Oncogene (1999) 18, 2739 ± 2746 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc SSX and the synovial-sarcoma-speci®c chimaeric protein SYT-SSX co-localize with the human Polycomb group complex

Marielle Soulez1, Andrew J Saurin2, Paul S Freemont*,2 and Jennifer C Knight*,1

1Department of Cancer Medicine and Imperial Cancer Research Fund Laboratories, Imperial College of Science, Technology and Medicine, Hammersmith campus, Du Cane Road, London W12 0NN, UK; 2Molecular Structure and Function Laboratory, Imperial Cancer Research Fund Laboratories, PO Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

Chromosome translocation t(X;18)(p11.2;q11.2) is unique tumours arise in mesenchymal tissue but display the to synovial sarcomas and results in an `in frame' fusion features of epithelial di€erentiation including keratins, of the SYT gene with the SSX1 or closely-related SSX2 basal lamina formation, and glandular architecture gene. Wild-type SYT and SSX proteins, and the SYT- (Fisher, 1986). Synovial sarcoma is most prevalent in SSX chimaeric proteins, can modulate transcription in the 15 ± 40 year age group, and a€ects both males and gene reporter assays. To help elucidate the role of these females (Enzinger and Weiss, 1995). The t(X;18) proteins in cell function and neoplasia we have performed translocation results in the fusion `in frame' of 3' immunolabelling experiments to determine their sub- sequences of the SYT (SYnovial sarcoma Transloca- cellular localization in three cell types. Transient tion) gene on chromosome 18, to 5' sequences of either expression of epitope-tagged proteins produced distinc- of two closely-related genes SSX1 or SSX2 (Synovial tive nuclear staining patterns. The punctate staining of Sarcoma X chromosome breakpoint) in chromosome SYT and SYT-SSX proteins showed some similarities. band Xp11.2 (Clark et al., 1994; Crew et al., 1995; de We immunolabelled a series of endogenous nuclear Leeuw et al., 1995) (Figure 1). The fusion gene is antigens and excluded the SYT and SYT-SSX focal transcribed and translated as a chimaeric SYT-SSX1 or staining from association with these domains (e.g. sites SYT-SSX2 protein, and it is these novel proteins that of active transcription, snRNPs). In further experiments are considered to underly the pathogenesis of synovial we immunolabelled the Polycomb group (PcG) proteins sarcoma. The SSX and SYT genes were unknown until RING1 or BMI-1 and showed that SSX and SYT-SSX identi®ed as fusion partners in t(X;18), and their proteins, but not SYT, co-localized with these markers. functions have yet to be elucidated. Consistent with this we show that SSX and SYT-SSX Transcripts of wild-type SYT are expressed in a wide associate with chromatin, and also associate with range of human tissues and cell lines including those condensed chromatin at metaphase. Noteably, SSX derived from synovial sarcoma (Clark et al., 1994; J produced a dense signal over the surface of metaphase Knight, unpublished observations). SYT is ubiqui- chromosomes whereas SYT-SSX produced discrete focal tously expressed in early mouse embryogenesis, and staining. Our data indicate that SSX and SYT-SSX in later stages expression becomes con®ned to cartilage proteins are recruited to nuclear domains occupied by tissues, speci®c neuronal cells and some epithelial- PcG complexes, and this provides us with a new insight derived tissues (de Bruijn et al., 1996). The SYT into the possible function of wild-type SSX and the protein shows few primary structural features. It mechanism by which the aberrant SYT-SSX protein comprises 387 amino acids rich in glutamine (19%), might disrupt fundamental mechanisms controlling cell proline (16%), and glycine (14%) residues, and division and cell fate. contains several putative SH2 and SH3 binding domains (Clark et al., 1994). There is no recognisable Keywords: chromosome translocation; SSX; SYT; DNA binding domain, although recently Brett and co- polycomb; chromatin; nucleus workers showed that SYT is able to transactivate transcription when targeted to a reporter gene (Brett et al., 1997). However, the functional signi®cance of this ®nding is unclear. The chromosome translocation t(X;18)(p11.2;q11.2) is The identi®cation of SSX1 and SSX2 has led to the a unique cytogenetic marker for the diagnosis of cloning of a further three family members (Gure et al., synovial sarcoma. It is observed in approximately 1997) with very high sequence similarity and unknown 75 ± 80% of these tumours (Sandberg and Bridge, function. SSX1 and SSX2 transcripts are abundant in 1994). The name is a misnomer for these tumours do human adult testis, expressed at low levels in the not originate from synovial membrane, although they thyroid, and not detectable in other normal tissues do predominently occur in close association with joint (Crew et al., 1995), although SSX expression during structures in the limbs. t(X;18) positive synovial embryogenesis has not been documented. SSX1 and sarcomas have also been described in the tongue, SSX2 encode closely-related proteins of 188 amino larynx, heart, jaw, pleura, and abdominal wall. The acids sharing 81% identity. SSX proteins contain no recognisable DNA binding sequence, but they can act as transcriptional repressors when targeted to a reporter gene (Brett et al., 1997; Lim et al., 1998). A *Correspondence: PS Freemont and JC Knight Received 12 August 1998; revised 14 December 1998; accepted 14 KruÈ ppel-associated box (KRAB) domain is present at December 1998 the amino-terminus of SSX proteins (Crew et al., SYT-SSX protein co-localize with Polycomb bodies MSoulezet al 2740

Figure 1 Schematic diagram of epitope-tagged proteins. The predicted protein product of the SYT gene is 387 residues in length and has no recognisable sequence motifs. Putative Src-homology domains (SH2/SH3) are shown. SSX genes encode small proteins of 188 residues characterized by an amino-terminal KRAB domain and a carboxy-terminal transcription repression domain (SSXRD). In t(X;18) the point of fusion between SYT and SSX1 or SSX2 is identical. The translated protein comprises (aa 1 ± 379) SYT and (aa 111 ± 188) SSX (Crew et al., 1994). Transcripts of the reciprocal fusion gene are not detected (Clark et al., 1994). To facilitate immunolabelling, proteins were expressed `in frame' with an amino-terminal haemagglutanin (HA) epitope (black shading)

1995). Unlike the typical KRAB domains found in approach of studying exogenously expressed proteins. KruÈ ppel-type zinc ®nger proteins, the KRAB domain Whilst this work was in progress similar approaches of SSX proteins does not potentiate a potent towards de®ning the sub-cellular localization of these transcriptional repression e€ect and does not interact proteins were undertaken by Brett et al. (1997) and dos with the KRAB co-repressor TIF1b (Lim et al., 1998). Santos et al. (1997). However, a strong transcriptional repression domain, In addition to the COS7 cell line, we analysed termed the SSXRD (SSX Repression Domain), is protein expression in a synovial sarcoma derived cell present at the carboxy-terminus of SSX proteins (Lim line CME-1 (Renwick et al., 1995) and in a clonal et al., 1998). derivative of the human ®brosarcoma cell line HT1080 In the majority of tumours the fusion point between designated 2C4 (Saurin et al., 1998). HA-tagged SYT, the SYT and SSX genes is consistent with the SSX1, SSX2, SYT-SSX1 and SYT-SSX2 each localized production of a chimaeric protein in which the C- to the nuclei of these cells, producing distinctive terminal eight amino acids of SYT are replaced by the staining patterns (data for CME-1 and 2C4 are shown seventy-eight C-terminal amino acids of SSX1 or SSX2 in Figure 2). The protein distributions we describe were (Crew et al., 1995). The chimaeric protein retains those consistently observed in experiments repeated three sequences of SYT shown to possess transactivation times and in which a minimum of 30 nuclei of each cell potential (Brett et al., 1997) and the SSXRD line were scored. SYT accumulated in the nucleus as transcription repression domain of SSX, but lacks the discrete toroidal-shaped foci. The number of foci SSX KRAB domain (Figure 1). To further understand varied, but averaged six per transfected cell. The the normal function of wild-type SYT and SSX nuclear distribution of SYT has previously been proteins and the mechanism by which aberrant SYT- described as `speckled' (Brett et al., 1997) and SSX protein could disrupt cell function and lead to `punctate' (dos Santos et al., 1997). We could not neoplasia, we have studied the sub-cellular localization make any distinction between the nuclear distributions of each protein in immunolabelling experiments. Here of HA-SSX1 and HA-SSX2 proteins (Figure 2). We we report our observations and conclusions. observed non-uniform microparticulate and di€use We performed immunolabelling experiments on staining throughout the nucleus but excluded from proteins expressed exogenously by transfection of the nucleoli. In addition we observed patches of DNA into mammalian cells. We constructed mamma- stronger signal that were particularly apparent in 2C4 lian expression vectors containing the appropriate full- cells as two prominent foci (Figure 2). The distribution length cDNA sequences (con®rmed by sequencing) of HA-SYT-SSX1 and HA-SYT-SSX2 in the nucleus fused to an amino-terminal haemagglutanin (HA) produced a distinctive punctate staining. A proportion epitope tag to facilitate immunodetection (Figure 1). of the protein accumulated as toroidal-like structures, Following transient transfection into cells, protein reminiscent of the pattern of HA-SYT, but SYT-SSX expression and integrity was con®rmed by immuno- foci were abundant particularly in the nuclei of COS7 blotting with an anti-HA monoclonal antibody (mAb; and CME-1 cells (Figure 2). In 2C4 nuclei, we Boehringer) (data not shown). The sub-cellular observed two prominent foci (Figure 2) and failed to localization was determined on paraformaldehyde- see any similar signal with HA-SYT. ®xed and permeabilized cells by indirect immunofluor- The nuclear distribution of transiently expressed escence labelling with anti-HA mAb (see legend to GFP, VSV, FLAG tagged or untagged synovial Figure 2) that was then localized by imaging 1 mm sarcoma proteins has been described recently by Brett thick sections. Our rabbit polyclonal antisera to et al. (1997) and dos Santos et al. (1997), their data endogenous SYT and SSX failed to show sucient indicating that sub-cellular localization is consistent in speci®city in these experiments thus necessitating our the presence or absence of a peptide tag. SYT and SYT-SSX protein co-localize with Polycomb bodies MSoulezet al 2741

Figure 2 Nuclear localization of the SYT, SSX and SYT-SSX proteins. Expression constructs for HA-SYT, HA-SSX, and HA- SYT-SSX were transiently expressed in 2C4 and CME-1 cells by transfection using the calcium phosphate method. Cells were harvested and ®xed for 10 min (4% (w/v) paraformaldehyde in bu€er A: 10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3mM MgCl2,2mM EDTA) and permeabilized (0.5% Triton X100 in bu€er A, 4 min incubation) after 16 h (2C4) or 36 h (COS7 and CME-1). The proteins were then immunolabelled using antibodies to the HA tag (Boehringer mouse mAb 12CA5 or Autogen Bioclear UK mAb AB101). Non-speci®c staining was reduced by diluting antibodies in 5% calf serum/0.2% ®sh gelatin, the same mix being used for washing away unbound antibody. Primary antibody was incubated for 1 h at room temperature, secondary antibody for 45 min. Images were captured with a BioRad MRC 1000 inverted confocal microscope. The images shown of CME-1 cells represent a projection of multiple confocal optical sections; the images of 2C4 cells represent a single confocal optical section 1 mm thick. Scale bars represent 10 mm in each panel

SYT-SSX proteins showed a nuclear speckled appear- produced a di€use nuclear distribution in NIH3T3 ance in all cell lines transfected including NIH3T3, cells and COS1 cells. 2C4 cells (and the parent cell line COS1, and HT1080, and similarities between the HT1080) have not been used previously in the analysis distribution of the wild-type and chimaeric proteins of SSX distribution. dos Santos and co-workers (1997) were emphasised. FLAG- or GFP-tagged SSX showed that endogenous SYT-SSX1 protein in synovial SYT-SSX protein co-localize with Polycomb bodies MSoulezet al 2742 sarcomas and SYT-SSX1 expressed exogenously in Polycomb group proteins form multimeric com- transfected cells, have a similar punctate nuclear plexes (PcG bodies) which can be associated with distribution. Both groups (Brett et al., 1997; dos pericentromeric chromatin (Saurin et al., 1998). Two Santos et al., 1997) suggested that SYT-SSX and components of mammalian PcG complexes are RING1 SYT produce signals that overlap, but did not provide and BMI-1 which interact in vivo (Satijn et al., 1997), the data to underscore this assumption and failed to although the total number of PcG gene products is at identify any known nuclear structure or domain with present unknown. Immuno¯uorescence labelling of which the signals correspond. endogenous RING1 has previously revealed that 2C4 To de®ne the nuclear compartment(s) with which cells have prominent PcG bodies highly concentrated SYT, SSX and SYT-SSX proteins associate, we into two foci per nucleus (Saurin et al., 1998). These performed double immuno¯uorescence labelling using PcG bodies do not co-localize with any of the nuclear antibodies against known nuclear antigens to label antigens screened earlier in this or previous studies endogenous proteins and anti-HA antibodies to label (Saurin et al., 1998). By labelling RING1 as a marker exogenously expressed HA-tagged protein in 2C4 cells. for PcG bodies we have found that the signal for SYT- We have recently carried out a rigorous analysis of the SSX2 overlaps with the signal for RING1 in all cells distribution of nuclear antigens/nuclear structures in examined (Figure 3c and inset; yellow signal). We have 2C4 cells (Saurin et al., 1998), thus making this an also demonstrated that SYT-SSX1 and SYT-SSX2 co- appropriate system to use for the present study. All of localize with endogenous BMI-1 (Figure 4d and e). In the antibodies used have been characterized and addition, partial ¯uorescence overlap was observed validated previously (Saurin et al., 1998; see legend to between SYT-SSX2 and centromere-associated protein Figure 3 for references). structures called kinetochores (labelled with antibody We found no co-localization or association between H33; Figure 3d and inset; yellow signal). This is SYT-SSX2 and PML nuclear bodies, SC-35 `inter- consistent with the recent ®nding that PcG bodies can chromatin granules', small nuclear riboproteins be juxtaposed with kinetochores bound to peri- (snRNPs), or p80 coilin and coiled bodies. Similarly centromeric heterochromatin (Saurin et al., 1998; we could not demonstrate any association between Wang et al., 1997). Thus in interphase 2C4 cells the SYT and PML bodies or snRNPs. These data (not SYT-SSX2 protein accumulates preferentially with PcG shown) con®rm conclusions drawn from the analysis of proteins in the vicinity of the centromere. We repeated other cell lines (Brett et al., 1997; dos Santos et al., these immunolabelling experiments with HA-tagged 1997). In addition to these data our results indicate SSX1, SSX2, and SYT transfected into 2C4 cells. There that SYT-SSX2 does not co-localize with polypyrimi- was no association between SYT and BMI-1, as can be dine tract binding protein (PTB) (Figure 3a), and that seen by the absence of yellow signal in Figure 4a. neither SYT-SSX2, SYT, nor SSX proteins accumulate However, the results with SSX proteins were striking. at sites of active transcription (Figure 3b and data not In addition to the microparticulate staining pattern shown). throughout the nucleus we observed that the two prominent SSX1 or SSX2 signals that we had noted previously co-localized with endogenous BMI-1, as seen by the yellow signals in Figure 4b and c. These data suggest that the SSX portion of the chimaeric protein is the determinant for targetting the protein within the nucleus, and that SYT-SSX is recruited to the nuclear compartment occupied by PcG complexes. Our results di€er from the conclusions of two previous studies which suggested that the SYT portion of the chimaeric protein recruits it to so-called SYT bodies (Brett et al., 1997; dos Santos et al., 1997). These conclusions were based on some similarities in the immunostaining patterns of SYT and SYT-SSX proteins, but these studies did not present co- localization data for SYT and SYT-SSX. Our data supports the opposite model, whereby SSX is dominant in terms of targetting the sub-cellular localization of the SYT-SSX protein. We are able to form such a Figure 3 Localization of SYT-SSX2 relative to known nuclear conclusion because we can study the co-localization of structures and domains. HA-SYT-SSX2 was transiently expressed in 2C4 cells and its distribution compared with that of each individual SYT, SSX, or SYT-SSX protein with a endogenous nuclear antigens. SYT-SSX2 was immunolocalized well-de®ned nuclear component, namely the PcG with a polyclonal antibody against HA (Y11; Autogen Bioclear complex. UK Ltd.) (Green channel, a, b and d) or a monoclonal antibody PcG bodies are known to form distinct nuclear foci, against HA (red channel, c) and compared with signals obtained the numbers of which di€er from cell type to cell type with anti-PTB (Huang et al., 1997; gift of D Spector), anti- RING1 (ASA3) (Saurin et al., 1998) and anti-H33 (kinetochore (Alkema et al., 1997a; Satijn et al., 1997; Saurin et al., antigen; Stuurman et al., 1992; gift of A Otte) antibodies. Sites of 1998). In 2C4 cells the PcG bodies are highly active RNA transcription were visualized using an anti-BrUTP prominent. In the synovial sarcoma cell line CME-1, antibody (Sigma) following in vivo incorporation of the nucleotide endogenous BMI-1 (marking the PcG complexes) analogue bromo-UTP into nascent RNA (protocol according to Wansink et al., 1994). All images comprise a digital overlay of appears as dense speckling throughout the nucleus two optical channels (red and green) obtained from a single (centre panels in Figure 5), thus making the interpreta- optical confocal section (1 mm thick) tion of images more dicult. However it can be seen SYT-SSX protein co-localize with Polycomb bodies MSoulezet al 2743 clear, since SSX2 was highly expressed throughout the nuclear volume of CME-1 cells (see also Figure 2, bottom right panel). However we could distinguish a signi®cant association with BMI-1 in regions where SSX2 was most concentrated (Figure 5f). The heterogeneity of distribution of PcG complexes between cell lines may explain why previous studies, using di€erent cell lines, have described SSX proteins as producing a di€use nuclear staining (Brett et al., 1997; dos Santos et al., 1997). In Drosophila large multimeric PcG complexes have a fundamental role as a stable repressor system that maintains the embryonic segmentation program of homeotic gene silencing, essentially ensuring the correct execution of developmental programmes (for reviews see Bienz and Muller, 1995; Pirrotta, 1998). Many PcG proteins lack DNA binding activity and the available evidence suggests they assemble via protein- protein interactions. These large protein complexes are believed to function epigenetically by modifying higher order chromatin structure (Paro, 1990). Recent ®ndings indicate strong evolutionary conservation of PcG- mediated repression, with several vertebrate homolo- gues now described (for reviews see Gould, 1997; Schumacher and Magnuson, 1997), but little is known about PcG function in vertebrates. Speci®c interactions have been de®ned between components of mammalian PcG complexes, including RING1 and BMI-1 (Alkema et al., 1997a; Gunster et al., 1997; Satijn et al., 1997; Schoorlemmer et al., 1997; Hashimoto et al., 1998). Although we have shown that SSX and SYT-SSX can be recruited to regions containing RING1 and BMI-1, we have no evidence for a direct interaction between SSX or SYT-SSX with these PcG components. Western blots using antibodies speci®c for RING1 and BMI-1 after co-immunopreci- pitation of tagged SSX or SYT-SSX gave negative results, suggesting that SSX and SYT-SSX do not interact directly with RING1 and/or BMI-1 (data not shown). This result is perhaps not surprising since the exact number of mammalian PcG proteins is unknown (in Drosophila the estimate is 30 ± 40 PcG genes) and SSX and SYT-SSX may interact with as yet unidenti®ed components of these complexes. Our co- immunoprecipitation experiments using radio-labelled cell extracts (preliminary data, not shown) showed that both SSX and SYT-SSX interact speci®cally with a 65 kDa protein, the identi®cation of which is currently being investigated. We have previously reported that in a number of Figure 4 Co-localization of HA-SSX and HA-SYT-SSX with the mammalian cell lines PcG bodies remain chromatin- human Polycomb group complex in 2C4 cells. Epitope-tagged associated throughout mitosis (Saurin et al., 1998). SYT, SSX and SYT-SSX were transiently expressed in 2C4 cells (see legend to Figure 2) and immunolabelled with antibody to the During mitosis chromatin undergoes condensation, HA tag and FITC-conjugated secondary antibody (green there is a general inhibition of transcriptional activity, channel). Polycomb group complexes were visualized (red and many sequence-speci®c transcription factors are channel) by labelling the endogenous PcG protein BMI-1 (anti- displaced (Martinez-Balba s et al., 1995). Given that BMI-1; Satijn et al., 1997; gift of A Otte). Each image comprises a SSX1, SSX2, and the SYT-SSX chimaeric proteins digital overlay of the two channels from a single optical confocal section 1 mm thick. Note the yellow signal where green and red appear to co-localize with PcG bodies, we wanted to signals overlap in (b, c, d), and (e) reinforce this ®nding by determining whether these proteins associate with chromatin in mitotic cells. To address this question we transiently expressed HA- tagged SYT, SSX1, SSX2, or SYT-SSX2 in COS7 cells, that exogenous SYT-SSX2 (Figure 5g) co-localizes with immunolabelled the cells with anti-HA and counter- endogenous BMI-1 (Figure 5i, yellow signal), whereas stained the chromatin with propidium iodide (Figure SYT (Figure 5a) does not co-localize with BMI-1 6). We looked at mitotic cells for evidence of (Figure 5c). The data for SSX2 (Figure 5d) was less association between the exogenous protein and the SYT-SSX protein co-localize with Polycomb bodies MSoulezet al 2744

Figure 5 Co-localization of HA-SSX and HA-SYT-SSX with the human Polycomb group complex in CME-1 cells. Epitope-tagged SYT, SSX2 and SYT-SSX2 were transiently expressed in the t(X;18) positive synovial sarcoma cell line CME-1 (see legend to Figure 2) and immunolabelled with antibody to the HA tag and Texas Red-conjugated secondary antibody. PcG complexes were visualized (green channel) by labelling the endogenous PcG protein BMI-1 (b, e, h). These images were obtained from a single optical confocal section 1 mm thick. The image in (c, f, and i) is a digital overlay of the two optical channels, indicating where the distribution of proteins overlaps (yellow signal)

condensed chromatin. There was no correlation responses and di€erentiation of progenitor cells in the between SYT and the distribution of chromatin mouse haematopoietic system, for example overexpres- (Figure 6c). However the microparticulate staining of sion of BMI-1 predisposes to the development of SSX1 and SSX2 was clearly distributed over the lymphomas (Core et al., 1997; Haupt et al., 1991; van chromatin at mitosis. At prometaphase/metaphase Lohuizen et al., 1991, van Lohuizen, 1998). To date SSX proteins produced dense staining on the chromo- there are no examples of human disease-associated somes, presumably accessing sites exposed over the chromosome translocations or other somatic mutations entire surface of the chromosomes (Figures 6f and i). that directly disrupt a PcG gene. SSX genes do not This highlighted a di€erence in the characteristics of share any sequence homology with known PcG genes, wild-type SSX and the chimaeric SYT-SSX. At but SSX proteins and known PcG proteins all share a prometaphase/metaphase SYT-SSX2 signal also corre- common functional phenotype whereby they have the lated with areas of chromatin, but it appeared as ability to repress transcription when targeted to a discrete foci (Figure 6j and l) similar to the pattern we reporter gene, and lack speci®c DNA binding domains have observed previously for RING1 and BMI-1 (Alkema et al., 1997b; Bunker and Kingston, 1994; (Saurin et al., 1998). These observations support the Satijn et al., 1997; Schoorlemmer et al., 1997; Lim et contention that SSX and SYT-SSX proteins are al., 1998). Clearly the identi®cation of proteins recruited to the vicinity of PcG complexes, and argue interacting with SSX and SYT-SSX proteins is a against the possibility that SYT-SSX is recruited to the priority. location of wild-type SYT in the nucleus, since wild- In this report we have provided evidence that the type SYT is clearly not a chromatin-associated protein. chimaeric protein SYT-SSX generated by the t(X;18) The biological signi®cance of the association of SSX translocation can be recruited to sites on chromatin, and SYT-SSX proteins with the PcG complex is at that it can interact with available sites on condensed present unclear, but it would be plausible to propose chromatin at mitosis, and can occupy sites in the that there is a connection with the pathogenesis of vicinity of PcG complexes near centromeres in synovial sarcoma. Many aspects of the function of interphase nuclei. These characteristics seem to be a mammalian PcG proteins are still unknown, but there function of SSX rather than SYT. The interaction of is increasing evidence that they play a role in regulating SYT-SSX with condensed chromatin at mitosis di€ers cell cycle progression and di€erentiation, beyond their in at least one respect from wild-type SSX as revealed role in regulating homeotic boundaries (Schumacher by their di€erent staining patterns on metaphase and Magnuson, 1997; van Lohuizen et al., 1998). chromosomes. This may have functional signi®cance. Transgenic expression and gene knockout approaches In addition the chimaeric protein may have acquired have implicated certain PcG genes in proliferative new functions from SYT which is inappropriately SYT-SSX protein co-localize with Polycomb bodies MSoulezet al 2745

Figure 6 HA-SSX and HA-SYT-SSX proteins associate with mitotic chromosomes. Epitope-tagged SYT, SSX1, SSX2 and SYT- SSX2 were transiently expressed in COS7 cells (see legend to Figure 2) and immunolabelled with anti-HA antibody and FITC- conjugated secondary antibody (green channel). Propidium iodide (0.1 mg/ml) was added during the secondary antibody incubation to counterstain the chromosomes (visualized with rhodamine ®lters, shown in middle panels). The images represent a single optical confocal section 1 mm thick. A digital overlay of the two optical channels is shown in the right hand panels, producing a yellow signal where red and green signals overlap

expressed in the vicinity of PcG complexes and Acknowledgements chromatin. Furthermore, the fusion between the SYT We are particularly indebted to AP Otte for the generous and SSX genes puts the expression of the chimaeric gift of the BMI-1 antibody and to our other colleagues protein under the control of the SYT promoter such who kindly provided antibodies for use in these studies. We that the expression of SSX itself is inappropriately thank P Jordan for technical support in the use of the confocal microscope. J Knight gratefully acknowledges the regulated within the cell. We have provided a new funding support of the British Medical Research Council insight into the possible role that SSX genes play in cell for this project. M Soulez was supported by a grant from function that should contribute to an understanding of Imperial College of Science Technology and Medicine. PcG function in vertebrates and to understanding the Facilities and support for the work were also provided by molecular basis of synovial sarcoma. the Imperial Cancer Research Fund.

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