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As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes., (Copyright 2001 S. Karger AG, Basel.)

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 in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus., (Copyright 2000 Academic Press.)

Synovial sarcoma is characterized by a prevalent chromosomal translocation, t(X;18)(p11;q11). As a result of this translocation the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome. In this study, we generated polyclonal antibodies against the SYT and SSX2 proteins. These antibodies specifically detected both these proteins and the SYT-SSX fusion proteins in transfected COS-1 cell extracts. Indirect immunofluorescence analysis of COS-1 cells expressing tagged or untagged SYT, SSX2, SYT-SSX1 or SYT-SSX2 indicated that all these proteins are localized in the nucleus, excluding the nucleoli. The SSX2 protein exhibited a diffuse staining pattern whereas both the SYT and SYT-SSX proteins appeared in several nuclear dots. Similar nuclear dots were also detected in primary synovial sarcoma cells growing in a short-term in vitro culture. Double immunofluorescence in conjunction with confocal laser-scanning microscopy revealed that the SYT and SYT-SSX nuclear dots do not co-localize with known nuclear structures as e.g. coiled bodies, SC35 interchromatin granules or PML bodies. The similar nuclear localization patterns of SYT and SYT-SSX suggest that the SYT-SSX fusion proteins are directed to SYT-associated nuclear domains where an abnormal function may be exerted.

In a previous study we reported the isolation of the human synovial sarcoma-associated t(X;18) breakpoint. As a result of this translocation, the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome, depending on the exact location of the breakpoint within band Xp11.2. As yet, little is known about the modes of action of the SYT and SSX genes and their respective (fusion) products. Here we report the isolation of the mouse homolog of SYT, its full length cDNA sequence, its chromosomal localization, and its spatio-temporal expression patterns in adult and embryonic tissues. The SYT gene was found to be well conserved during evolution and is part of a region of synteny between the human and mouse chromosomes 18. In early embryogenesis, Syt is ubiquitously expressed. In later stages, the expression becomes confined to cartilage tissues, specific neuronal cells and some epithelial derived tissues. In mature testis, expression was specifically observed in primary spermatocytes.

As a result of the synovial sarcoma-associated t(X;18) translocation, the SS18 gene on chromosome 18 is fused to either one of the three closely related SSX genes on the X chromosome. The SS18 protein is thought to act as a transcriptional co-activator, whereas the SSX proteins are thought to act as transcriptional corepressors. The main SSX-repression domain is located in its C terminus, a domain that is retained in the respective SS18–SSX fusion proteins. Both the SS18 and SSX proteins lack DNA-binding domains. Previously, we found that the SS18 and SS18–SSX fusion proteins may be tethered to DNA targets via the SS18-interacting protein AF10. Here, we set out to isolate proteins that interact with the SSX C-terminal repression domain using a yeast two-hybrid interaction trap. Of the positive clones isolated, two corresponded to the LIM homeobox protein LHX4, a DNA-binding protein that is involved in transcription regulation. An endogenous interaction was subsequently established in mammalian cells via colocalization and coimmunoprecipitation of the respective proteins. Interestingly, the LHX4 gene was previously found to be deregulated in various human leukemias. In addition, it was previously found that LIM homeobox proteins may bind to and activate the glycoprotein-α (CGA) promoter. Using LHX4 chromatin immunoprecipitation and CGA-promoter assays, we found that endogenous LHX4 binds to the CGA promoter and that LHX4-mediated CGA activation is enhanced by the SS18–SSX protein, but not by the SSX protein. Taken together, we conclude that this novel protein – protein interaction may have direct consequences for the (de)regulation of SSX and/or SS18–SSX target genes and, thus, for the development of human synovial sarcomas.Oncogene (2008) 27, 653–662; doi:10.1038/sj.onc.1210688; published online 30 July 2007 [ABSTRACT FROM AUTHOR]

The highly conserved synovial sarcoma associated protein SS18 is thought to act as a transcriptional co-activator through interactions with various proteins involved in (epigenetic) gene regulation. The SS18 SNH domain appears to act as a major interface for these protein-protein interactions. Previously, we used this SNH domain to identify SS18 paralogs (SS18L1 and SS18L2) and orthologs in various species. The functional significance of these SS18-like proteins is embodied by the observations that SS18L1 and SS18L2 can replace SS18 in its various protein-protein interactions, and that SS18L1 may act as a fusion partner of SSX in synovial sarcoma. In the current study, we performed a comprehensive comparison of SNH-containing loci in several distinct species. By doing so, we found that the vertebrate SS18 and SS18L1 genes map within co-linear DNA segments that may have evolved through a relatively recent genomic duplication event. An additional phylogenetic study indicated that the more divergent SS18L2 gene is most likely derived from an earlier gene duplication event, again in the vertebrate lineage. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]