hypothesized that SYT could also function as a coactivator. Subsequently, we isolated a cDNA encoding a larger isoform of SYT, SYT-long (SYT-L), from the brain and liver total RNA using RT-PCR. SYT-L possesses an additional 31 amino acids in its C terminus compared with SYT, suggesting that these two SYT isoforms may be expressed from two mRNAs produced by alternative splicing of a transcript from a single gene. By Northern blot analysis, we found that SYT-L mRNA is expressed in several human embryonic tissues, such as the brain, liver, and kidney. However, we could not detect SYT-L in adult tissues. Glutathione-S-transferase pull-down studies showed that SYT binds to the C-terminus of CoAA, but not to the coactivator modulator. Both isoforms of SYT function as transcriptional coactivators of nuclear hormone receptors in a ligand- and dose-dependent manner in CV-1, COS-1, and JEG-3 cells. However, the pattern of transactivation was different between SYT and SYT-L among these cells. SYT synergistically activates transcription with CoAA. In addition, SYT activates transcription through activator protein-1, suggesting that SYT may function as a general coactivator. These results indicate that SYT activates transcription, possibly through CoAA, to interact with the histone acetyltransferase complex.
Introduction
TRANSCRIPTIONAL REGULATION by nuclear hormone receptors (NRs) is dependent on their interactions with multiple coactivators and corepressors (1, 2). In particular, p160 coactivators, such as steroid receptor coactivator-1 (SRC-1) (2) and SRC-3 (3), have been shown to bind with ligand-bound NRs through their LXXLL motifs (1). SRC-1, SRC-3, cAMP response element binding protein (CREB)-binding protein (CBP)/p300 (4), and P/CAF (5) each possess histone acetyltransferase (HAT) activity and modify nucleosome structure to activate transcription. In addition, SWI/SNF, an ATP-dependent chromatin remodeling complex (6), and DRIP/thyroid hormone receptor-associated protein/mediator, a complex that associates with the basal transcriptional machinery (7, 8, 9), also play important roles in transcription control. Recently, thyroid hormone receptor (TR)-binding protein (TRBP) (10), also named ASC-2/RAP250/PRIP/NRA/AIB3 (11, 12, 13, 14, 15), was cloned and characterized as a general coactivator (10). TRBP interacts with a series of nuclear complexes, such as HAT complex, DRIP/TRAP /mediator complex, and DNA-dependent protein kinase complex, through its C terminus (10).
We have recently cloned and characterized a coactivator activator (CoAA) using the Sos-Ras yeast two-hybrid screening system and TRBP C terminus as bait (16). CoAA functions as a general coactivator through thyroid hormone, glucocorticoid, estrogen, activator protein-1 (AP-1), and nuclear factor-B response elements, similar to the activities of TRBP and CBP/p300 (16). CoAA possesses two consensus RNA recognition motifs (RRMs) in its N terminus and a TRBP-interacting domain that contains repeating amino acid (aa) regions with multiple XYXXQ motifs in its middle region. Subsequently, it has been reported that CoAA functions as a modulator of mRNA splicing and affects both transcription and splicing in a promoter-preferential manner (17, 18, 19). We have also cloned and characterized coactivator modulator (CoAM), an alternative splice form of CoAA that functions as a transcriptional repressor (16). A database search showed that CoAA is identical with the previously described synovial sarcoma translocation (SYT)-interacting protein (SIP).
Human synovial sarcoma often exhibits a specific chromosomal translocation, t(X;18)(p11.2;q11.2) (20, 21). The fusion gene formed by this translocation encodes chimeric SYT-SSX (synovial sarcoma X breakpoint) proteins, which are considered to underlie the pathogenesis of synovial sarcoma. The SYT genes are ubiquitously expressed in early mouse embryogenesis (22). In later stages, their expression is confined to cartilage, specific neuronal cells, and some epithelium-derived tissues (22). Mouse and human genomic structures of SYT, also described as synovial sarcoma translocation chromosome 18, has been predicted by in silico methods and confirmed by RT-PCR (23). The human SYT gene is composed of 11 exons with intron-exon boundaries similar to those of the mouse, extending over about 70 kb of genomic sequence (23). It has been speculated that SYT may serve as a transcriptional coactivator by interacting with a yet unidentified sequence-specific, DNA-binding transcription factor, inasmuch as SYT itself does not possess a DNA-binding motif (24). These findings raise the possibility that NRs may interact with SYT, which directly or indirectly functions as a coactivator.
The transcriptional coactivator activity of SYT is augmented by the deletion of N-terminal 60 aa, suggesting that the N terminus of SYT may act as transcriptional suppressor (24). One possible role of SYT in transcriptional regulation may be mediated by ATP-dependent chromatin remodeling. Recent studies have shown that the N terminus of SYT binds to BRM, a human homologue of Drosophila protein brahma (brm) (24), and to brm/SWI2-related gene-1 (BRG-1) protein product (25, 26, 27). BRM and BRG-1 belong to a family of SWI/SNF, ATP-dependent chromatin remodeling factors, that positively and negatively regulate gene expression (6), because BRM, but not SIP/CoAA, is colocalized with SYT and SYT-SSX in nuclear speckles (24). Therefore, BRM and BRG-1 could negatively regulate the transcriptional activity of SYT (27).
Taken together, we hypothesize that SYT may recruit various coactivator complexes via interaction with CoAA. Furthermore, we found that SYT functions as a general coactivator that synergistically activates NR-mediated transcription with SRC-1, TRBP, and CoAA.
Materials and Methods
Plasmids
Mouse CBP, human TRBP, CoAA, CoAM, SRC-1, and their derived plasmids have been previously described (16). Human BRG-1 was a gift from Dr. H. Kato (University of Tokyo, Tokyo, Japan) (26). Glutathione-S-transferase (GST)-CoAA, GST-CoAM, GST-N-CoAA (aa150–669), and GST-SYT-long (SYT-L) were subcloned into pGEX-4T using the EcoRI/XhoI sites (Amersham Biosciences, Little Chalfont, UK). The expression vectors for glucocorticoid receptor (GR), estrogen receptor (ER), TR1, MAPK kinase (MEK) kinase (MEKK) and the reporter plasmid, the mouse mammary tumor virus (MMTV) promoter fused to luciferase reporter (MMTV-LUC) (28), 2x estrogen response element (ERE)-LUC, F2-thyroid hormone response element (TRE)-LUC, and 7x AP-1-LUC have also been described previously (10, 16). Deletion mutants of SYT and SYT-L, N-SYT (aa 73–387), N-SYT-L (aa 73–418), and C-SYT (aa 1–186), were constructed by inserting the PCR-amplified fragments from SYT or SYT-L into the HindIII and XhoI sites of pcDNA3 (Invitrogen Life Technologies, Inc., Carlsbad, CA).
Isolation of SYT and SYT-L
Using an RT-PCR Kit (Invitrogen Life Technologies, Inc.) and SYT-specific primers, 5'-GAT TCC AAG CTT ACC ATG GAC TAC AAA GAC GAT GAC GAC AAG ATG GGC GGC AAC ATG TCT GTG (forward), and 3'-AGT AGG CCT CGA GTC ACT GCT GGT AAT TTC CAT A (reverse), which were designed based on sequences from GenBank (GenBank accession no. X79201), RT-PCR was performed using total RNA from human liver and brain tissues (OriGene Technologies, Rockville, MD). Full-length SYT and SYT-L were inserted into the HindIII/XhoI sites of pcDNA3. Sequences were subsequently confirmed, and amino acid sequences were predicted using Swiss Institute of Bioinformatics software (Geneva, Switzerland). The sequence of SYT-L cDNA was presented at 83rd Annual Meeting of The Endocrine Society, Denver, CO, by Iwasaki and Chin.
Northern blot analysis
Several different tissues from human embryos and adults were obtained for Northern blots from BD Clontech (Palo Alto, CA) and Ambion, Inc. (Austin, TX), respectively. Each blot contained 2 μg polyadenylated RNA from the adult brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and colon or from embryonic brain, lung, liver, and kidney tissues. The SYT-L probe was prepared with random-primed 32P-labeled, SYT-L-specific cDNA (aa 295–325). Northern blot analysis was performed according to the manufacturer’s protocol. Cyclophilin was used as an internal control.
GST pull-down studies
GST and GST fusion proteins were produced in Escherichia coli BL21 (DE3) and purified by glutathione-Sepharose resin chromatography (Amersham Biosciences). The GST-fusion protein pull-down assay was performed as previously described (16). Briefly, GST resin and [35S]methionine-labeled, in vitro-translated proteins produced by rabbit reticulocyte lysate (Promega Corp., Madison, WI) were incubated at room temperature for 1 h in binding buffer [20 mM HEPES (pH 7.4), 50 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% (vol/vol) Triton X-100, and 10% (vol/vol) glycerol]. After extensive washes, the mixture was subjected to SDS-PAGE and autoradiography.
Cell culture and transient transfection-based reporter assay
CV-1, COS-1, JEG-3, and SW-13 cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 C in 5% CO2. Cells were plated in 24-well plates 2 d before transfection. Each cell was transfected using Lipofectamine 2000 reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s protocol. Sixteen to 24 h after transfection, cells were replenished with fresh medium containing the indicated concentration of specific ligand. After 24 h, cells were harvested for measurement of luciferase activities as previously described (16). Total amounts of DNA for each well were normalized by adding vector pcDNA3 (Invitrogen Life Technologies). Data shown represent the mean ± SE of the average of three transfection studies. Statistical comparisons were made using two- or three-way ANOVA. Post hoc comparisons were made using the Bonferroni test.
Results
Isolation of SYT and SYT-L
Using total RNA from human liver and brain, we isolated cDNAs encoding SYT and SYT-L by RT-PCR (Fig. 1A). Sequence analysis predicted that SYT-L possesses an extra 31 aa at aa residue 294 without a frame shift (Fig. 1, B and C). Comparison of SYT and SYT-L sequences indicated the addition of 93 nucleotides between 880 and 974 in SYT-L relative to SYT cDNA (Fig. 1, B and C). The boundaries of the spliced sequence were flanked by GT-AG consensus acceptor-donor sequences, suggesting that an alternative splicing event led to the production of SYT-L.
The human SYT protein is composed of 387 aa and contains a conserved 54-aa domain called SYT N-terminal homology (SNH) domain (function unknown) at the N terminus. A C-terminal domain, rich in glutamine, proline, glycine, and tyrosine (QPGY-rich region) containing possible transcriptional activator sequences was also observed (Fig. 1C). The SYT-L protein is composed of 418 aa.
Genomic structure of the human SYT/SYT-L gene
The cDNA sequences obtained by RT-PCR in the present study were used to identify the human SYT-L genomic sequences from the GenBank htg database (accession no. NC_000018). The human SYT gene is located on chromosome 18q11.2 and contains 11 exons spanning approximately 74 kb (Fig. 1D) (23). An additional exon was found after exon 7 (Fig. 1D). Alternative splicing of exon 8 of SYT-L (cDNA nucleotides 881–973) resulted in the lack of 31 aa and the formation of SYT mRNA.
SYT-L mRNA is expressed in human embryonic, but not adult, tissues
We examined the expression pattern of endogenous SYT-L mRNA in different tissues from human embryos and adults using Northern blots. The blots were probed with a cDNA probe encoding the SYT-L-specific, 31-aa region. A predominant 4.8-kb transcript was detected in human embryonic brain, liver, and kidney, with lower amounts in embryonic lung (Fig. 2). However, in adult brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and colon, hybridization signals were not detected, although SYT-L was initially isolated from adult brain by RT-PCR.
SYT interacts with the general coactivator CoAA, but not with CoAM, in vitro
GST pull-down studies were performed with recombinant GST fusion CoAA, CoAM, N-terminal deletion mutant of CoAA, N-CoAA (aa 150–669), and 35S-labeled, in vitro-translated SYT. SYT bound to CoAA and N-CoAA, but not to CoAM (Fig. 3A). When GST-SYT-L and 35S-labeled in vitro-transcribed and -translated CoAA, CoAM, and N-CoAA (aa 150–669) were incubated together, SYT-L bound to CoAA and N-CoAA, but not to CoAM (Fig. 3B). These results suggest that both SYT and SYT-L interact with the CoAA C terminus, but not with CoAM. LUC served as a negative control (data not shown).
SYT and SYT-L function as NR coactivators
We then investigated the role of SYT in NR-mediated transcriptional regulation. MMTV-LUC was cotransfected with the expression vector encoding GR and SYT or SYT-L into CV-1 cells in the presence or absence of dexamethasone (DEX; Fig. 4). Cotransfection of SYT together with CoAA synergistically activated MMTV promoter in the presence of DEX (Fig. 4A). Both SYT and SYT-L transactivated MMTV promoter in a dose-dependent manner up to 10-fold (Fig. 4). These transcriptional activities were ligand and receptor dose dependent (Fig. 4, B and C). We observed a dose-related difference in transcription between SYT and SYT-L. Transcription was more efficiently activated by SYT-L at lower doses and by SYT at higher doses (Fig. 4D). Cotransfection of SYT and SYT-L with SRC-1, which possesses HAT activity, or TRBP showed synergistic activation of MMTV promoter (Fig. 5). These results indicate that SYT and SYT-L are potent NR coactivators and suggest that SYT and SYT-L may activate transcription at least in part via SRC-1-containing HAT complex.
SYT and SYT-L function as general coactivators
Because the general coactivator CoAA activates NRs as well as other transcriptional factors, such as AP-1 (16), we investigated whether SYT and/or SYT-L possess similar activity. CoAA and SYT or SYT-L were cotransfected with a number of luciferase reporters containing TRE, ERE, and AP-1 enhancer-binding sites. SYT and SYT-L synergistically activated transcription in the presence of CoAA and the response elements (Fig. 6). Consistent with in vitro binding studies with CoAA, these results suggest that SYT and SYT-L are potent NR coactivators as well as general coactivators.
Activation domain of SYT
To determine the activation domain of SYT and SYT-L, we constructed a series of truncation mutants of SYT and SYT-L (Fig. 7A). Deletion of the C terminus of SYT or SYT-L, C-SYT (aa 1–186), produced a significant loss of transactivation function, suggesting that the activation domain is located in this region (Fig. 7B). In contrast, the deletion of the N terminus, N-SYT (aa 73–387) and N-SYT-L (aa 73–418), showed a tendency to activate, suggesting the existence of a repressor domain in the N-terminal region (Fig. 7B).
Activation of SYT depends on the cell line
We investigated whether SYT and/or SYT-L function differently in different cell lines, such as COS-1, JEG-3, and SW-13. Although the magnitude of transactivation induced by SYT and SYT-L was similar in CV-1 cells (Fig. 4D), SYT activated transcription more efficiently than SYT-L in COS-1, JEG-3, and SW-13 cells. The magnitude of activation by both SYT and SYT-L was much less in SW-13 cells, which lack SWI/SNF proteins (Fig. 8). Cotransfection of BRG-1 did not enhance SYT or SYT-L action (data not shown), suggesting that factors other than the SWI/SNF complex could be involved in the activation of transcription via SYT.
Discussion
Recently, we cloned CoAA, a coactivator of TRBP-interacting protein (16), which is identical with SIP. We hypothesized that SYT might function as an NR coactivator. Therefore, we isolated and characterized cDNAs encoding human SYT and its splicing variant, SYT-L. SYT and SYT-L function as NR coactivators and general coactivators, and both interact with CoAA.
Two SYT RT-PCR products were reproducibly observed in human brain and liver using different sets of primers. A comparison of the sequences revealed that SYT-L possesses an insert relative to SYT that contains a splice donor and an acceptor consensus GT-AG sequence. We also showed SYT-L mRNA expression in different tissues from human embryos using Northern blot analysis with SYT-L-specific primer. These results indicate that SYT-L is not a PCR artifact, but a natural splice variant of SYT. Furthermore, translocated SYT isoforms, SYT-SSX, SYT-SSX2v, and SYT-SSX4v, isolated from human synovial sarcoma tissues (29), all contain the region identical with SYT-L. Comparing the cDNA and chromosome 18q11.2 genomic sequences, we confirmed the presence of an additional exon specific for SYT-L that is flanked by GT-AG consensus acceptor-donor sequences. These data provide additional support for the idea that SYT-L is a naturally occurring SYT isoform.
RT-PCR using total RNA from adult human tissues and Northern blot analysis of different tissues from human embryos confirmed SYT isoform mRNA expression. The relative amounts of SYT-L expression in brain were greater than those in liver, indicating that SYT-L may be involved in transcriptional regulation of neuronal genes. Additional investigations are required to clarify this hypothesis. Of note, we could not detect the expression of SYT-L mRNA in adult tissues using Northern blot analysis. Thus, as shown for mouse SYT (22), human SYT mRNA expression may occur largely during development, including possible regulation of organogenesis in the brain.
Functional analyses indicate that at low concentrations of isoform cDNA, SYT-activated GR-mediated transcription through the MMTV promoter was more efficient than SYT-L; however, at high concentrations, their relative activities were reversed. Although there was no essential difference between SYT and SYT-L in terms of transcriptional activation, this dose-related reversal of relative activities could contribute to subtleties of SYT-mediated transactivation regulation. The transcriptional activation was dose, ligand, and NR dependent, indicating that SYT and SYT-L are potent NR coactivators. Furthermore, SYT and SYT-L activated transcription through an AP-1 enhancer-binding site. These results suggest that SYT and SYT-L may function as general coactivators.
CoAA, but not CoAM, interacts with SYT in vitro. In reporter gene assays, CoAA, SRC-1, and TRBP synergistically activated transcription with SYT. These results suggest that SYT-associated transcriptional activation may be mediated through SRC-1-containing HAT complexes. This could occur by interactions with CoAA, because TRBP, a CoAA-interacting protein, has been also reported to interact with SRC-1-containing HAT complexes (10). These results indicate that SYT may function in collaboration with CoAA and HAT complex in vivo.
The present study also indicates that CoAA interacts with SYT through the TRBP-interacting domain located in the middle region of CoAA, which is absent in the CoAM (16). This domain contains more than 20 XYXXQ motifs (X denotes a small amino acid residue including G, A, S, and P) (16). Through this domain, CoAA also interacts with CBP/p300. Although the function of this motif has not been fully clarified, previous studies have shown that this motif is important for permitting protein-protein interactions (16, 30, 31). The present study confirmed that this motif plays an important role in such interactions, particularly with transcriptional coactivators. Additionally, because CoAA/CoAM have been reported as promoter-dependent RNA splicing modulators (17, 18, 19), the interaction between SYT and CoAA might affect posttranscriptional regulation, especially the control of alternative RNA splicing. This raises the possibility that an abnormal balance of cellular protein products caused by SYT-SSX due to aberrant mRNA splicing might affect the development and/or progression of synovial sarcoma.
We have determined that the activation domain of SYT, the QPGY-rich region (aa 73–387) is located at the C terminus, which contains several glutamine (Q), proline (P), glycine (G), and tyrosine (T) residues in an XXYXX motif. In this sequence, the tyrosine residue is surrounded by at least three X residues (Q, P, or G) (24). A previous study reported that GAL4-DNA-binding domain-coupled SYT (aa 158–387) showed the greatest transcriptional activity (24). However, because SYT does not contain a potential DNA-binding domain, SYT is involved in transcriptional events through an indirect mechanism. Our study not only confirmed the function of this region (24), but also showed that SYT may work with CoAA to interact with TRBP. This complex then may interact with various native NRs, such as TR, ER, and GR, as well as other transcription factors to regulate transcription.
SYT contains an SNH domain at its N terminus. This domain is conserved across a wide range of species, although the role of this domain is not fully understood (24). SYT binds to BRG-1, the human homologue of SWI2/SNF2, through the SNH domain (25, 26, 27). The SWI/SNF is a large family of ATP-dependent chromatin remodeling complex proteins that are associated with transcriptional regulation. The N terminus of SYT has been reported to be a repression domain, and our results support this concept. Thus, our data are consistent with the hypothesis that the SWI/SNF complex is involved in transcriptional repression via interaction with SYT. Furthermore, our data show that the effects of SYTs are greatly diminished in SW-13 cells that lack the SWI/SNF complex. Our results, however, do not support a recent report showing that BRM and BRG-1 suppress GAL4-fused SYT-activated transcription in a dose-dependent manner in SW-13 cells (24). It has been reported that GAL4-SYT directly interacts with DNA through an upstream activating sequence (24), whereas we show that SYT interacts indirectly with DNA (present study). Although SYT is capable of binding to SWI/SNF proteins, including BRG-1, this complex probably does not play an important role in transcriptional activation through GR. However, the possible involvement of SYT in repression in a different promoter context cannot be excluded. In contrast, cotransfection of SRC-1, TRBP, or CoAA with SYT synergistically activated GR-mediated MMTV promoter-driven transcription. Therefore, SRC-1-containing HAT complexes, but not BRG-1-containing SWI/SNF complexes, may be the dominant forms involved in SYT-associated transcriptional activation by NRs.
It has been reported that the pathogenesis of soft tissue sarcomas is highly related to translocation of chromosomes (20, 21). As a result, translocations, such EWS-FLI1 in Ewing’s sarcoma, SYT-SSX in synovial sarcoma, and CHOP-FUS in myxoid liposarcoma, are used for clinical diagnosis (32). Besides these fusion proteins, a series of chromosomal translocations, including PAX3/7-FKHR, EWS-ETF1/WT1/CHN/CHOP, RBP56-CHN, ETV6-NTRK3 and COL1A1-platelet-derived growth factor-B, have been observed as pathogens of soft tissue tumors (33). Among these sarcoma-related proteins, EWS, the product of a gene commonly translocated in Ewing sarcoma; TLS/FUS, (translocated in liposarcoma); and RBP56 (RNA-binding protein 56) possess the domains analogous to the QPGY region in SYT, suggesting that these wild-type proteins could interact with coactivators to modulate transcriptional regulation. Interestingly, EWS, TLS/FUS, and RBP56 possess RGG repeats (RNA-binding motifs), in their C termini, which suggest that RNA-binding proteins related to transcriptional regulation could also be involved in tumorigenesis.
In summary, our study shows that SYT may function as a transcriptional coactivator that interacts with SRC-1-containing HAT complexes through CoAA. It has been reported that SYT interacts with p300, which promotes cell adhesion to a fibronectin matrix (34), and with cyclin D1, which is related to the cell cycle (35, 36). Furthermore, CoAA, an SYT-interacting protein, may be associated with other cellular activities, such as DNA repair via interaction with DNA-dependent protein kinase complex (16) or alternative RNA splicing (17, 18, 19). Taken together, the SYT may serve key roles involving diverse nuclear events, leading to the control of multiple cellular functions.
Acknowledgments
We thank Drs. C. S. Cooper, H. Kato, and A. Takeshita for kindly preparing the constructs. We thank Drs. C. S. Suen and S. Nagpal for critical discussions and comments during the entire project. We thank M. Ohta and W. Miyazaki for technical assistance throughout the study.
Footnotes
This work was supported by Lilly postdoctoral research fellowships; Grant-in-Aid for Scientific Research 14370020 from the Japanese Ministry of Education, Science, Sports, and Culture; and a grant from CREST, Japan Science and Technology Corp. (to J.S.T.).
Abbreviations: aa, Amino acid; AP-1, activator protein-1; BRG-1, brm/SWI2-related gene-1; BRM, brahma; CBP, cAMP response element binding protein (CREB)-binding protein; CoAA, coactivator activator; CoAM, coactivator modulator; DEX, dexamethasone; ER, estrogen receptor; ERE, estrogen response element; GR, glucocorticoid receptor; GRE, glucocorticoid receptor response element; GST, glutathione-S-transferase; HAT, histone acetyltransferase; LUC, luciferase; MEK, MAPK/extracellular signal-regulated kinase kinase; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; MMTV, mouse mammary tumor virus; NR, nuclear receptor; RBP56, RNA-binding protein 56; RRM, RNA recognition motif; SIP, SYT-interacting protein; SNH, synovial sarcoma translocation N-terminal homology; SRC-1, steroid receptor coactivator-1; SSX, synovial sarcoma X break point; SYT, synovial sarcoma translocation; SYT-L, larger isoform of synovial sarcoma translocation; TLS, translocated in liposarcoma; TR, thyroid hormone receptor; TRBP, thyroid receptor-binding protein; TRE, thyroid hormone response element.
References
Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1997 Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389:194–198
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a histone acetyltransferase. Nature 384:641–643
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382:319–324
Peterson CL, Workman JL 2000 Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr Opin Genet Dev 10:187–192
Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828
Yuan CX, Ito M, Fondell JD, Fu ZY, Roeder RG 1998 The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci USA 95:7939–7944
Jiang YW, Veschambre P, Erdjument-Bromage H, Tempst P, Conaway JW, Conaway RC, Kornberg RD 1998 Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc Natl Acad Sci USA 95:8538–8543
Ko L, Cardona GR, Chin WW 2000 Thyroid hormone receptor-binding protein, an LXXLL motif-containing protein, functions as a general coactivator. Proc Natl Acad Sci USA 97:6212–6217
Lee SK, Anzick SL, Choi JE, Bubendorf L, Guan XY, Jung YK, Kallioniemi OP, Kononen J, Trent JM, Azorsa D, Jhun BH, Cheong JH, Lee YC, Meltzer PS, Lee JW 1999 A nuclear factor, ASC-2, as a cancer-amplified transcriptional coactivator essential for ligand-dependent transactivation by nuclear receptors in vivo. J Biol Chem 274:34283–34293
Caira F, Antonson P, Pelto-Huikko M, Treuter E, Gustafsson JA 2000 Cloning and characterization of RAP250, a novel nuclear receptor coactivator. J Biol Chem 275:5308–5317
Zhu Y, Kan L, Qi C, Kanwar YS, Yeldandi AV, Rao MS, Reddy JK 2000 Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR. J Biol Chem 275:13510–13516
Mahajan MA, Samuels HH 2000 A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREB-binding protein. Mol Cell Biol 20:5048–5063
Guan XY, Xu J, Anzick SL, Zhang H, Trent JM, Meltzer PS 1996 Hybrid selection of transcribed sequences from microdissected DNA: isolation of genes within amplified region at 20q11–q13.2 in breast cancer. Cancer Res 56:3446–3450
Iwasaki T, Chin WW, Ko L 2001 Identification and characterization of RRM-containing coactivator activator (CoAA) as TRBP-interacting protein, and its splice variant as a coactivator modulator (CoAM). J Biol Chem 276:33375–33383
Auboeuf D, Dowhan DH, Kang YK, Larkin K, Lee JW, Berget SM, O’Malley BW 2004 Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes. Proc Natl Acad Sci USA 101:2270–2274
Auboeuf D, Dowhan DH, Li X, Larkin K, Ko L, Berget SM, O’Malley BW 2004 CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing. Mol Cell Biol 24:442–453
Auboeuf D, Honig A, Berget SM, O’Malley BW 2002 Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science 298:416–419
dos Santos NR, de Bruijn DR, van Kessel AG 2001 Molecular mechanisms underlying human synovial sarcoma development. Genes Chromosomes Cancer 30:1–14
Clark J, Rocques PJ, Crew AJ, Gill S, Shipley J, Chan AM, Gusterson BA, Cooper CS 1994 Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat Genet 7:502–508
de Bruijn DR, Baats E, Zechner U, de Leeuw B, Balemans M, Olde Weghuis D, Hirning-Folz U, Geurts van Kessel AG 1996 Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas. Oncogene 13:643–648
de Bruijn DR, Kater-Baats E, Eleveld M, Merkx G, Geurts Van Kessel A 2001 Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene. Cytogenet Cell Genet 92:310–319
Thaete C, Brett D, Monaghan P, Whitehouse S, Rennie G, Rayner E, Cooper CS, Goodwin G 1999 Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus. Hum Mol Genet 8:585–591
Perani M, Ingram CJ, Cooper CS, Garrett MD, Goodwin GH 2003 Conserved SNH domain of the proto-oncoprotein SYT interacts with components of the human chromatin remodelling complexes, while the QPGY repeat domain forms homo-oligomers. Oncogene 22:8156–8167
Kato H, Tjernberg A, Zhang W, Krutchinsky AN, An W, Takeuchi T, Ohtsuki Y, Sugano S, de Bruijn DR, Chait BT, Roeder RG 2002 SYT associates with human SNF/SWI complexes and the C-terminal region of its fusion partner SSX1 targets histones. J Biol Chem 277:5498–5505
Ishida M, Tanaka S, Ohki M, Ohta T 2004 Transcriptional co-activator activity of SYT is negatively regulated by BRM and Brg1. Genes Cells 9:419–428
Takeshita A, Yen PM, Ikeda M, Cardona GR, Liu Y, Koibuchi N, Norwitz ER, Chin WW 1998 Thyroid hormone response elements differentially modulate the interactions of thyroid hormone receptors with two receptor binding domains in the steroid receptor coactivator-1. J Biol Chem 273:21554–21562
Brodin B, Haslam K, Yang K, Bartolazzi A, Xie Y, Starborg M, Lundeberg J, Larsson O 2001 Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression. Gene 268:173–182
Cartegni L, Maconi M, Morandi E, Cobianchi F, Riva S, Biamonti G 1996 hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins. J Mol Biol 259:337–348
Biamonti G, Ruggiu M, Saccone S, Della Valle G, Riva S 1994 Two homologous genes, originated by duplication, encode the human hnRNP proteins A2 and A1. Nucleic Acids Res 22:1996–2002
Uchida A, Seto M, Hashimoto N, Araki N 2000 Molecular diagnosis and gene therapy in musculoskeletal tumors. J Orthop Sci 5:418–423
Skapek SX, Chui CH 2000 Cytogenetics and the biologic basis of sarcomas. Curr Opin Oncol 12:315–322
Eid JE, Kung AL, Scully R, Livingston DM 2000 p300 Interacts with the nuclear proto-oncoprotein SYT as part of the active control of cell adhesion. Cell 102:839–848
Xie Y, Skytting B, Nilsson G, Gasbarri A, Haslam K, Bartolazzi A, Brodin B, Mandahl N, Larsson O 2002 SYT-SSX is critical for cyclin D1 expression in synovial sarcoma cells: a gain of function of the t(X;18)(p11.2;q11.2) translocation. Cancer Res 62:3861–3867
Xie Y, Skytting B, Nilsson G, Grimer RJ, Mangham CD, Fisher C, Shipley J, Bjerkehagen B, Myklebost O, Larsson O 2002 The SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1. A link between t(X;18)(p11.2; q11.2) and the cell cycle machinery. Oncogene 21:5791–5796(Toshiharu Iwasaki, Noriyu)
Department of Integrative Physiology, Gunma University Graduate School of Medicine (T.I., N.K.), Maebashi, Gunma 371-8511, Japan
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corp. (T.I., N.K.), Kawaguchi, Saitama 332-0012, Japan
Abstract
We previously cloned and characterized a novel RNA-binding motif-containing coactivator, named coactivator activator (CoAA), as a thyroid hormone receptor-binding protein-interacting protein using a Sos-Ras yeast two-hybrid screening system. A database search revealed that CoAA is identical with synovial sarcoma translocation (SYT)-interacting protein. Thus, we hypothesized that SYT could also function as a coactivator. Subsequently, we isolated a cDNA encoding a larger isoform of SYT, SYT-long (SYT-L), from the brain and liver total RNA using RT-PCR. SYT-L possesses an additional 31 amino acids in its C terminus compared with SYT, suggesting that these two SYT isoforms may be expressed from two mRNAs produced by alternative splicing of a transcript from a single gene. By Northern blot analysis, we found that SYT-L mRNA is expressed in several human embryonic tissues, such as the brain, liver, and kidney. However, we could not detect SYT-L in adult tissues. Glutathione-S-transferase pull-down studies showed that SYT binds to the C-terminus of CoAA, but not to the coactivator modulator. Both isoforms of SYT function as transcriptional coactivators of nuclear hormone receptors in a ligand- and dose-dependent manner in CV-1, COS-1, and JEG-3 cells. However, the pattern of transactivation was different between SYT and SYT-L among these cells. SYT synergistically activates transcription with CoAA. In addition, SYT activates transcription through activator protein-1, suggesting that SYT may function as a general coactivator. These results indicate that SYT activates transcription, possibly through CoAA, to interact with the histone acetyltransferase complex.
Introduction
TRANSCRIPTIONAL REGULATION by nuclear hormone receptors (NRs) is dependent on their interactions with multiple coactivators and corepressors (1, 2). In particular, p160 coactivators, such as steroid receptor coactivator-1 (SRC-1) (2) and SRC-3 (3), have been shown to bind with ligand-bound NRs through their LXXLL motifs (1). SRC-1, SRC-3, cAMP response element binding protein (CREB)-binding protein (CBP)/p300 (4), and P/CAF (5) each possess histone acetyltransferase (HAT) activity and modify nucleosome structure to activate transcription. In addition, SWI/SNF, an ATP-dependent chromatin remodeling complex (6), and DRIP/thyroid hormone receptor-associated protein/mediator, a complex that associates with the basal transcriptional machinery (7, 8, 9), also play important roles in transcription control. Recently, thyroid hormone receptor (TR)-binding protein (TRBP) (10), also named ASC-2/RAP250/PRIP/NRA/AIB3 (11, 12, 13, 14, 15), was cloned and characterized as a general coactivator (10). TRBP interacts with a series of nuclear complexes, such as HAT complex, DRIP/TRAP /mediator complex, and DNA-dependent protein kinase complex, through its C terminus (10).
We have recently cloned and characterized a coactivator activator (CoAA) using the Sos-Ras yeast two-hybrid screening system and TRBP C terminus as bait (16). CoAA functions as a general coactivator through thyroid hormone, glucocorticoid, estrogen, activator protein-1 (AP-1), and nuclear factor-B response elements, similar to the activities of TRBP and CBP/p300 (16). CoAA possesses two consensus RNA recognition motifs (RRMs) in its N terminus and a TRBP-interacting domain that contains repeating amino acid (aa) regions with multiple XYXXQ motifs in its middle region. Subsequently, it has been reported that CoAA functions as a modulator of mRNA splicing and affects both transcription and splicing in a promoter-preferential manner (17, 18, 19). We have also cloned and characterized coactivator modulator (CoAM), an alternative splice form of CoAA that functions as a transcriptional repressor (16). A database search showed that CoAA is identical with the previously described synovial sarcoma translocation (SYT)-interacting protein (SIP).
Human synovial sarcoma often exhibits a specific chromosomal translocation, t(X;18)(p11.2;q11.2) (20, 21). The fusion gene formed by this translocation encodes chimeric SYT-SSX (synovial sarcoma X breakpoint) proteins, which are considered to underlie the pathogenesis of synovial sarcoma. The SYT genes are ubiquitously expressed in early mouse embryogenesis (22). In later stages, their expression is confined to cartilage, specific neuronal cells, and some epithelium-derived tissues (22). Mouse and human genomic structures of SYT, also described as synovial sarcoma translocation chromosome 18, has been predicted by in silico methods and confirmed by RT-PCR (23). The human SYT gene is composed of 11 exons with intron-exon boundaries similar to those of the mouse, extending over about 70 kb of genomic sequence (23). It has been speculated that SYT may serve as a transcriptional coactivator by interacting with a yet unidentified sequence-specific, DNA-binding transcription factor, inasmuch as SYT itself does not possess a DNA-binding motif (24). These findings raise the possibility that NRs may interact with SYT, which directly or indirectly functions as a coactivator.
The transcriptional coactivator activity of SYT is augmented by the deletion of N-terminal 60 aa, suggesting that the N terminus of SYT may act as transcriptional suppressor (24). One possible role of SYT in transcriptional regulation may be mediated by ATP-dependent chromatin remodeling. Recent studies have shown that the N terminus of SYT binds to BRM, a human homologue of Drosophila protein brahma (brm) (24), and to brm/SWI2-related gene-1 (BRG-1) protein product (25, 26, 27). BRM and BRG-1 belong to a family of SWI/SNF, ATP-dependent chromatin remodeling factors, that positively and negatively regulate gene expression (6), because BRM, but not SIP/CoAA, is colocalized with SYT and SYT-SSX in nuclear speckles (24). Therefore, BRM and BRG-1 could negatively regulate the transcriptional activity of SYT (27).
Taken together, we hypothesize that SYT may recruit various coactivator complexes via interaction with CoAA. Furthermore, we found that SYT functions as a general coactivator that synergistically activates NR-mediated transcription with SRC-1, TRBP, and CoAA.
Materials and Methods
Plasmids
Mouse CBP, human TRBP, CoAA, CoAM, SRC-1, and their derived plasmids have been previously described (16). Human BRG-1 was a gift from Dr. H. Kato (University of Tokyo, Tokyo, Japan) (26). Glutathione-S-transferase (GST)-CoAA, GST-CoAM, GST-N-CoAA (aa150–669), and GST-SYT-long (SYT-L) were subcloned into pGEX-4T using the EcoRI/XhoI sites (Amersham Biosciences, Little Chalfont, UK). The expression vectors for glucocorticoid receptor (GR), estrogen receptor (ER), TR1, MAPK kinase (MEK) kinase (MEKK) and the reporter plasmid, the mouse mammary tumor virus (MMTV) promoter fused to luciferase reporter (MMTV-LUC) (28), 2x estrogen response element (ERE)-LUC, F2-thyroid hormone response element (TRE)-LUC, and 7x AP-1-LUC have also been described previously (10, 16). Deletion mutants of SYT and SYT-L, N-SYT (aa 73–387), N-SYT-L (aa 73–418), and C-SYT (aa 1–186), were constructed by inserting the PCR-amplified fragments from SYT or SYT-L into the HindIII and XhoI sites of pcDNA3 (Invitrogen Life Technologies, Inc., Carlsbad, CA).
Isolation of SYT and SYT-L
Using an RT-PCR Kit (Invitrogen Life Technologies, Inc.) and SYT-specific primers, 5'-GAT TCC AAG CTT ACC ATG GAC TAC AAA GAC GAT GAC GAC AAG ATG GGC GGC AAC ATG TCT GTG (forward), and 3'-AGT AGG CCT CGA GTC ACT GCT GGT AAT TTC CAT A (reverse), which were designed based on sequences from GenBank (GenBank accession no. X79201), RT-PCR was performed using total RNA from human liver and brain tissues (OriGene Technologies, Rockville, MD). Full-length SYT and SYT-L were inserted into the HindIII/XhoI sites of pcDNA3. Sequences were subsequently confirmed, and amino acid sequences were predicted using Swiss Institute of Bioinformatics software (Geneva, Switzerland). The sequence of SYT-L cDNA was presented at 83rd Annual Meeting of The Endocrine Society, Denver, CO, by Iwasaki and Chin.
Northern blot analysis
Several different tissues from human embryos and adults were obtained for Northern blots from BD Clontech (Palo Alto, CA) and Ambion, Inc. (Austin, TX), respectively. Each blot contained 2 μg polyadenylated RNA from the adult brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and colon or from embryonic brain, lung, liver, and kidney tissues. The SYT-L probe was prepared with random-primed 32P-labeled, SYT-L-specific cDNA (aa 295–325). Northern blot analysis was performed according to the manufacturer’s protocol. Cyclophilin was used as an internal control.
GST pull-down studies
GST and GST fusion proteins were produced in Escherichia coli BL21 (DE3) and purified by glutathione-Sepharose resin chromatography (Amersham Biosciences). The GST-fusion protein pull-down assay was performed as previously described (16). Briefly, GST resin and [35S]methionine-labeled, in vitro-translated proteins produced by rabbit reticulocyte lysate (Promega Corp., Madison, WI) were incubated at room temperature for 1 h in binding buffer [20 mM HEPES (pH 7.4), 50 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% (vol/vol) Triton X-100, and 10% (vol/vol) glycerol]. After extensive washes, the mixture was subjected to SDS-PAGE and autoradiography.
Cell culture and transient transfection-based reporter assay
CV-1, COS-1, JEG-3, and SW-13 cells were maintained in DMEM supplemented with 10% fetal bovine serum at 37 C in 5% CO2. Cells were plated in 24-well plates 2 d before transfection. Each cell was transfected using Lipofectamine 2000 reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s protocol. Sixteen to 24 h after transfection, cells were replenished with fresh medium containing the indicated concentration of specific ligand. After 24 h, cells were harvested for measurement of luciferase activities as previously described (16). Total amounts of DNA for each well were normalized by adding vector pcDNA3 (Invitrogen Life Technologies). Data shown represent the mean ± SE of the average of three transfection studies. Statistical comparisons were made using two- or three-way ANOVA. Post hoc comparisons were made using the Bonferroni test.
Results
Isolation of SYT and SYT-L
Using total RNA from human liver and brain, we isolated cDNAs encoding SYT and SYT-L by RT-PCR (Fig. 1A). Sequence analysis predicted that SYT-L possesses an extra 31 aa at aa residue 294 without a frame shift (Fig. 1, B and C). Comparison of SYT and SYT-L sequences indicated the addition of 93 nucleotides between 880 and 974 in SYT-L relative to SYT cDNA (Fig. 1, B and C). The boundaries of the spliced sequence were flanked by GT-AG consensus acceptor-donor sequences, suggesting that an alternative splicing event led to the production of SYT-L.
The human SYT protein is composed of 387 aa and contains a conserved 54-aa domain called SYT N-terminal homology (SNH) domain (function unknown) at the N terminus. A C-terminal domain, rich in glutamine, proline, glycine, and tyrosine (QPGY-rich region) containing possible transcriptional activator sequences was also observed (Fig. 1C). The SYT-L protein is composed of 418 aa.
Genomic structure of the human SYT/SYT-L gene
The cDNA sequences obtained by RT-PCR in the present study were used to identify the human SYT-L genomic sequences from the GenBank htg database (accession no. NC_000018). The human SYT gene is located on chromosome 18q11.2 and contains 11 exons spanning approximately 74 kb (Fig. 1D) (23). An additional exon was found after exon 7 (Fig. 1D). Alternative splicing of exon 8 of SYT-L (cDNA nucleotides 881–973) resulted in the lack of 31 aa and the formation of SYT mRNA.
SYT-L mRNA is expressed in human embryonic, but not adult, tissues
We examined the expression pattern of endogenous SYT-L mRNA in different tissues from human embryos and adults using Northern blots. The blots were probed with a cDNA probe encoding the SYT-L-specific, 31-aa region. A predominant 4.8-kb transcript was detected in human embryonic brain, liver, and kidney, with lower amounts in embryonic lung (Fig. 2). However, in adult brain, placenta, skeletal muscle, heart, kidney, pancreas, liver, lung, spleen, and colon, hybridization signals were not detected, although SYT-L was initially isolated from adult brain by RT-PCR.
SYT interacts with the general coactivator CoAA, but not with CoAM, in vitro
GST pull-down studies were performed with recombinant GST fusion CoAA, CoAM, N-terminal deletion mutant of CoAA, N-CoAA (aa 150–669), and 35S-labeled, in vitro-translated SYT. SYT bound to CoAA and N-CoAA, but not to CoAM (Fig. 3A). When GST-SYT-L and 35S-labeled in vitro-transcribed and -translated CoAA, CoAM, and N-CoAA (aa 150–669) were incubated together, SYT-L bound to CoAA and N-CoAA, but not to CoAM (Fig. 3B). These results suggest that both SYT and SYT-L interact with the CoAA C terminus, but not with CoAM. LUC served as a negative control (data not shown).
SYT and SYT-L function as NR coactivators
We then investigated the role of SYT in NR-mediated transcriptional regulation. MMTV-LUC was cotransfected with the expression vector encoding GR and SYT or SYT-L into CV-1 cells in the presence or absence of dexamethasone (DEX; Fig. 4). Cotransfection of SYT together with CoAA synergistically activated MMTV promoter in the presence of DEX (Fig. 4A). Both SYT and SYT-L transactivated MMTV promoter in a dose-dependent manner up to 10-fold (Fig. 4). These transcriptional activities were ligand and receptor dose dependent (Fig. 4, B and C). We observed a dose-related difference in transcription between SYT and SYT-L. Transcription was more efficiently activated by SYT-L at lower doses and by SYT at higher doses (Fig. 4D). Cotransfection of SYT and SYT-L with SRC-1, which possesses HAT activity, or TRBP showed synergistic activation of MMTV promoter (Fig. 5). These results indicate that SYT and SYT-L are potent NR coactivators and suggest that SYT and SYT-L may activate transcription at least in part via SRC-1-containing HAT complex.
SYT and SYT-L function as general coactivators
Because the general coactivator CoAA activates NRs as well as other transcriptional factors, such as AP-1 (16), we investigated whether SYT and/or SYT-L possess similar activity. CoAA and SYT or SYT-L were cotransfected with a number of luciferase reporters containing TRE, ERE, and AP-1 enhancer-binding sites. SYT and SYT-L synergistically activated transcription in the presence of CoAA and the response elements (Fig. 6). Consistent with in vitro binding studies with CoAA, these results suggest that SYT and SYT-L are potent NR coactivators as well as general coactivators.
Activation domain of SYT
To determine the activation domain of SYT and SYT-L, we constructed a series of truncation mutants of SYT and SYT-L (Fig. 7A). Deletion of the C terminus of SYT or SYT-L, C-SYT (aa 1–186), produced a significant loss of transactivation function, suggesting that the activation domain is located in this region (Fig. 7B). In contrast, the deletion of the N terminus, N-SYT (aa 73–387) and N-SYT-L (aa 73–418), showed a tendency to activate, suggesting the existence of a repressor domain in the N-terminal region (Fig. 7B).
Activation of SYT depends on the cell line
We investigated whether SYT and/or SYT-L function differently in different cell lines, such as COS-1, JEG-3, and SW-13. Although the magnitude of transactivation induced by SYT and SYT-L was similar in CV-1 cells (Fig. 4D), SYT activated transcription more efficiently than SYT-L in COS-1, JEG-3, and SW-13 cells. The magnitude of activation by both SYT and SYT-L was much less in SW-13 cells, which lack SWI/SNF proteins (Fig. 8). Cotransfection of BRG-1 did not enhance SYT or SYT-L action (data not shown), suggesting that factors other than the SWI/SNF complex could be involved in the activation of transcription via SYT.
Discussion
Recently, we cloned CoAA, a coactivator of TRBP-interacting protein (16), which is identical with SIP. We hypothesized that SYT might function as an NR coactivator. Therefore, we isolated and characterized cDNAs encoding human SYT and its splicing variant, SYT-L. SYT and SYT-L function as NR coactivators and general coactivators, and both interact with CoAA.
Two SYT RT-PCR products were reproducibly observed in human brain and liver using different sets of primers. A comparison of the sequences revealed that SYT-L possesses an insert relative to SYT that contains a splice donor and an acceptor consensus GT-AG sequence. We also showed SYT-L mRNA expression in different tissues from human embryos using Northern blot analysis with SYT-L-specific primer. These results indicate that SYT-L is not a PCR artifact, but a natural splice variant of SYT. Furthermore, translocated SYT isoforms, SYT-SSX, SYT-SSX2v, and SYT-SSX4v, isolated from human synovial sarcoma tissues (29), all contain the region identical with SYT-L. Comparing the cDNA and chromosome 18q11.2 genomic sequences, we confirmed the presence of an additional exon specific for SYT-L that is flanked by GT-AG consensus acceptor-donor sequences. These data provide additional support for the idea that SYT-L is a naturally occurring SYT isoform.
RT-PCR using total RNA from adult human tissues and Northern blot analysis of different tissues from human embryos confirmed SYT isoform mRNA expression. The relative amounts of SYT-L expression in brain were greater than those in liver, indicating that SYT-L may be involved in transcriptional regulation of neuronal genes. Additional investigations are required to clarify this hypothesis. Of note, we could not detect the expression of SYT-L mRNA in adult tissues using Northern blot analysis. Thus, as shown for mouse SYT (22), human SYT mRNA expression may occur largely during development, including possible regulation of organogenesis in the brain.
Functional analyses indicate that at low concentrations of isoform cDNA, SYT-activated GR-mediated transcription through the MMTV promoter was more efficient than SYT-L; however, at high concentrations, their relative activities were reversed. Although there was no essential difference between SYT and SYT-L in terms of transcriptional activation, this dose-related reversal of relative activities could contribute to subtleties of SYT-mediated transactivation regulation. The transcriptional activation was dose, ligand, and NR dependent, indicating that SYT and SYT-L are potent NR coactivators. Furthermore, SYT and SYT-L activated transcription through an AP-1 enhancer-binding site. These results suggest that SYT and SYT-L may function as general coactivators.
CoAA, but not CoAM, interacts with SYT in vitro. In reporter gene assays, CoAA, SRC-1, and TRBP synergistically activated transcription with SYT. These results suggest that SYT-associated transcriptional activation may be mediated through SRC-1-containing HAT complexes. This could occur by interactions with CoAA, because TRBP, a CoAA-interacting protein, has been also reported to interact with SRC-1-containing HAT complexes (10). These results indicate that SYT may function in collaboration with CoAA and HAT complex in vivo.
The present study also indicates that CoAA interacts with SYT through the TRBP-interacting domain located in the middle region of CoAA, which is absent in the CoAM (16). This domain contains more than 20 XYXXQ motifs (X denotes a small amino acid residue including G, A, S, and P) (16). Through this domain, CoAA also interacts with CBP/p300. Although the function of this motif has not been fully clarified, previous studies have shown that this motif is important for permitting protein-protein interactions (16, 30, 31). The present study confirmed that this motif plays an important role in such interactions, particularly with transcriptional coactivators. Additionally, because CoAA/CoAM have been reported as promoter-dependent RNA splicing modulators (17, 18, 19), the interaction between SYT and CoAA might affect posttranscriptional regulation, especially the control of alternative RNA splicing. This raises the possibility that an abnormal balance of cellular protein products caused by SYT-SSX due to aberrant mRNA splicing might affect the development and/or progression of synovial sarcoma.
We have determined that the activation domain of SYT, the QPGY-rich region (aa 73–387) is located at the C terminus, which contains several glutamine (Q), proline (P), glycine (G), and tyrosine (T) residues in an XXYXX motif. In this sequence, the tyrosine residue is surrounded by at least three X residues (Q, P, or G) (24). A previous study reported that GAL4-DNA-binding domain-coupled SYT (aa 158–387) showed the greatest transcriptional activity (24). However, because SYT does not contain a potential DNA-binding domain, SYT is involved in transcriptional events through an indirect mechanism. Our study not only confirmed the function of this region (24), but also showed that SYT may work with CoAA to interact with TRBP. This complex then may interact with various native NRs, such as TR, ER, and GR, as well as other transcription factors to regulate transcription.
SYT contains an SNH domain at its N terminus. This domain is conserved across a wide range of species, although the role of this domain is not fully understood (24). SYT binds to BRG-1, the human homologue of SWI2/SNF2, through the SNH domain (25, 26, 27). The SWI/SNF is a large family of ATP-dependent chromatin remodeling complex proteins that are associated with transcriptional regulation. The N terminus of SYT has been reported to be a repression domain, and our results support this concept. Thus, our data are consistent with the hypothesis that the SWI/SNF complex is involved in transcriptional repression via interaction with SYT. Furthermore, our data show that the effects of SYTs are greatly diminished in SW-13 cells that lack the SWI/SNF complex. Our results, however, do not support a recent report showing that BRM and BRG-1 suppress GAL4-fused SYT-activated transcription in a dose-dependent manner in SW-13 cells (24). It has been reported that GAL4-SYT directly interacts with DNA through an upstream activating sequence (24), whereas we show that SYT interacts indirectly with DNA (present study). Although SYT is capable of binding to SWI/SNF proteins, including BRG-1, this complex probably does not play an important role in transcriptional activation through GR. However, the possible involvement of SYT in repression in a different promoter context cannot be excluded. In contrast, cotransfection of SRC-1, TRBP, or CoAA with SYT synergistically activated GR-mediated MMTV promoter-driven transcription. Therefore, SRC-1-containing HAT complexes, but not BRG-1-containing SWI/SNF complexes, may be the dominant forms involved in SYT-associated transcriptional activation by NRs.
It has been reported that the pathogenesis of soft tissue sarcomas is highly related to translocation of chromosomes (20, 21). As a result, translocations, such EWS-FLI1 in Ewing’s sarcoma, SYT-SSX in synovial sarcoma, and CHOP-FUS in myxoid liposarcoma, are used for clinical diagnosis (32). Besides these fusion proteins, a series of chromosomal translocations, including PAX3/7-FKHR, EWS-ETF1/WT1/CHN/CHOP, RBP56-CHN, ETV6-NTRK3 and COL1A1-platelet-derived growth factor-B, have been observed as pathogens of soft tissue tumors (33). Among these sarcoma-related proteins, EWS, the product of a gene commonly translocated in Ewing sarcoma; TLS/FUS, (translocated in liposarcoma); and RBP56 (RNA-binding protein 56) possess the domains analogous to the QPGY region in SYT, suggesting that these wild-type proteins could interact with coactivators to modulate transcriptional regulation. Interestingly, EWS, TLS/FUS, and RBP56 possess RGG repeats (RNA-binding motifs), in their C termini, which suggest that RNA-binding proteins related to transcriptional regulation could also be involved in tumorigenesis.
In summary, our study shows that SYT may function as a transcriptional coactivator that interacts with SRC-1-containing HAT complexes through CoAA. It has been reported that SYT interacts with p300, which promotes cell adhesion to a fibronectin matrix (34), and with cyclin D1, which is related to the cell cycle (35, 36). Furthermore, CoAA, an SYT-interacting protein, may be associated with other cellular activities, such as DNA repair via interaction with DNA-dependent protein kinase complex (16) or alternative RNA splicing (17, 18, 19). Taken together, the SYT may serve key roles involving diverse nuclear events, leading to the control of multiple cellular functions.
Acknowledgments
We thank Drs. C. S. Cooper, H. Kato, and A. Takeshita for kindly preparing the constructs. We thank Drs. C. S. Suen and S. Nagpal for critical discussions and comments during the entire project. We thank M. Ohta and W. Miyazaki for technical assistance throughout the study.
Footnotes
This work was supported by Lilly postdoctoral research fellowships; Grant-in-Aid for Scientific Research 14370020 from the Japanese Ministry of Education, Science, Sports, and Culture; and a grant from CREST, Japan Science and Technology Corp. (to J.S.T.).
Abbreviations: aa, Amino acid; AP-1, activator protein-1; BRG-1, brm/SWI2-related gene-1; BRM, brahma; CBP, cAMP response element binding protein (CREB)-binding protein; CoAA, coactivator activator; CoAM, coactivator modulator; DEX, dexamethasone; ER, estrogen receptor; ERE, estrogen response element; GR, glucocorticoid receptor; GRE, glucocorticoid receptor response element; GST, glutathione-S-transferase; HAT, histone acetyltransferase; LUC, luciferase; MEK, MAPK/extracellular signal-regulated kinase kinase; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; MMTV, mouse mammary tumor virus; NR, nuclear receptor; RBP56, RNA-binding protein 56; RRM, RNA recognition motif; SIP, SYT-interacting protein; SNH, synovial sarcoma translocation N-terminal homology; SRC-1, steroid receptor coactivator-1; SSX, synovial sarcoma X break point; SYT, synovial sarcoma translocation; SYT-L, larger isoform of synovial sarcoma translocation; TLS, translocated in liposarcoma; TR, thyroid hormone receptor; TRBP, thyroid receptor-binding protein; TRE, thyroid hormone response element.
References
Glass CK, Rosenfeld MG 2000 The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141
Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1997 Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 389:194–198
Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580
Bannister AJ, Kouzarides T 1996 The CBP co-activator is a histone acetyltransferase. Nature 384:641–643
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1A. Nature 382:319–324
Peterson CL, Workman JL 2000 Promoter targeting and chromatin remodeling by the SWI/SNF complex. Curr Opin Genet Dev 10:187–192
Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828
Yuan CX, Ito M, Fondell JD, Fu ZY, Roeder RG 1998 The TRAP220 component of a thyroid hormone receptor-associated protein (TRAP) coactivator complex interacts directly with nuclear receptors in a ligand-dependent fashion. Proc Natl Acad Sci USA 95:7939–7944
Jiang YW, Veschambre P, Erdjument-Bromage H, Tempst P, Conaway JW, Conaway RC, Kornberg RD 1998 Mammalian mediator of transcriptional regulation and its possible role as an end-point of signal transduction pathways. Proc Natl Acad Sci USA 95:8538–8543
Ko L, Cardona GR, Chin WW 2000 Thyroid hormone receptor-binding protein, an LXXLL motif-containing protein, functions as a general coactivator. Proc Natl Acad Sci USA 97:6212–6217
Lee SK, Anzick SL, Choi JE, Bubendorf L, Guan XY, Jung YK, Kallioniemi OP, Kononen J, Trent JM, Azorsa D, Jhun BH, Cheong JH, Lee YC, Meltzer PS, Lee JW 1999 A nuclear factor, ASC-2, as a cancer-amplified transcriptional coactivator essential for ligand-dependent transactivation by nuclear receptors in vivo. J Biol Chem 274:34283–34293
Caira F, Antonson P, Pelto-Huikko M, Treuter E, Gustafsson JA 2000 Cloning and characterization of RAP250, a novel nuclear receptor coactivator. J Biol Chem 275:5308–5317
Zhu Y, Kan L, Qi C, Kanwar YS, Yeldandi AV, Rao MS, Reddy JK 2000 Isolation and characterization of peroxisome proliferator-activated receptor (PPAR) interacting protein (PRIP) as a coactivator for PPAR. J Biol Chem 275:13510–13516
Mahajan MA, Samuels HH 2000 A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREB-binding protein. Mol Cell Biol 20:5048–5063
Guan XY, Xu J, Anzick SL, Zhang H, Trent JM, Meltzer PS 1996 Hybrid selection of transcribed sequences from microdissected DNA: isolation of genes within amplified region at 20q11–q13.2 in breast cancer. Cancer Res 56:3446–3450
Iwasaki T, Chin WW, Ko L 2001 Identification and characterization of RRM-containing coactivator activator (CoAA) as TRBP-interacting protein, and its splice variant as a coactivator modulator (CoAM). J Biol Chem 276:33375–33383
Auboeuf D, Dowhan DH, Kang YK, Larkin K, Lee JW, Berget SM, O’Malley BW 2004 Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes. Proc Natl Acad Sci USA 101:2270–2274
Auboeuf D, Dowhan DH, Li X, Larkin K, Ko L, Berget SM, O’Malley BW 2004 CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing. Mol Cell Biol 24:442–453
Auboeuf D, Honig A, Berget SM, O’Malley BW 2002 Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science 298:416–419
dos Santos NR, de Bruijn DR, van Kessel AG 2001 Molecular mechanisms underlying human synovial sarcoma development. Genes Chromosomes Cancer 30:1–14
Clark J, Rocques PJ, Crew AJ, Gill S, Shipley J, Chan AM, Gusterson BA, Cooper CS 1994 Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat Genet 7:502–508
de Bruijn DR, Baats E, Zechner U, de Leeuw B, Balemans M, Olde Weghuis D, Hirning-Folz U, Geurts van Kessel AG 1996 Isolation and characterization of the mouse homolog of SYT, a gene implicated in the development of human synovial sarcomas. Oncogene 13:643–648
de Bruijn DR, Kater-Baats E, Eleveld M, Merkx G, Geurts Van Kessel A 2001 Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene. Cytogenet Cell Genet 92:310–319
Thaete C, Brett D, Monaghan P, Whitehouse S, Rennie G, Rayner E, Cooper CS, Goodwin G 1999 Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus. Hum Mol Genet 8:585–591
Perani M, Ingram CJ, Cooper CS, Garrett MD, Goodwin GH 2003 Conserved SNH domain of the proto-oncoprotein SYT interacts with components of the human chromatin remodelling complexes, while the QPGY repeat domain forms homo-oligomers. Oncogene 22:8156–8167
Kato H, Tjernberg A, Zhang W, Krutchinsky AN, An W, Takeuchi T, Ohtsuki Y, Sugano S, de Bruijn DR, Chait BT, Roeder RG 2002 SYT associates with human SNF/SWI complexes and the C-terminal region of its fusion partner SSX1 targets histones. J Biol Chem 277:5498–5505
Ishida M, Tanaka S, Ohki M, Ohta T 2004 Transcriptional co-activator activity of SYT is negatively regulated by BRM and Brg1. Genes Cells 9:419–428
Takeshita A, Yen PM, Ikeda M, Cardona GR, Liu Y, Koibuchi N, Norwitz ER, Chin WW 1998 Thyroid hormone response elements differentially modulate the interactions of thyroid hormone receptors with two receptor binding domains in the steroid receptor coactivator-1. J Biol Chem 273:21554–21562
Brodin B, Haslam K, Yang K, Bartolazzi A, Xie Y, Starborg M, Lundeberg J, Larsson O 2001 Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression. Gene 268:173–182
Cartegni L, Maconi M, Morandi E, Cobianchi F, Riva S, Biamonti G 1996 hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins. J Mol Biol 259:337–348
Biamonti G, Ruggiu M, Saccone S, Della Valle G, Riva S 1994 Two homologous genes, originated by duplication, encode the human hnRNP proteins A2 and A1. Nucleic Acids Res 22:1996–2002
Uchida A, Seto M, Hashimoto N, Araki N 2000 Molecular diagnosis and gene therapy in musculoskeletal tumors. J Orthop Sci 5:418–423
Skapek SX, Chui CH 2000 Cytogenetics and the biologic basis of sarcomas. Curr Opin Oncol 12:315–322
Eid JE, Kung AL, Scully R, Livingston DM 2000 p300 Interacts with the nuclear proto-oncoprotein SYT as part of the active control of cell adhesion. Cell 102:839–848
Xie Y, Skytting B, Nilsson G, Gasbarri A, Haslam K, Bartolazzi A, Brodin B, Mandahl N, Larsson O 2002 SYT-SSX is critical for cyclin D1 expression in synovial sarcoma cells: a gain of function of the t(X;18)(p11.2;q11.2) translocation. Cancer Res 62:3861–3867
Xie Y, Skytting B, Nilsson G, Grimer RJ, Mangham CD, Fisher C, Shipley J, Bjerkehagen B, Myklebost O, Larsson O 2002 The SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1. A link between t(X;18)(p11.2; q11.2) and the cell cycle machinery. Oncogene 21:5791–5796(Toshiharu Iwasaki, Noriyu)