delineation of the protein domains responsible for syt, ssx, and syt-ssx nuclear localization

Experimental Cell Research 256, 192–202 (2000)doi:10.1006/excr.2000.4813, available online at http://www.idealibrary.com on

Delineation of the Protein Domains Responsible for SYT, SSX,and SYT-SSX Nuclear Localization

Nuno R. dos Santos,*,1 Diederik R. H. de Bruijn,* Ellen Kater-Baats,* Arie P. Otte,†and Ad Geurts van Kessel*

*Department of Human Genetics, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands; and
†E. C. Slater Instituut, BioCentrum Amsterdam, University of Amsterdam, 1018 TV Amsterdam, The Netherlands
In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2.The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized inthe nucleus. Recent studies in experimental systemsindicated that the SYT protein may function as a tran-scriptional activator whereas the SSX proteins mayact as transcriptional repressors. In the present workwe created a series of deletion mutants and found thatSYT and SSX depend on N-terminal and highly con-served C-terminal domains for nuclear localization,respectively. Our results also show that the SYT-SSXproteins colocalize with SSX2, a feature that dependson the presence of the C-terminal SSX sequences in thechimeric proteins. Absence of these sequences led toan altered subcellular localization, coinciding withthat of SYT. Besides, we found that endogenously ex-pressed SSX proteins colocalize with polycomb-groupproteins and condensed chromosomes during mitosis,features that are also conferred by the C-terminus ofSSX. Taken together, these results led us to concludethat the SSX moiety, especially the most C-terminal 34amino acids, of the SYT-SSX fusion proteins is crucialfor aberrant spatial targeting and transcriptional con-trol within the nucleus. © 2000 Academic Press

Key Words: SYT; SSX; SYT-SSX; synovial sarcoma;nuclear localization; polycomb-group proteins.

INTRODUCTION

Recurrent chromosomal translocations are frequentphenomena in leukemias and lymphomas [1]. Cytoge-netic studies on solid tumors demonstrated that theseaberrations are also frequent in sarcomas and, to alesser extent, in carcinomas [2]. Recently, several chro-mosomal breakpoints have been molecularly charac-terized, not only in various soft tissue sarcomas [3], but

1 To whom reprint requests should be addressed at Department ofHuman Genetics, P.O. Box 9101, 6500 HB Nijmegen, The Nether-
lands. Fax: 131-24-3540488.
1920014-4827/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

also in at least one subtype of renal cell carcinoma [4].Consistently, these endeavors have shown that nearlyall solid tumor-associated chromosomal translocations,in parallel with several leukemias, result in gene fu-sions. These fused genes, in turn, encode chimeric on-coproteins.

Synovial sarcomas are no exception to this rule. Arecurrent and very frequent chromosomal transloca-tion has been identified in these tumors, t(X;18)(p11.2;q11.2) [5]. The cloning of the respective breakpointsrevealed a fusion between the SYT gene on chromo-some 18 and an SSX gene on the X chromosome [6].Two mutually exclusive breakpoints on Xp11.2 may beencountered in t(X;18) translocations [7], resulting intwo different gene fusions, SYT-SSX1 and SYT-SSX2[8, 9]. SYT-SSX fusions are frequent, even occurring insynovial sarcomas that do not show t(X;18) transloca-tions at the cytogenetic level [10]. Three other highlyhomologous SSX genes, SSX3, SSX4, and SSX5, havebeen identified [11, 12]. So far, only in a single case hasSSX4 been found to be involved in a fusion with SYT[13]. Furthermore, ectopic expression of SSX4 andSSX5, as well as SSX1 and SSX2, has been found inseveral malignancies, most frequently in melanomas[12, 14]. The pathological implications of the alterna-tive SYT-SSX fusions for synovial sarcoma develop-ment are still unclear, yet a putative correlation hasbeen found with the histological classification; i.e., al-most all biphasic synovial sarcomas studied carriedSYT-SSX1 gene fusions whereas SYT-SSX2-positivetumors were consistently found to be monophasic innature [7, 15]. Furthermore, Kawai et al. [15] found intheir series of patients that, in comparison with SYT-SSX1, the occurrence of SYT-SSX2 fusion was associ-ated with a statistically significant better metastasis-free survival.

A common theme that has emerged from the studyof many chimeric oncoproteins is that they act asaberrant transcription factors. Typically, an activa-tor or repressor domain from one protein is juxta-posed to the DNA-binding domain of another protein,
thereby creating a transcription factor which dereg-

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193NUCLEAR LOCALIZATION DOMAINS OF SYT AND SSX

ulates the activity of genes normally controlled bythe latter protein [3]. Accordingly many chimericoncoproteins, as well as their normal counterparts,are localized in the nucleus. It has also become ap-parent that usually their nuclear distribution is notuniform. Recent studies have unveiled that the nu-cleus is a complex and compartmentalized organellewhere structure and function are intimately related[16, 17]. Several proteins, including several cancer-related proteins, were found to reside in so-callednuclear bodies, protein structures usually visualizedas dots by immunofluorescence microscopy [17]. Af-ter raising polyclonal antibodies against the SYTand SSX proteins, we found that these proteins, aswell as their chimeric derivatives, are also localizedin the nucleus [19]. In addition, these studies showedthat the SYT and SYT-SSX proteins are mainlypresent in nuclear dots whereas SSX is diffuselydistributed throughout the nucleus [19, 20]. All pro-teins showed nucleolar exclusion. No colocalizationwas found between SYT or SYT-SSX and other nu-clear bodies such as the PML oncogenic domains(PODs) and coiled bodies [19, 20]. Regarding thefunction of the synovial sarcoma-related proteins,only a few clues have been gathered up till now.Studies in mice have shown that Syt is expressed

idely throughout development, yet more promi-ently in cartilaginous, neuronal, and some epithe-

ial tissues at later stages of embryogenesis [21]. Indult life Syt is ubiquitously expressed, but some-hat more abundantly in heart, kidney, and testis

21]. Human SYT is also ubiquitously expressed [6].n contrast, SSX expression is confined to the adult

testis and thyroid, in the latter at low levels [9, 23].In keeping with its nuclear localization, it has beenfound that SYT has transcriptional activating capac-ity [20, 23]. In contrast, the SSX proteins show tran-scriptional repressing activity mediated by the C-terminally localized SSXRD domain [24]. However,since no DNA-binding domain has been identified ineither of these proteins, the transcriptional regula-tory functions are presumably exerted through inter-actions with other proteins. Recently, the hBRM pro-tein, a component of the transcriptional coactivatorSNF complex [25], was found to colocalize and inter-act in vitro with the SYT and SYT-SSX proteins [23].These findings suggest that SYT may coactivatetranscription by recruiting SNF protein complexes toDNA-binding transcription factors.

In this report we set out to identify and characterizethe domains of SYT and SSX that are responsible fortheir nuclear targeting, and to relate the (aberrant)SYT-SSX subnuclear distribution to that of SYT and
SSX.
MATERIALS AND METHODS

Cloning of deletion mutants and site-directed mutagenesis. cDNAdeletion mutants were constructed using adequate restriction endo-nucleases or polymerase chain reaction (PCR) amplification andin-frame cloning in eukaryotic expression vectors containing epitopetags, either pSG8-VSV (a gift from Dr. Edwin Cuppen) or pCATCH[27], or the green fluorescent protein, pEGFP (Clontech). Site-di-rected mutagenesis of SSX2 was performed using a two-step PCRprotocol [28]. Briefly, in the first step, specific mutations were intro-duced by a mutagenic primer (SSX2mut1, 59-GGACCCAT-AGGGGGGGAGCATGCCTGGACC-39; or SSX2mut2, 59-ACCCA-

ATACTGCGTGATCAAAATCAGCTGGTG-39) and a flankingrimer (T7) on PfuI polymerase PCR, using as template a pT7T3

plasmid (Pharmacia) containing the SSX2 cDNA [19]. The resultantPCR product was then used as a primer (“megaprimer”) along withan opposite end flanking primer (T3) for a second PCR step. The finalPCR products were digested with DpnI to remove methylated tem-plate plasmid DNA and digested with appropriate restriction en-zymes prior to subcloning in pEGFP. All clones were sequenced inboth orientations using vector-specific oligonucletides and the ABIPRISM Dye Terminator Cycle Ready Reaction kit (Perkin–Elmer)and an automated sequencer (ABI 373A, Applied Biosystems).

Cell culture and transfections. COS-1, HeLa, and 518A2 cellswere grown in DMEM medium containing 10% fetal calf serum.COS-1 and HeLa cells were transiently transfected by either elec-troporation in 0.4-cm cuvettes, using a Bio-Rad Gene Pulser appa-ratus, or DOSPER liposomal reagent according to manufacturer’sinstructions (Boeringher Mannheim).

Indirect immunofluorescence analysis. Immunofluorescenceanalyses were carried out as previously described [19]. As primaryantibodies we used the anti-SYT polyclonal antibodies C44 andRA2009 [19], anti-SSX polyclonal antibody B39 [19], and the E3ASanti-SSX monoclonal antibody [29]. Tagged proteins were detectedwith anti-VSV (P5D4) and anti-FLAG (M5; Sigma) antibodies. Forcoimmunolabeling of other nuclear proteins we used antibodiesagainst PML (5E10 [30]), NDP55 (mAb138 [31]), SC35 [32], hRad51[33], WT1 (WTc8 [34]), lamin B2 (X223 [35]), NuMA (TransductionLaboratories), RING1, BMI1, and HPC2 [36]. As secondary antibod-ies, FITC-conjugated swine anti-rabbit, rabbit anti-mouse IgG(Dako), and Texas red-conjugated goat anti-mouse IgG and IgM(Jackson Immunoresearch) were used. Fixed cells were counter-stained with DAPI (Serva). Slides were visualized under a ZeissAxiophot epifluorescence microscope equipped with appropriate fil-ters, and digital images were recorded using a Photometrics high-performance cooled CCD camera interfaced onto a Macintosh com-puter. The images were displayed in RGB pseudocolors using theimage analysis and processing software program BDS-image (Bio-logical Detection Systems).

RESULTS

The N-terminus of SYT determines its nuclear local-ization. We have previously found that SYT proteinsare localized in the nucleus, in a punctate patternexcluding nucleoli [19]. These results were obtainedboth in COS-1 and HeLa cells after short-term post-transfection incubations, using both protein taggingand anti-SYT polyclonal antibodies. Transfection ofother cell lines resulted in similar nuclear distributions(data not shown and [20]). Long-term posttransfectionincubations yielded cells with large, round, and hollowspeckles in both the nucleus and the cytoplasm (data
not shown).

f,h,

194 DOS SANTOS ET AL.

The SYT protein comprises 387 amino acids and hasa molecular weight of 53 kDa. An inspection of itsamino acid sequenced did not reveal any consensualnuclear localization signal (NLS). A few basic aminoacids are present at the N-terminus of SYT (aminoacids 9 to 14), but their amino acid substitution bysite-directed mutagenesis did not abolish nuclear local-ization (data not shown). To identify the domain(s)responsible for SYT nuclear targeting we created aseries of deletion mutants fused to epitope tags, eitherFLAG or VSV, or to the green fluorescent protein(GFP) (Fig. 1A). COS-1 or HeLa cells were transfectedwith these constructs and the corresponding proteinswere detected by indirect immunofluorescence micros-

FIG. 1. SYT nuclear localization is determined by an N-terminalan N-terminal tag, either FLAG, VSV, or EGFP, and their respectiveC, cytoplasm). C-terminal deletions up to amino acid 90 and N-termiof SYT. In contrast, larger N-terminal deletions resulted in nuclearcells transfected with the SYT deletion mutants as indicated on the pthe corresponding anti-tag antibodies. SYTD1-157 and SYT(158-238)SYTD91-387, SYTD160-387, and SYTD208-387 were localized exclusautofluorescence, is localized in the nucleus (panels k,l). Panels b,d,

copy using anti-tag monoclonal antibodies or by GFP

autofluorescence. We also used two polyclonal antibod-ies that recognize distinct segments of SYT: C44,raised against the N-terminus, and RA2009, raisedagainst a C-terminal peptide [19]. These antibodiesenabled the differential detection of the various trun-cated proteins. As depicted in Fig. 1B, deletion ofamino acids 1 to 157 of SYT resulted in both nuclearand cytoplasmic localization. In contrast, sequentialC-terminal SYT deletions up to amino acid 91 did notaffect nuclear localization. Conversely, N-terminal de-letions up to amino acid 50 resulted in nuclear local-ization. From these results, we conclude that an N-terminal domain of SYT (amino acids 51 to 90) isresponsible for its nuclear localization, although it does

ain. (A) Scheme depicting SYT deletion mutant constructs fused tocellular localization in transfected HeLa or COS-1 cells (N, nucleus;deletions up to amino acid 50 did not affect the nuclear localizationcytoplasmic localization. (B) Immunofluorescence analysis of HeLa

els. Panels a,c,e,g,i: Tagged SYT deleted proteins were detected withre localized in both the nucleus and the cytoplasm (a–d). In contrast,ly in the nucleus (e–j). In addition, EGFP-SYTD1-50, as detected byj,l: DAPI nuclear counterstaining.

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not exhibit any consensual NLS.


SSX nuclear localization is mediated by a definedC-terminal domain. We and others have previously

FIG. 2. The C-terminus of SSX2 is necessary and sufficient for nufused to peptide tags, either FLAG, VSV or GFP, and their respectivboxes indicate putative bipartite NLSs as reported previously [19(B) PCR site-directed mutagenised SSX2 constructs showing amino a(C) Protein sequence alignment of the C-terminal domain of all SSDetection by indirect immunofluorescence or autofluorescence of COon the panels. The FLAG-SSX2D1-24 construct, which excludes theSSX2D155-188, which excludes the most C-terminal putative NLSconstruct was localized in the nucleus (e,f). Amino acid substitutaffect localization (g,h) whereas substitution of basic amino acids ofin both N-terminally deleted (GFP-SSX2D1-154mut2) and full-lencounterstaining.

reported that the SSX proteins are diffusely distrib-

uted in the nuclei, excluding nucleoli, of transfectedCOS-1 cells [19, 20]. However, further careful observa-

r localization. (A) Scheme depicting SSX deletion mutant constructsubcellular localization in transfected HeLa or COS-1 cells. Hatchedsterisks indicate location of introduced amino acid substitutions.substitutions (underlined) in two basic amino acid clusters (boxed).roteins so far identified. Boxes denote amino acid differences. (D)(a–d) or HeLa (e–l) cells transfected with the constructs indicated

erminal putative NLS, was localized in the nucleus (a,b). Constructas localized in the cytoplasm (c,d) whereas the GFP-SSX2D1-154of the N-terminal basic cluster (GFP-SSX2D1-154mut1) did not

C-terminal cluster resulted in nuclear and cytoplasmic localization,(GFP-SSX2mut2) protein (i–l). Panels b,d,f,h,j,l: DAPI nuclear

cleae s

]. AcidX pS-1N-t, wionthegth

tions revealed nuclear dots in addition to diffuse nu-


clear staining in a subset of transfected HeLa cells (see,e.g., Fig. 3, panel f). The size and number of dots foundper cell were variable, ranging from 2 to 50. Two mainstaining patterns could be discerned. About one-fourthof the cells exhibited few large dots above diffuse stain-ing whereas about three-fourths showed abundant finedots in addition to diffuse staining. Recently similarobservations have also been reported by Soulez et al.[37] in transfected synovial sarcoma and fibrosarcomacells. In addition, we found that the subnuclear distri-bution of both SSX3 and SSX4 in transfected HeLacells is indistinguishable from that of SSX1 and SSX2proteins, including punctated patterns in a low per-centage of cells (data not shown). Furthermore, using aspecific monoclonal antibody, we detected endogenousSSX expression in several melanoma cell lines andprimary melanoma lesions [29]. Again, the majority ofSSX-expressing cells showed a diffuse nuclear stainingpattern, whereas a small percentage displayed nucleardots, ranging in number from 1 to 20 (see, e.g., Fig. 5,panel m). No cells with abundant fine dots were ob-served, thus suggesting that such staining patternsmay be the result of exogenous SSX overexpression intransfected cells.

All currently known SSX proteins are composed of188 amino acids and exhibit molecular weights of 29kDa. Closer inspection of the SSX amino acid se-quences disclosed three regions with a high content inbasic amino acids that may function as bipartite NLS[19]. In order to verify whether any of these putativeNLSs are responsible for nuclear localization, we cre-ated a set of SSX2 deletion mutants fused to an epitopetag (Fig. 2A). Deletion of the most N-terminal putativeNLS had no effect on nuclear localization, thereforeindicating that the latter is not necessary for nucleartargeting (Fig. 2D, panel a). In contrast, deletion of the34 most C-terminal amino acids of SSX2, including theC-terminal putative NLS, led to a predominant cyto-plasmic staining of transfected cells (Fig. 2D, panel c).Supporting the notion that the 34 C-terminal aminoacids, the so-called SSXRD domain, are necessary andsufficient for nuclear targeting, we found that thisSSX2 segment was also able to translocate fused GFPto the nucleus (GFP-SSX2D1-154; Fig. 2D, panel e).Furthermore, we observed a punctate nuclear patternidentical to the one observed with full-length SSX2,suggesting that this segment is also responsible for thepunctated nuclear distribution.

To test whether the SSX C-terminal basic stretchfunctions as a bipartite or simple NLS [38], we intro-duced several amino acid substitutions in each of thetwo clusters of basic amino acids (Fig. 2B). Amino acidsubstitutions in the N-terminal basic cluster (mut1;K158I and R159G) had no effect on the localization ofGFP-SSX2D1-154 (Fig. 2D, panel g). Conversely, non-
conservative amino acid substitutions in the larger
C-terminal basic cluster (mut2; R167I, R171Q andK172N) resulted in homogeneous cellular staining en-compassing both the nucleus and the cytoplasm (Fig.2D, panel i). Similar localization was observed using afull-length SSX2 protein fused to GFP and carrying the“mut2” mutations (Fig. 2D, panel k), thus indicatingthat the substituted amino acids are necessary for ef-ficient nuclear targeting. However, the fact that GFP-SSX2mut2, a protein of predicted 50 kDa, was alsofound in the nucleus suggests that the SSXRD domaindoes not contain a typical NLS and that other (nonba-sic) amino acids may be involved in nuclear localiza-tion. The C-terminal region of SSX2 is highly con-served between the five SSX members (Fig. 2C),indicating that all SSX proteins may rely on theSSXRD domain for nuclear localization and punctateddistribution.

The chimeric SYT-SSX proteins colocalize with SSX2in nuclear dots. As previously reported, the SYT-SSXfusion proteins also display nuclear punctated patterns[19, 20]. The finding that both SYT and, to a lesserextent, SSX are distributed in nuclear dots promptedus to investigate whether any of these wild-type pro-teins colocalize with the SYT-SSX fusion proteins. Tothis end, COS-1 cells were cotransfected with VSV-SYTand SYT-SSX2 constructs and the respective proteinswere detected using anti-VSV and anti-SSX2 antibod-ies. Several cells expressing both proteins showed par-tial colocalization while others showed no colocaliza-tion at all (Fig. 3, panels a–d). Similarly, HeLa cellswere cotransfected with SSX2 and SYT-SSX1 or SYT-SSX2 constructs. The respective expressed proteinswere detected with an anti-SYT polyclonal antibodyand an anti-SSX monoclonal antibody that only recog-nizes the N-terminus of SSX and thus not the SYT-SSXproteins [29]. By doing so, we found that the SYT-SSXfusion proteins and SSX2 colocalize in nuclear dots.Two different patterns could be discerned. Approxi-mately 80% of the transfected cells showed fine andnumerous nuclear dots (data not shown) whereasabout 20% displayed less than 10 larger nuclear dots(Fig. 3, panels e–h). These results suggest that the SSXC-terminal domain prevails over SYT in determiningthe subnuclear localization of the chimeric SYT-SSXproteins. Supporting this notion, we found that dele-tion of the last 34 C-terminal amino acids of SYT-SSX2(SYT-SSX2D155-188), and, therefore, deletion of theSSXRD domain of SSX2 (see above), gave rise to largerdots located in both the nucleus and the cytoplasm(Fig. 3, panel i). Since this latter localization resemblesthat of SYT, we cotransfected the SYT-SSX2D155-188mutant with either VSV-SYT or SSX2 into HeLa cells.Interestingly, this C-terminally truncated SYT-SSX2protein completely colocalized with SYT in nuclear and
cytoplasmic dots (Fig. 3, panels j–l), and not with SSX2

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(data not shown). Taken together, these results indi-cate that the SSX C-terminus (SSXRD domain) plays apivotal role in determining the subnuclear localizationof the SYT-SSX chimeric oncoproteins.

The SSXRD domain mediates SSX protein associa-tion with mitotic chromosomes. It was recently ob-served that the SSX proteins are associated with chro-matin during mitosis [37]. We made similar observa-tions not only in SSX2-transfected HeLa cells (Fig. 4,panels a and b) but also in melanoma cells expressingendogenous SSX protein (Fig. 4, panels c and d). Fur-thermore, we found that GFP-SSX2D1-154 protein alsolabeled chromatin, indicating that this feature islinked to the most C-terminal 34 amino acids of SSX2(Fig. 4, panels e and f). Furthermore, like Soulez et al.37] we observed that during mitosis the SYT protein iselocalized to cytoplasmic dots, typically two (data nothown).The chimeric SYT-SSX and SSX proteins colocalize

n the nucleus with polycomb-group repressor proteins.reviously, we found that neither SYT nor SYT-SSXolocalized with PML, snRNP, or SC35 nuclear bodies19]. We have now extended these studies and foundhat several other nuclear proteins with a punctatedistribution, such as hRAD51 (involved in DNA re-air), NDP55 (a POD component), WT1 (a putativeumor-suppressor protein), PRCC (a putative tran-criptional coactivator), lamin B2 (a structural nuclearrotein), and NuMA (a nuclear matrix constituent), doot colocalize with either SYT, SSX, or SYT-SSX pro-eins (data not shown). Yet, further careful observationsevealed that a small percentage of transfected HeLaells showed colocalization between a few SYT-SSXuclear dots, typically 1 to 4, and PML dots (Fig. 5,anels a–d). In addition, preliminary data have indi-ated that the transcription factor TFE3 may colocalizeith SSX2 in nuclear dots in a subset of cells exog-nously coexpressing both proteins (data not shown).Double labeling of SSX2 and polycomb-group (PcG)

roteins (HPC2, RING1, or BMI1), however, revealedomplete colocalization in SSX2-transfected HeLa cellsFig. 5, panels e–h). In keeping with the above-men-ioned results, we also found that the PcG proteinsolocalized with the SYT-SSX1 and SYT-SSX2 proteinsFig. 5, panels i–l), although in some cells this colocal-zation was only partial (data not shown). Similar re-ults were recently obtained by Soulez et al. [37] inransfected fibrosarcoma 2C4 cells. Furthermore, welso found that the C-terminus of SSX2 (GFP-SSX2D1-54) colocalized with PcG dots in transfected cells (Fig., panels q–t), indicating that the SSXRD domain isot only responsible for nuclear targeting and chroma-in association but also localization in PcG domains. Toest whether the SSX and PcG proteins colocalize when
xpressed at endogenous levels, we immunostained
518A2 melanoma cells using anti-SSX monoclonal andanti-RING1 polyclonal antibodies. By doing so, wefound that indeed SSX colocalized with RING1 in nu-clear dots (Fig. 5, panels m–p). To evaluate if thiscolocalization reflected a physical interaction betweenthe SSX and RING1 proteins we performed coimmuno-precipitation assays. SSX protein was immunoprecipi-tated from 518A2 cell extracts using the anti-SSXmonoclonal antibody, yet no RING1 protein was de-tected in association with SSX after immunoblotting(data not shown). In addition, we found in yeast two-hybrid assays that SSX fused to the DNA-binding do-main of GAL4 does not interact with PcG proteins(RING1, BMI1, HPC2, HPH1, Enx, or EED) fused tothe transactivation domain of GAL4 [39, 40] (data notshown).

DISCUSSION

As a corollary of our previous observation that theSYT, SSX, and SYT-SSX proteins exhibit distinct butspecific nuclear localization patterns [19], we now setout to identify and characterize the protein domainsresponsible for these patterns. To this end we createdseveral deletion mutants of both SYT and SSX2 andevaluated their subcellular localization in transfectedcells. By doing so, we found that the localization of SYTin the nucleus depends on an N-terminal domain lim-ited to amino acids 51 to 90. Likewise, we found thatSSX2 nuclear localization relies on a 34 amino acid-long C-terminal domain, also known as SSXRD do-main, that is highly conserved in the SSX family ofproteins. Taken together, our results indicate that,besides transcriptional repression [24], the SSXRD do-main confers several properties to the SSX proteins.First, it is required for the SSX colocalization with PcGnuclear domains; second, it is required for the SYT-SSX colocalization with SSX and PcG nuclear domains;and finally, it mediates the association of SSX proteinswith chromatin.

To carry out their normal functions, proteins must belocalized in the appropriate subcellular compartments.Several examples of altered protein localization due toeither mutation or gene fusion are known to be associ-ated with human disease [18, 41–43]. Previously, wefound three basic amino acidic regions in SSX proteinswhich could function as nuclear localization signals[19]. After deletion mapping, we found that the C-terminal segment of SSX2 is sufficient for nuclear tar-geting. To assess whether a functional NLS waspresent in the SSXRD domain, we substituted its basicamino acids by site-directed mutagenesis. Substitutionof two N-terminally located basic amino acids (GFP-SSX2D1-154mut1) had no effect on nuclear localization(Fig. 2). In contrast, amino acid substitutions in theC-terminal basic amino acid cluster resulted in a
change in subcellular localization, from nuclear to both

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FIG. 3. SYT-SSX proteins colocalize with SSX2 but not with SYT. Panels a–d: COS-1 cell cotransfected with VSV-SYT and SYT-SSX2and immunolabeled with B39 anti-SSX (a) and P5D4 anti-VSV (b) antibodies showed no colocalization between these proteins (overlay, panelc). Panels e–h: HeLa cell transfected with SSX2 and SYT-SSX1 and immunolabeled with C44 anti-SYT (e) and monoclonal anti-SSX (f)antibodies showed complete colocalization between these proteins, as denoted by yellow color in overlay panel (g). Panels i–l: HeLa celltransfected with C-terminally truncated SYT-SSX2 (SYT-SSX2D155-188) and VSV-SYT and immunolabeled with B39 anti-SSX (i) and P5D4nti-VSV (j) antibodies showed complete colocalization between these proteins in nuclear and cytoplasmic dots (overlay, panel k). Panels,h,l: DAPI nuclear counterstaining.FIG. 4. SSX protein association with chromatin is mediated by a C-terminal domain. Panels a,b: SSX2-transfected HeLa cell in telophase

howing colocalization of SSX2, immunolabeled with B39 anti-SSX2 antibody (a), and DAPI-stained condensed chromosomes (b). Panels c,d:18A2 melanoma cell in telophase and immunolabeled with monoclonal anti-SSX antibody showing colocalization between SSX (c) andAPI-stained condensed chromatin (d). Panels e,f: Transfected HeLa cell in metaphase expressing GFP-SSX2D1-154 fusion protein (e)

howing colocalization between this protein and the DAPI-stained condensed chromatin (f).

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nuclear and cytoplasmic (Fig. 2), indicating that theseamino acids are essential for correct nuclear targeting.It was found that a hybrid protein composed of twoGFPs and weighing 54 kDa could diffuse through thenuclear pore [44], so the possibility remains that theGFP-SSX2mut2 proteins (with 50 kDa) may also dif-fuse to the nucleus regardless of the introduced muta-

FIG. 5. SSX protein colocalization with the PcG protein complex iHeLa cells were immunolabeled with C44 anti-SYT (a) and 5E10 antas denoted by yellow color in overlay panel (c). Panels e–h: SSX2-tanti-BMI1 (f) antibodies. These proteins colocalize in nuclear dots,transfected HeLa cells were immunolabeled with C44 anti-SYT (i) aas denoted by yellow color in overlay panel (k). Panels m–p: 518A2 manti-RING1 (n) antibodies. These proteins colocalized in nuclear dotsexpressing GFP-SSX2D1-154 (q) were immunolabeled with anti-BMIyellow color in overlay panel (s). Panels d,h,l,p,t: DAPI nuclear coun

tions. However, the fact that the SSX2D155-188 pro-

tein, which is a small protein (approximately 26 kDa),was predominantly localized in the cytoplasm, sug-gests that the nuclear localization of GFP-SSX2mut2, alarger protein, is not due to diffusion. In any case, theobserved difference in localization between GFP-SSX2and GFP-SSX2mut2 supports that the SSXRD domainis important for the nuclear localization of SSX pro-

ediated by a C-terminal domain. Panels a–d: SYT-SSX2-transfectedL (b) antibodies. These proteins partially colocalize in nuclear dots,

sfected HeLa cells were immunolabeled with B39 anti-SSX (e) andenoted by yellow color in overlay panel (g). Panels i–l: SYT-SSX2-

anti-HPC2 (j) antibodies. These proteins colocalized in nuclear dots,noma cells were immunolabeled with monoclonal anti-SSX (m) anddenoted by yellow color in overlay panel (o). Panels q–t: HeLa cells

) antibody. These proteins colocalized in nuclear dots, as denoted bystaining.

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tdtmpfpittv


The SYT protein does not contain any canonical NLSsequence. A cluster of basic amino acids is present atthe N-terminus but mutagenesis experiments showedthat those are not essential for nuclear localization(data not shown). Therefore, we created deletion mu-tants to test whether the nuclear targeting of SYTcould be assigned to any particular sequence or do-main. As it became apparent from our experiments, theSYT protein relies on an N-terminal sequence, includ-ing amino acids 51 to 90, to be localized in the nucleus(Fig. 1). Protein constructs that lacked this domainwere localized in both the nucleus and the cytoplasm.Since no NLS is present in this sequence, SYT maytranslocate to the nucleus through association with anNLS-containing interacting protein, a mechanism towhich several other nuclear proteins resort [45–47].The observed presence of C-terminal SYT fragments inthe nucleus next to cytoplasmic may be due to passivediffusion to the nucleus since these truncated proteinsare relatively small (below 28 kDa). Also, the smallsizes of VSV- and FLAG-tagged proteins may haveprecluded the consistent observation of nuclear dots.

In our previous report it was suggested that theSYT-SSX nuclear domains might be identical to thoseof SYT [19]. Here we show that the SYT-SSX proteinscolocalize preferentially with SSX rather than withSYT. These data fit well with our observations andthose of Soulez et al. [37] that both SSX and SYT-SSXare present in Polycomb-group nuclear bodies, at leastin the majority of transfected cells. However, the factthat SYT-SSX proteins may also partially colocalizewith SYT and, like SYT, interact with hBRM [23],suggests that the SYT-SSX protein localization is notfixed and may be modulated by its macromolecularenvironment or cellular metabolic state. Analysis ofthe distribution of endogenous SYT-SSX proteins inprimary synovial sarcomas may be crucial to clarifythese issues.

The finding of endogenous colocalization betweenSSX and PcG proteins in melanoma cells substantiatedthe SSX/SYT-SSX colocalization results in transfectedcells obtained by us and others [37] and excluded thepossibility of overexpression artifacts. Interestingly,we also found that SYT-SSX proteins, like PcG pro-teins [48], partially colocalize with PML in HeLa cells.This colocalization may be coincidental. Alternatively,this phenomenon may be explained by the presence ofheterochromatin protein 1 (HP1) in PODs [48, 49] andthe simultaneous PcG/SSX association with hetero-chromatin [48]. PcG proteins form multiprotein com-plexes that function as transcriptional repressors andare involved in stable gene silencing [36, 51]. In Dro-sophila, PcG proteins maintain the inactive state ofhomeotic genes initiated by segmentation repressorproteins [52, 53]. Several mammalian PcG homologues
have been identified and also these proteins are most
likely involved in the repression of homeobox and othergenes [26]. The recent finding that SSX proteins arecapable of repressing transcription in experimentalsystems [24] suggests that PcG complexes mediate thisfunction. This hypothesis is supported by our findingthat the SSX repressor domain (SSXRD) alone is ableto colocalize with PcG proteins. In the present reportwe found association of SSX proteins with condensedchromosomes, both endogenously and in transfectedHeLa cells, thus reinforcing the findings of Soulez et al.37]. Interestingly, PcG proteins also colocalize with

itotic chromosomes [48, 54]. Additionally, we foundhat the SSXRD domain mediates the association ofSX with chromatin. Taken together, these findings

ndicate that the SSX proteins are present in PcGomains, in both interphase and mitotic cells. How-ver, no evidence has been provided yet for a directnteraction between SSX and PcG proteins (this reportnd [37]).Our findings and those of other investigators suggest

hat the SYT-SSX oncoproteins may cause cancer byeregulating transcription through changes in chroma-in structure. Interference with chromatin remodelingay be crucial for neoplastic development [55]. In the

resent work we delineated the domains responsibleor nuclear localization of SYT, SSX, and SYT-SSXroteins and found that the SSX C-terminus (SSXRD)s responsible for the localization of SYT-SSX and SSXo PcG nuclear domains. Elucidation of its exact role inumorigenesis awaits investigation in primary syno-ial sarcoma cells.

We thank Jos Broers, Daniel A. Haber, Thomas Haaf, Tom Ma-niatis, Luitzen de Jong, and Gerd G. Maul for kindly providingantibodies. Marian Weterman is acknowledged for providing TFE3and PRCC expression constructs. Bert Janssen and Jose Thyssen areacknowledged for expert technical contributions. This work was sup-ported by the Fundacao para a Ciencia e Tecnologia of Portugal(Grant PRAXIS XXI/BD/3232/94 to N.R.d.S.) and the Dutch CancerSociety (Koningin Wilhelmina Fonds).

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Received August 16, 1999Revised version received December 29, 1999

delineation of the protein domains responsible for syt, ssx, and syt-ssx nuclear localization

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