Research Article The Synovial Sarcoma–Associated SS18-SSX2 Fusion Protein Induces Epigenetic Gene (De)Regulation Diederik R.H. de Bruijn,1 Susanne V. Allander,2 Anke H.A. van Dijk,1 Marieke P. Willemse,1 Jose Thijssen,1 Jan J.M. van Groningen,1 Paul S. Meltzer,2 and Ad Geurts van Kessel1 1Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands and 2Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, Maryland Abstract The human and mouse SS18 genes and their encoded proteins are highly homologous and are expressed widely, both in Fusion of the SS18 and either one of the SSX genes is a embryonic and in adult tissues (7, 8). The SS18 protein resides in hallmark of human synovial sarcoma. The SS18 and SSX genes the nucleus, displaying a punctate pattern, distinct from nuclear encode nuclear proteins that exhibit opposite transcriptional domains such as coiled bodies, splicing factor speckles, or PML activities. The SS18 protein functions as a transcriptional oncogenic domains (9, 10). Within the SS18 protein, two functional coactivator and is associated with the SWI/SNF complex, domains have been identified, the SS18 NH -terminal homology whereas the SSX proteins function as transcriptional core- 2 (SNH) domain, and the glutamine-, proline-, glycine-, and tyrosine- pressors and are associated with the polycomb complex. The rich QPGY domain (11, 12). Previously, we found that the SS18 domains involved in these opposite transcriptional activities protein interacts directly with the AF10 protein, the fusion are retained in the SS18-SSX fusion proteins. Here, we set out partner to MLL in t(10;11)-positive acute leukemias (12). Through to determine the direct transcriptional consequences of this interaction, the SS18 protein associates with the glioma- conditional SS18-SSX2 fusion protein expression using com- amplified protein GAS41 (13) and the rhabdomyosarcoma-associated plementary DNA microarray-based profiling. By doing so, SWI/SNF protein INI1 (14). SWI/SNF proteins form multimeric we identified several clusters of SS18-SSX2–responsive complexes that activate gene transcription through ATP-dependent genes, including a group of genes involved in cholesterol chromatin remodeling mechanisms (15). Interestingly, the SS18 synthesis, which is a general characteristic of malignancy. protein also interacts and colocalizes with two SWI/SNF ATPase In addition, we identified a group of SS18-SSX2–responsive subunits, i.e., the Brahma (BRM) and Brahma-related gene 1 genes known to be specifically deregulated in primary synovial (BRG1) proteins (11, 16). In addition, the SS18 protein interacts sarcomas, including IGF2 and CD44. Furthermore, we ob- directly with the histone acetyltransferase p300 (17) and the served an uncoupling of EGR1, JUNB, and WNT signaling in histone deacetylase–associated corepressor SIN3A (18). Both latter response to SS18-SSX2 expression, suggesting that the SWI/ proteins are involved in epigenetic gene regulation through SNF-associated coactivation functions of the SS18 moiety are covalent chromatin modifications, in particular (de-)acetylation impaired. Finally, we found that SS18-SSX2 expression affects of histone tails. Because all these interactions are mediated by the histone modifications in the CD44 and IGF2 promoters and SS18 SNH domain, this domain emerges as a versatile protein- DNA methylation levels in the IGF2 imprinting control region. protein interaction domain. The SS18 QPGY domain acts as a Together, we conclude that the SS18-SSX2 fusion protein may transcriptional activation domain and is able to mediate multi- act as a so-called transcriptional ‘‘activator-repressor,’’ which merization of the SS18 protein (10, 11, 16). In addition, this domain induces downstream target gene deregulation through epige- shares structural characteristics with two SWI/SNF proteins, i.e., netic mechanisms. Our results may have implications for both ARID1A (BAF250a/SMARCF1) and ARID1B (ELD/OSA1/BAF250b; the development and clinical management of synovial ref. 14). Interestingly, the SS18-SWI/SNF association is also found in sarcomas. (Cancer Res 2006; 66(19): 9474-82) the Caenorhabditis elegans protein-protein interaction map (19), indicating that its functional significance is conserved during Introduction evolution. The lack of obvious DNA-binding domains, combined Synovial sarcomas are aggressive soft tissue tumors that account with the QPGY-mediated transcriptional activation capacities, for up to 10% of all human sarcomas (1). Cytogenetically, synovial indicate that the SS18 protein acts as a transcriptional coactivator. sarcomas are characterized by a recurring chromosomal translo- The SNH domain–mediated protein-protein interactions may serve cation, t(X;18)(p11.2;q11.2), which is found in >95% of all cases (2). to tether SS18 to its DNA targets. As a result of this translocation, the SS18 gene (previously called Currently, nine human SSX genes, encoding highly similar SYT or SSXT) on chromosome 18 is fused to one of three closely proteins, have been identified (20). The NH2-terminal moieties of related SSX genes on the X chromosome, SSX1, SSX2,orSSX4 (3–6). the SSX proteins exhibit homology to the Kru¨ppel-associated box (KRAB) domain (5), a domain that is known to be involved in transcriptional repression (21). Although SSX proteins are able to repress the transcription of reporter genes (5, 11), only part of this Note: Supplementary data for this article are available at Cancer Research Online activity has been attributed to the KRAB-like domain. Considerably (http://cancerres.aacrjournals.org/). Requests for reprints: A. Geurts van Kessel, Department of Human Genetics, 855 stronger transcriptional repression was exerted by the highly acidic Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, the COOH-terminal 34 amino acids of SSX (22). Therefore, this Netherlands. Phone: 31-24-3614107; Fax: 31-24-3540488; E-mail: a.geurtsvankessel@ sequence was designated SSX repression domain. The SSX proteins antrg.umcn.nl. I2006 American Association for Cancer Research. are localized in the nucleus, being distributed both diffusely and in doi:10.1158/0008-5472.CAN-05-3726 nuclear speckles (9, 10). These speckles were found to also harbor Cancer Res 2006; 66: (19). October 1, 2006 9474 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2006 American Association for Cancer Research. Synovial Sarcoma–Associated SS18-SSX Fusion Proteins several polycomb group (PcG) proteins, i.e., HPC2, BMI1, and constituted the raw data from which differential gene expression ratio values RING1 (23, 24). PcG proteins form multimeric protein complexes were calculated. For all targets, the ratios of the red (Cy5) to green (Cy3) that induce the repression of target genes through modulation of intensities (R/G) were determined, and ratio normalizations were done chromatin structures (25). based on 88 preselected internal controls as previously described (26). Spots were included for further analyses when either the quality scores (assigned Because the wild-type SS18 and SSX proteins exhibit apparently by the DeArray software) were >0.5, or the signal-to-background ratios in opposite transcriptional activities, and because the domains both channels were >3. In addition, data were excluded when the normalized critical to these activities are retained in the synovial sarcoma– ratios between duplicate experiments varied by more than a factor of 2. associated SS18-SSX fusion proteins, we questioned what the The ratio threshold values, indicating down-regulation or up-regulation of transcriptional consequences of SS18-SSX expression are. There- the genes represented by the cDNA clones on the array, were set to À0.6 and fore, we set out to assess the SS18-SSX2downstream effects using 0.6, respectively, which corresponds to a 50% difference in gene expression. an inducible cell system in conjunction with microarray-based gene Western blotting and immunoprecipitation. Protein extracts were expression profiling, chromatin immunoprecipitation (ChIP), and prepared from the HEK-FUS cells after 0, 2, 6, and 24 hours of tetracyclin global and gene-specific histone modification and DNA methyla- induction in extraction buffer containing 150 mmol/L of NaCl, 0.5% NP40, tion assays. Our data indicate that the SS18-SSX2fusion protein 50 mmol/L of HEPES (pH 7), 5 mmol/L of EDTA, 1 mmol/L of phenyl- methylsulfonyl fluoride, 0.5 mmol/L of DTT, 2mg/mL of leupeptin, and induces the deregulation of downstream target genes through 2.5 mg/mL of aprotinin. Protein extracts were analyzed on a Western blot, epigenetic mechanisms. incubated with a mouse monoclonal anti-myc antibody (9E10; Santa Cruz Biotechnology, Santa Cruz, CA) and a horseradish peroxidase–coupled rabbit Materials and Methods anti-mouse IgG antibody (Dako, Glostrup, Denmark) as a secondary antibody. Cloning and sequencing procedures. All cloning procedures were Signals were detected with the enhanced chemiluminescence system essentially as described before (7, 8, 12). The sequence of all oligos used are (Amersham, Piscataway, NJ) and X-omat R X-ray films (Kodak, New Haven, available upon request. Sequence analyses were done at the DNA CT). For the SS18-SSX2/BRG1 immunoprecipitations, fresh nuclear extracts Sequencing Facility of the Radboud University Nijmegen Medical Centre. were prepared from HEK-FUS cells, according to the Lamond lab protocol A DNA and protein databases