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Human Molecular Genetics Pages 585-591  

Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus
Introduction
Results
   Domain structure of the SYT protein
   The SSX C-terminal domain in SYT-SSX has repressing activity
   SYT and SYT-SSX co-localize with BRM in the cell nucleus
   SYT and BRM bind directly and specifically with one another in vitro
Discussion
Materials And Methods
Acknowledgements
References
Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus

Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus

C. Thaete+, D. Brett+, P. Monaghan, S. Whitehouse, G. Rennie, E. Rayner, C. S. Cooper and G. Goodwin*

Institute of Cancer Research, Molecular Carcinogenesis Section, The Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey SM2 5NG, UK

Received September 8, 1998; Revised and Accepted January 21, 1999

The t(X;18)(p11.2;q11.2) chromosomal translocation commonly found in synovial sarcomas fuses the SYT gene on chromosome 18 to either of two similar genes, SSX1 or SSX2, on the X chromosome. The SYT protein appears to act as a transcriptional co-activator and the SSX proteins as co-repressors. Here we have investigated the functional domains of the proteins. The SYT protein has a novel conserved 54 amino acid domain at the N-terminus of the protein (the SNH domain) which is found in proteins from a wide variety of species, and a C-terminal domain, rich in glutamine, proline, glycine and tyrosine (the QPGY domain), which contains the transcriptional activator sequences. Deletion of the SNH domain results in a more active transcriptional activator, suggesting that this domain acts as an inhibitor of the activation domain. The C-terminal SSX domain present in SYT-SSX translocation protein contributes a transcriptional repressor domain to the protein. Thus, the fusion protein has transcriptional activating and repressing domains. We demonstrate that the human homologue of the SNF2/Brahama protein BRM co-localizes with SYT and SYT-SSX in nuclear speckles, and also interacts with SYT and SYT-SSX proteins in vitro. This interaction may provide an explanation of how the SYT protein activates gene transcription.

INTRODUCTION

Chromosomal translocations that result in oncogenic transformation frequently involve the fusion of two genes to create a hybrid gene that expresses a novel chimeric protein. In many cases, this protein is an aberrant transcription factor. In the case of synovial sarcomas, we have described the characterization of the genes involved in the commonly observed t(X;18)(p11.2;q11.2) translocation. This results in the fusion of the SYT gene on chromosome 18 to either of two closely related genes SSX1 and SSX2 on chromosome X. The resulting chimaeric genes express SYT-SSX1 or SYT-SSX2 fusion proteins in which the C-terminal amino acids of SYT are replaced by amino acids from the C-terminus of the SSX proteins (1-3).

The normal SYT gene is expressed in a wide variety of cell types during embryogenesis (4) and in the adult, but the function of the protein is not known. The protein is localized in the cell nucleus but it has no recognizable nucleic acid-binding motifs. Although the functional domains within SYT currently have not been characterized, the C-terminal two-thirds of the protein is rich in glutamine, proline, glycine and tyrosine residues, resembling the composition of a number of transcription activators. In agreement with this, we have shown that the SYT protein acts as a potent transcriptional activator (5).

The N-terminal half of both the normal SSX proteins (which is not retained in the fusion proteins) has a domain related to the KRAB domain which has been described previously in a number of transcription repressors containing zinc finger DNA-binding domains (6). We have shown in previous experiments (5) that the entire SSX proteins have transcriptional repressing activity when assayed as GAL4 fusions, but the precise region responsible for this activity has not been established. Since both the SYT and SSX proteins do not have recognizable DNA-binding domains, they presumably function to modulate gene transcription through association with other nuclear proteins that recruit them to their target promoters. In this study, we have carried out deletion analyses of the SYT and SSX proteins to investigate the function of the different domains in more detail.

Using green fluorescent protein (GFP) fusions, we have demonstrated that the SYT, SSX and SYT-SSX proteins are nuclear proteins (5). Similar results have been obtained by immunofluorescent studies (7). Whilst the SSX1 protein has a uniform nuclear distribution, the SYT protein has a speckled distribution in the cell nucleus, and this distribution is retained with the SYT-SSX2 fusion protein. The SYT speckles did not co-localize with the promyelocitic leukaemia bodies or spliceosomes. In this study, we have extended our analysis to examine whether SYT co-localizes with other nuclear proteins known to exhibit heterogeneous or speckled distribution, including the protein BRM (8), one of the human homologues of the Drosophila Brahma and the yeast SNF2 proteins (9-12). BRM is one of components of the multiprotein complexes called the SNF complexes (13,14). The SNF complexes function as transcriptional activators of genes that are repressed by chromatin structural proteins such as histones and polycomb-group proteins (11). One model for their mode of action is that sequence-specific DNA-binding proteins [e.g. nuclear hormone receptors (9,10,15)] can recruit the SNF complex to target promoters and, once bound, the complex disrupts the nucleosomes in its vicinity, thereby allowing the binding of other transcription factors required to initiate transcription. The mammalian SNF complex is heterogeneous and the complexes are composed of 9-12 proteins, termed BAFs, many of which have now been cloned (13,14). It is likely that the SNF/BAF proteins interact with other nuclear proteins, either to target the SNF complexes to specific promoters or, once bound to these promoters, to interact with other factors that regulate gene expression. As we show here, the SYT protein may be one of these proteins or may be a core component of the SNF complex (i.e. a BAF).

RESULTS

Domain structure of the SYT protein

Examination of the SYT protein sequence reveals three regions within the protein that may have functional significance. A database search revealed 11 protein sequences and translations of expressed sequence tag (EST) sequences with significant homology with a domain near the N-terminus of SYT comprising amino acids 20-73 (Fig. 1, the SNH domain). One of these is contained in what appears to be a new human SYT family member (SYT homologue 1), the homology with SYT spanning the whole length of the two proteins (~60% homology). A second human homologue (SYT homologue 2) was also identified in these searches. The SNH domain was identified in predicted protein sequences from plants, nematodes and fish, as well as mammals, demonstrating that this region is conserved across a broad range of species. The function of this novel domain in these proteins has not been described.


Figure 1. Sequence of the SYT protein and homologues. (a) The SNH domain identified by homology to sequences in the databases: 1, Brassica rapa (EST 887272); 2, Arabidopsis thaliana (gi22552866); 3, rice (EST 702013); 4, human SYT; 5, mouse SYT (4); 6, zebra fish (EST 2225596); 7, human SYT homologue 1 (gi 3327200); 8, human SYT homologue 2 (AA86859); 9, rat (EST 2862851); 10, Brugia malayi (EST 1172331); 11, Caenorhabditis elegans (EST 2388279). EST homologies were from the EST database at NCBI and the full-length sequences were from GenBank (the accession numbers are GenBank IDs). (b) The human SYT sequence showing the SNH domain and the QPGY domain that encompasses the region of the protein that has the high concentration of the four amino acids glutamine (27%), proline (19%), glycine (19%) and tyrosine (12%). The arrows represent the positions of translocations in synovial sarcoma.

In the C-terminal half of SYT, between amino acids 187 and 387, lies a transcriptional activation domain. The sequence is composed predominantly of glutamine, proline and glycine, with tyrosine residues occurring at variable intervals (Fig. 1b, the QPGY domain). The abundance of glutamine, glycine and tyrosine in this region is similar to that observed in the N-terminal activating region of the EWS/FUS/TLS family of proteins, which contain (S/G)YQQ(S/Q) repeats (16). However, we could not identify a specific repeat peptide around the tyrosine residues as found in the EWS/FUS/TLS proteins. Nevertheless, the C-terminal tyrosines in the two human and the mouse SYT homologues (4) are totally conserved, as are most of the glutamines. The SNH and QPGY domains are separated by a sequence of 114 amino acids which is highly conserved (96%) in the mouse SYT and characterized by an unusually high content of methionine (16%). Although this region is not closely related to sequences in other proteins, it appears to be functionally significant, as we describe below.

We have shown previously that when the N- and C-terminal halves of SYT are fused to GAL4 and assayed for their ability to activate a luciferase reporter containing a 5× upstream activating sequence (UAS) upstream of the thymidine kinase (TK) promoter (consisting of TATA and CAAT boxes plus two Sp1-binding sites), the C-terminal half has strong transactivating activity whilst the N-terminal has little activating or repressing activity. A deletion analysis of the C-terminal half of SYT revealed that the activating activity was spread throughout this region of the protein (data not shown). This was confirmed with a different reporter promoter: when two halves of the SYT C-terminal domain were assayed for their ability to activate a promoter consisting of the adenovirus TATA box region plus 5×UAS upstream of luciferase, both constructs exhibited transactivating activity (Fig. 2, constructs C and D). The results of Figure 2 also reveal that removal of the N-terminal 60 amino acids from the full-length SYT protein, which removes most of the SNH domain, results in a 30-fold increase in the ability of the protein to activate the adenovirus promoter (construct A), strongly suggesting that the SNH domain may have an inhibitory role. A further deletion, removing most of the methionine-containing region, results in little further change in transactivating activity (construct B).


Figure 2. Transcriptional activation of SYT deletions. Expression constructs of GAL4-SYT deletions of the full-length SYT (387 amino acids) and GAL4 DNA-binding domain (GAL4 DBD) were transfected into SW480 cells together with the 5×UAS-adenovirus TATA box-firefly luciferase reporter and the pRL-SV40 Renilla luciferase reporter. Firefly luciferase activities of the SYT constructs were corrected for transfection efficiency, and these values have been normalized between different experiments such that all constructs are compared with the activity of the full-length SYT which is given the value 100. (The absolute firefly luciferase activity of the full-length GAL4-SYT was 16-22-fold above the background obtained with GAL4 DBD.) All six constructs were assayed in duplicate in two experiments, and the standard deviations are given in parentheses.

It is possible that the differences in activities of some of the constructs are due to differences in protein stability of the GAL4 fusions, but attempts to assess this using a number of commercial GAL4 antibodies were unsuccessful. Nevertheless, it is still apparent that the two non-overlapping C-terminal constructs C and D both have substantial activities, demonstrating that the activation domain is spread throughout the C-terminal half of the protein. It is also apparent that the N-terminal SNH domain may control the activity of the protein either as an inhibitory sequence or as an element that controls protein stability.

The SSX C-terminal domain in SYT-SSX has repressing activity

We have demonstrated previously that the SSX1 protein, when fused to GAL4, acts as transcriptional repressor (5). We have found that the C-terminal region of SSX that is involved in the translocation reproducibly represses the TK promoter 3-fold but, surprisingly, the N-terminal part of the protein containing the KRAB-like domain does not repress (Fig. 3). Thus, the SSX C-terminal addition to the SYT protein in the translocation may be contributing a repression domain. This may explain why the SYT-SSX fusion always has 3- to 5-fold lower activity when compared with full-length SYT (5). It would appear, therefore, that the SYT-SSX protein has both transcriptional activating and repressing domains. The histone deacetylase inhibitor trichostatin A (TSA) has no effect on the repressing activity of either the full-length SSX or the C-terminal SSX domain (data not shown), indicating that the repression is not mediated by histone or transcription factor deacetylation.


Figure 3. Repression domain in the C-terminus of the SSX1 protein. The C-terminal 78 amino acids (which are translocated to SYT in sarcomas), the remaining N-terminal domain and full-length SSX1 were expressed as GAL4 fusions in SW480 cells co-transfected with the 5×UAS TK-luciferase or with the TK-luciferase. Luciferase activities were divided by that obtained with GAL4 DBD alone to give the fold repression of the two reporters.

SYT and SYT-SSX co-localize with BRM in the cell nucleus

We (5) and dos Santos et al. (7) have shown previously that SYT and SYT-SSX, when expressed as GFP fusion proteins or detected by immunofluorescence, are localized in discrete speckles in the cell nucleus. In order to see whether any other transcription factors that are known to localize to discrete regions in the nucleus might co-localize with SYT in these bodies, we investigated a number of possible candidates in co-transfection experiments, including the protein BRM which recently has been shown to give a speckled distribution in the cell nucleus (8). As shown in Figure 4, we found that the human homologue of the SNF2 protein, BRM, co-localizes with GFP-SYT and with GFP-SYT-SSX. When a construct expressing epitope-tagged BRM protein is co-transfected into cells and detected by immunofluorescence, the BRM protein (red fluorescence) co-localizes with the green fluorescence of the SYT and SYT-SSX proteins in these speckles (Fig. 4A and B). These results were obtained using both the anti-haemagglutinin (HA) monoclonal antibody (Fig. 5) and anti-BRM polyclonal antibody (data not shown) in a number of cell types (SW480, kidney 293, Cos7 and HT1080). In control experiments, no red speckles are seen when cells transfected with the GFP-SYT-SSX alone are stained with the anti-HA monoclonal antibodies (Fig. 4C). In addition, when BRM is transfected into the cells with the GFP-SIP fusion protein, a protein that we have found also to give nuclear speckles, the red speckles of BRM do not co-localize with the green SIP speckles (Fig. 4D). The SIP protein is a nuclear protein that we recently have identified and shown to be similar to members of the EWS/TLS/FUS family (P. Antonson and G. Goodwin, unpublished data). Similarly, no co-localization was seen with the SSX protein (data not shown). Deletion of the N-terminal 60 amino acids from the SYT protein, which removes most of the SNH domain, had little effect on the ability of the protein to co-localize with BRM in nuclear speckles (Fig. 5). Deletion of the N-terminal half of SYT resulted in diminished association such that <30% of the SYT speckles had BRM associated, but we were able to detect some residual binding to some of the SYT speckles. These results suggest that BRM interacts principally with the methionine-containing central region of SYT, but there may also be a secondary binding site in the C-terminal half of the protein.

Figure 4. (A) Co-localization of GFP-SYT-SSX (green) and HA-tagged BRM (red). (B) Co-localization of GFP-SYT (green) and HA-tagged BRM (red). (C) Experiment carried out as in (A), except that the cells were transfected with GFP-SYT-SSX constructs only. (D) Localization of GFP-SIP (green) and HA-tagged BRM (red). Transfections were into SW480 cells. For all studies, the left hand panels shows GFP fluorescence, the central panel shows the results above with anti-HA monoclonal antibody and the right hand panel shows the combined image. The white scale bar is 10 µm.


Figure 5. Co-localization of BRM with N-terminal deletions of SYT. GFP fusions of SYT deletions were transfected with BRM into HT1080 cells and the extent of localization of the BRM with the SYT speckles observed. +++ indicates that >80% of SYT nuclear speckles are also stained with BRM, + indicates <30% co-localization.

These results therefore show that the SYT co-localizes with BRM in the cell nucleus, and the addition of the SSX portion or the removal of the inhibitory SNH domain, although altering its transcriptional activating properties, does not alter the association of SYT with BRM. No difference was detected between the SYT and the SYT-SSX fusion in their ability to bind BRM in the nuclear co-localization assay. However, it is important to point out that in experiments that were carried out with HT1080 and NIH 3T3 cells which have a larger cytoplasm than SW480 cells, it was apparent that whilst the SYT-SSX and BRM proteins were localized solely in the nucleus, a variable fraction of the SYT (10-50%) was observed as speckles in the cytoplasm. Thus, an additional role for the SSX C-terminus in the fusion protein may be to alter the cellular distribution of the SYT by conferring another nuclear localization signal. This is supported by the observation that a GFP fusion containing just the C-terminal translocated domain of SSX localizes to the cell nucleus (data not shown).

SYT and BRM bind directly and specifically with one another in vitro

In vitro protein-binding experiments provided evidence that the co-localization of SYT and BRM may in fact be explained by the direct interaction between the proteins. GST and GST-cBRM immobilized on glutathione beads were purified from Escherichia coli and incubated with radioactive SYT, SYT-SSX, and luciferase as a control. The beads were washed and the bound protein analysed by PAGE and detected with a phosphoimager (Fig. 6). The results show that both SYT and SYT-SSX bind to GST-cBRM and not to GST, and that luciferase binds to neither. Both proteins appeared to bind with similar affinity to the cBRM. Further evidence that the interaction is specific was provided by demonstrating that SYT and SYT-SSX do not bind to beads containing the N-terminal activation domain of TFE3 fused to GST (data not shown), and the TFIID protein TAF55 does not bind to GST-cBRM beads (see below).


Figure 6. Binding of SYT and SYT-SSX to chicken BRM in vitro. Labelled SYT, SSX and luciferase were incubated with GST or GST-cBRM beads and the bound proteins analysed by SDS-PAGE and phosphoimaging. Lanes 1-3 show the input proteins and lanes 4-9 the proteins bound to the beads.

To map the interacting domains, three portions of the cBRM protein, BRM-A, -B and -C, were expressed as GST fusions. BRM-A consists of amino acids 1-872, BRM-B amino acids 873-1433 (most of the central ATPase domain and the Rb-binding domain), and BRM-C, the C-terminal 135 amino acids containing part of the bromodomain. As can be seen (Fig. 7), the labelled SYT-SSX protein binds strongly only to the N-terminal BRM-A, as compared with the labelled BRM-B and -C and the luciferase and TAF55 controls.

DISCUSSION

When considered together, the data presented here demonstrate that the SYT protein co-localizes with BRM, probably as a consequence of direct interactions between the central region of SYT and the N-terminal half of the BRM protein. BRM is one of the human homologues of the Drosophila Brahma and yeast SNF2 proteins (9,10), proteins that are components of the multiprotein complexes called the SNF complexes. The SNF complexes function as transcriptional activators of genes that are repressed by chromatin structural proteins such as histones and polycomb-group proteins (11). One model for their mode of action is that certain sequence-specific DNA-binding proteins (such as nuclear hormone receptors) that are able to bind to their specific sequences even when complexed in nucleosomes can recruit the SNF complex and, once bound, the complex disrupts the nucleosomes in its vicinity, thereby allowing the binding of other transcription factors required to initiate transcription. An alternative model has been put forward from studies in yeast which suggest that the SNF complex may be recruited to promoters through its association with the RNA polymerase holoenzyme, and whether or not a particular promoter responds to the SNF complex is dependent on the nucleoprotein structure of the promoter (17). Evidence that the first model is operative in higher eukaryotes comes from studies that show that components of the SNF complex co-immunoprecipitate with the glucocorticoid receptors (15) and the recent demonstration that the human trithorax-related protein MLL, a DNA-binding protein, interacts with the hSNF5 protein, one of the components of the SNF complex (18). This is consistent with the findings in Drosophila that the trithorax-group proteins, which include trithorax and Brahma, are required to maintain the pattern of active homeobox genes of the ANT-C and BX-C loci during embryonic development by counteracting the chromatin repressor polycomb (12).

The mammalian SNF complex is composed of 9-12 proteins, termed BAFs, many of which have now been cloned (13,14). Biochemical analysis has revealed that the complex is in fact heterogeneous; the SNF complexes are present in multiple forms which differ in their protein composition. For example, complexes differ in whether they contain BRG1 or BRM (the two human Brahma homologues) and which of the different BAF60 proteins they contain. The functions of the proteins in the SNF complexes are not known other than that BRM and BRG1 have DNA-dependent ATPase activities required for nucleosome disruption, (11) that they interact with the transcriptional repressor Rb, co-operating to induce cell cycle arrest (19), and that the hSNF5 protein binds MLL (18). It seems likely that the SNF proteins interact with other nuclear proteins, either to target the complex to specific promoters, or, once bound to these promoters, to interact with other factors that regulate gene expression. The SYT protein may be one of these proteins or may be a core component of the SNF complex (i.e. a BAF). Since most of the mammalian BAFs have now been cloned and none has any sequence homology with SYT, the latter seems unlikely.

It is of interest to find that the SYT-SSX protein has a transcriptional activation domain in the C-terminal half and a repression domain contributed by the SSX C-terminus. After this manuscript was submitted, Lim et al. (21) also reported that the C-terminus of SSX has repressing activity. The mechanism whereby the SSX portion of the protein represses is not known. It does not appear to do so by binding histone deacetylases since the inhibitor TSA has no effect on the repressing activity of full-length SSX or the C-terminal SSX domain, and it is tempting to speculate that the SSX C-terminus counteracts the effect of BRM by recruiting the polycomb complex to the SNF complex. The effect of the translocation in synovial sarcomas may be the alteration of an SNF complex containing the SYT protein from an activating complex to an inactive complex or even a repressing complex, depending on the relative activating and repressing activities of the SYT-SSX fusion in synovial sarcoma cells. Thus, genes that normally are activated by SNF complexes containing the SYT protein may be down-regulated in synovial sarcoma cells by SNF complexes containing the SYT-SSX protein. Alternatively, genes that normally are repressed by SSX protein complexes could be activated by the recruitment of the SNF complexes containing the SYT-SSX proteins.

An alternative model for the function of the SYT and SYT-SSX proteins comes from studies that show that BRM and BRG1, as well as interacting with components of the SNF complex, are known to interact with the tumour repressor Rb to induce cell growth arrest (19) and with the nuclear hormone receptors to augment their transcriptional activities (9,10,15). It is possible, therefore, that the SYT and SYT-SSX proteins function by modulating the activities of these complexes or the association of these proteins with one another.

Analysis of the repression activities of the SSX1 constructs suprisingly showed that the N-terminal construct containing the KRAB domain had no repressing activity. Nevertheless the full-length SSX protein had higher repressing activity than the C-terminal translocated domain, and this suggests that the KRAB domain may augment the activity of the C-terminal domain even though it has no intrinsic activity of its own.

The function of the SNH domain within SYT may be to control the activity of the C-terminal activation domain. Deleting most of the SNH did not affect nuclear localization nor its association with BRM, but did result in a more potent transactivator. The N-terminal half of SYT, when assayed as a separate fragment fused to GAL4, does not exhibit repressing activity. Thus enhanced transcriptional activity of the SYT N-terminal deletions may be explained by postulating that the SNH domain interacts with and masks the C-terminal activation domain. Alternatively, the SNH domain could control turnover of the protein, so that removal of the domain results in a more stable protein; this was not obvious in the GFP fusions but remains to be analysed more carefully.

MATERIALS AND METHODS

Transient transfections of GAL4-SYT fusion constructs together with transcription reporter constructs into SW480 cells were carried out using SuperFect transfection reagent (Qiagen, Crawley, UK) using the manufacturer’s methods. All constructs were assayed in duplicate in each transfection experiment, normalized for transfection efficiency, and all experiments were repeated at least twice. Typically, Gal4-SYT construct (0.4 µg), pG5luc firefly luciferase reporter (1.0 µg) and pRL-SV40 Renilla luciferase (250 ng) were transfected into the cells grown in 6-well plates. After 48 h, the cells were harvested and the extracts were analysed for firefly luciferase and Renilla luciferase using the dual reporter assay system. GAL4-SYT constructs are plasmids expressing fragments of SYT fused to the DNA-binding domain of GAL4 (5). pG5luc (Promega, Southampton, UK) contains the adenovirus major late promoter TATA box and 5×UAS upstream of the firefly luciferase gene. pRL-SV40 contains the Renilla luciferase gene downstream of the SV40 enhancer-promoter and is used as a measure of transfection efficiency to normalize the firefly luciferase values.


Figure 7. SYT-SSX binds to the N-terminal half of cBRM. Labelled SYT-SSX and controls luciferase and TAF55 were incubated with GST or GST fused to cBRM or to three fragments of cBRM (A, B and C). Bound proteins were analysed by SDS-PAGE and phosphoimaging. Lanes 1-3, labelled input proteins. Proteins bound to GST (lanes 4-6), GST-BRM-A fragment (lanes 7-9), GST-BRM-B fragment (lanes 10-12), GST-BRM-C fragment (lanes 13-15) and full-length GST-BRM (lanes 16-18).

GAL4-SSX constructs were assayed for repression as described previously (5) using luciferase reporters driven by the TK promoter with and without 5×UAS.

Confocal microscopy was carried out as follows. SW480 cells were transfected with GFP fusion or HA-tagged BRM expression constructs using lipofectamine as described previously (5). The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min. After a PBS wash, the cells were permeabilized for 10 min in 0.5% Triton X-100 in PBS followed by a PBS wash. HA-BRM was detected with anti-HA monoclonal antibody diluted 1:40 or 1:200 in Leibovitz L15 medium containing 5% fetal calf serum (L15/FCS). After PBS washes (3 × 5 min), bound antibody was detected using anti-mouse IgG conjugated to Alexa568 (Molecular Probes, Cambridge Bioscience, Cambridge, UK) diluted 1:200 in L15/FCS. Coverslips were mounted after washing in Vectashield (Vector Laboratories, Peterborough, UK) and sealed with varnish. Slides were imaged with a Leica TCS SP confocal microscope with Ar and Kr lasers. GFP-labelled proteins and HA-BRM (Alexa 568) were imaged concurrently. No image processing was used. Controls were carried out to ensure there was no bleed-through between green (GFP) and red (Alexa568) signals. In addition, cells transfected with GFP-labelled constructs alone were labelled using the HA antibody and Alexa 568 to check that the HA antibody did not detect transfected GFP-SYT.

Protein binding experiments were carried out by incubating GST, GST-cBRM (20) or GST-TFE3-N-terminal activation domain immobilized on glutathione beads, with in vitro [35S]methionine-labelled SYT, SYT-SSX, TAF55 or luciferase in 100 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM MgCl2, 0.1% Triton X-100, 0.1% bovine serum albumin (BSA) for 1 h, washed four times with the same buffer without BSA and bound proteins eluted with SDS sample solvent. The eluted proteins were analysed by electrophoresis and detected using a phosphoimager.

ACKNOWLEDGEMENTS

We would like to thank M. Yaniv and C. Muchardt for supplying us with the vector expressing HA-tagged BRM and the anti-BRM antibodies. This work was supported by the Cancer Research Campaign

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*To whom correspondence should be addressed. Tel: +44 181 643 8901; Fax: +44 181 770 7290; Email: goodwin@icr.ac.uk
+These authors contributed equally to this work

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