A recurrent t(9;22) (q22;q12) chromosome translocation has been described in extraskeletal myxoid chondrosarcoma (EMC). Fluorescent in situ hybridization experiments performed on one EMC tumour indicated that the chromosome 22 breakpoint occurred in the EWS gene. Northern blot analysis revealed an aberrant EWS transcript which is cloned by a modified RT-PCR procedure. This transcript consists of an in-frame fusion of the 5' end of EWS to a previously unidentified gene, which was named TEC. This fusion transcript was detected in six of eight EMC studied, and three different junction types between the two genes were found. In all junction types, the putative translation product contained the amino-terminal transactivation domain of EWS linked to the entire TEC protein. Homology analysis showed that the predicted TEC protein contains a DNA-binding domain characteristic of nuclear receptors. The highest identity scores were observed with the NURR1 family of orphan nuclear receptors. These receptors are involved in the control of cell proliferation and differentiation by modulating the response to growth factors and retinoic acid. This work provides, after the PML/RAR alpha gene fusion, the second example of the oncogenic conversion of a nuclear receptor and the first example involving the orphan subfamily. Analysis of the disturbance induced by the EWS/TEc protein in the nuclear receptor network and their target genes may lead to new approaches for EMC treatment.

SSX genes show extensive nucleotide sequence conservation but little is known of their function. Disruption of SSX1 or SSX2, by chromosome translocation and`inframe' fusion to SYT, is a consistent feature of synovial sarcomas. The resulting SYT-SSX1/SSX2 proteins are activators of transcription; transactivation function is located in SYT. Unrearranged SSX1 can repress transcription, and this has been attributed to a putative KruÈ ppel associated box (KRAB) repression domain at the N-terminus. Here we isolated SSX-KRAB domains to speci®cally measure repression activity, using a previously characterized KOX1-KRAB domain as a control. In our repressor assay SSX1-and SSX2-KRAB domains down-modulated the transactivation of a reporter gene by threefold, compared with 83-fold repression achieved by KOX1-KRAB in the assay. Yeast two-hybrid analysis showed that SSX1-KRAB, unlike KOX1-KRAB, fails to interact with the KRAB corepressor TIF1b. These results raise questions about the evolutionary and functional relationship of SSX-KRAB and typical KRAB domains of KruÈ ppel zinc ®nger genes. We found that full-length SSX1 showed potent (74-fold) repression in our repressor assay, indicating the existence of a repression domain distinct from SSX-KRAB. By assaying deletion constructs of SSX1 we localized repression activity to 33 amino acids at the C-terminus. This novel domain is conserved between SSX family members, and, unlike the KRAB-related domain, is retained on fusion with SYT. This has important implications in understanding the mechanism by which the SYT-SSX fusion protein could contribute to neoplasia.Keywords: sarcoma; SSX; KRAB; transcription Chromosome translocation t(X;18)(p11.2;q11.2) (Clark et al., 1994;Crew et al., 1995) is a diagnostic feature of human synovial sarcomas, in some cases representing the sole cytogenetic abnormality (Sandberg and Bridge, 1994). All the available evidence indicates that this translocation is a key event in tumorigenesis. In all tumours characterized, the SYT gene on chromosome 18 is juxtaposed`in-frame' with either the SSX1 gene or SSX2 gene on chromosome X (Figure 1). SSX1 and SSX2 are now known to be members of a highly conserved multigene family Gure et al., 1997), and the SSX loci that have been mapped are all located in chromosome band Xp11.2 (Crew et al., 1995;. In contrast to SYT, which is a widely expressed gene (de Bruijn et al., 1996; J Knight., unpublished data), SSX transcripts show a very restricted distribution in adult human tissues. So far, SSX1 and SSX2 expression has only been detected in testis and thyroid (Crew et al., 1995;Tureci et al., 1996;Gure et al., 1997).As yet, little is known about the normal biological functions of the SYT and SSX gene products. No DNA binding sequences are recognizable in the SYT or SSX proteins. However, when coupled to a GAL4 DNA binding domain in in vitro reporter assays, SYT can activate transcription (70-fold activation) and SSX1 can repress transcription (50-fold repression) from a minimal promoter in NIH3T3 ®broblasts (Brett ...

Chromosome translocation t(X;18)(p11.2;q11.2) is unique to synovial sarcomas and results in an`in frame' fusion of the SYT gene with the SSX1 or closely-related SSX2 gene. Wild-type SYT and SSX proteins, and the SYT-SSX chimaeric proteins, can modulate transcription in gene reporter assays. To help elucidate the role of these proteins in cell function and neoplasia we have performed immunolabelling experiments to determine their subcellular localization in three cell types. Transient expression of epitope-tagged proteins produced distinctive nuclear staining patterns. The punctate staining of SYT and SYT-SSX proteins showed some similarities. We immunolabelled a series of endogenous nuclear antigens and excluded the SYT and SYT-SSX focal staining from association with these domains (e.g. sites of active transcription, snRNPs). In further experiments we immunolabelled the Polycomb group (PcG) proteins RING1 or BMI-1 and showed that SSX and SYT-SSX proteins, but not SYT, co-localized with these markers. Consistent with this we show that SSX and SYT-SSX associate with chromatin, and also associate with condensed chromatin at metaphase. Noteably, SSX produced a dense signal over the surface of metaphase chromosomes whereas SYT-SSX produced discrete focal staining. Our data indicate that SSX and SYT-SSX proteins are recruited to nuclear domains occupied by PcG complexes, and this provides us with a new insight into the possible function of wild-type SSX and the mechanism by which the aberrant SYT-SSX protein might disrupt fundamental mechanisms controlling cell division and cell fate.

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Prostate cancer is the most frequently diagnosed male cancer, and its clinical outcome is difficult to predict. The disease may involve the inappropriate expression of genes that normally control the proliferation of epithelial cells in the basal layer and their differentiation into luminal cells. Our aim was to identify novel basal cell markers and assess their prognostic and functional significance in prostate cancer. RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling. We identified 112 and 267 genes defining basal and luminal populations, respectively. The transcription factor TEAD1 and the ubiquitin ligase c-Cbl were identified as novel basal cell markers. Knockdown of either marker using siRNA in prostate cell lines led to decreased cell growth in PC3 and disrupted acinar formation in a 3D culture system of RWPE1. Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics. These data are consistent with basal features impacting on the development and clinical course of prostate cancers.