The Synovial Sarcoma–Associated SS18-SSX2 Fusion Protein Induces Epigenetic Gene (De)Regulation

Research Article

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

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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 were searched using BLAST and/or BLAT (http://www.lamondlab.com/). Approximately 400 g of nuclear proteins were A search algorithms at the National Center for Biotechnology Information subjected to immunoprecipitations with 1.6 g of 9E10 anti-myc antibody and or University of California, Santa Cruz (UCSC), respectively (http:// a mouse preimmune serum in the presence of protein A/G agarose beads A www.ncbi.nlm.nih.gov/; http://genome.ucsc.edu/). All SS18, SSX2, and (Santa Cruz). After washing, the whole immunoprecipitates, and 40 g of the SS18-SSX2 fragments used as probes on Northern blots and/or for the nuclear extract as an input control, were loaded on a Western blot. This blot construction of the T-Rex tetracyclin-inducible cell system were generated was incubated with an anti-BRG1 antibody (sc-10768, Santa Cruz), after which by PCR from full-length complementary DNAs (cDNA) and sequence- the signals were detected as described above. Next, the blot was stripped verified prior to probe preparation and/or cloning procedures. (5 minutes in 0.2mol/L of glycine, pH 2.8, and 0.5 mol/L of NaCl), neutralized Conditional expression of the SS18-SSX2 fusion protein and real-time (5 minutes in 0.5 mol/L of Tris and 1.5 mol/L of NaCl), and incubated with the PCR analysis. For the identification of SS18-SSX downstream target genes, anti-SS18 antibody RA2009 (9). The signals were detected as described above. we generated a cell line exhibiting conditional expression of the SS18-SSX2 ChIP analysis. Chromatin was isolated from HEK-FUS cells at 0 and fusion protein using the T-Rex system (Invitrogen, Carlsbad, CA). Human 24 hours of tetracyclin induction as described by Boyd et al. (27). Protein-DNA HEK293-T-Rex cells were purchased from Invitrogen and cultured in T-Rex complexes were immunoprecipitated with antibodies directed against BRG1 culture medium (DMEM with 10% fetal bovine serum and 1 Ag/mL (see above), histone H4 acetylated at lysine 16 (H4K16-Ac, ab-1762; Abcam, blasticidin). A full-length SS18-SSX2 cDNA was cloned into the pcDNA4-TO Cambridge, MA), histone H4 methylated at lysine 20 (H4K20-Me, ab9051; vector, thereby yielding a construct in which the T-Rex tetracyclin-inducible Abcam), histone H3 trimethylated at lysine 4 (H3K4-triMe, ab8580; Abcam), promoter controls the expression of a myc-tagged SS18-SSX2protein. This and a rabbit preimmune serum. The recovered genomic DNAs were analyzed construct, and the empty vector, were transfected into the HEK293-T-Rex by Q-PCR with primer pairs spanning the IGF2 or CD44 promoters. A primer cells using a gene-pulser electroporation apparatus (Bio-Rad, Hercules, CA). pair located 2to 3 kb in a downstream intronic region was used as a negative Transfected cells were selected in T-Rex culture medium supplemented control. All Q-PCR data are presented as promoter/intron ratios (P/I) to with 1 Ag/mL of zeocin (Invitrogen). After 2weeks of selection, positive indicate the enrichment of IGF2 and/or CD44 promoter sequences in the ChIP. clones were isolated and pooled, such that each pool was a mix of 10 to Bisulfite sequencing of genomic DNA. Genomic DNAs from the HEK- 15 independent clones, thereby minimizing the transcriptional effects of VEC and the HEK-FUS cell lines (after 0 and 24 hours of tetracyclin independent integration sites. For induction, 1 ng/mL of tetracyclin (Sigma- induction) were treated with bisulfite as described by Frommer et al. (28). A A Aldrich, St. Louis, MO) was added to the HEK293-T-Rex empty vector (HEK- Briefly, 2 g of total genomic DNA was diluted in 50 LofH2O, after which VEC) and HEK293-T-Rex SS18-SSX2 (HEK-FUS) cell lines for 2, 6, or 24 hours. 5.5 AL of 2mol/L NaOH was added and incubated at 37 jC for 10 minutes. Cells were harvested by scraping in PBS, and RNA was isolated using a Trizol Subsequently, 30 AL of 10 mmol/L hydroquinone (Sigma) and 520 ALof protocol (Boehringer, Ridgefield, CT) in conjunction with RNeasy midi 3 mol/L sodium bisulfite (Sigma), both freshly prepared, were added and the columns (Qiagen, Valencia, CA). Northern blots were prepared as described mixture was incubated at 50jC for 16 hours. Next, this (sulfonated) DNA previously (7). cDNA was prepared from 1 Ag of RNA, using the Omniscript was isolated using a wizard cleanup kit (Promega, Madison, WI) and reverse transcription kit (Qiagen) in a 20 AL volume. Real-time quantita- deaminated by the addition 1/10 volume of 3 mol/L NaOH and incubation tive reverse transcription-PCR (QRT-PCR) was done in duplicate with 0.2 AL for 5 minutes at 25jC. Finally, the bisulfite-adapted DNA was precipitated A A of reverse transcriptase material on an ABI 7700 sequence detection system, and resuspended in 20 LofH2O. For each PCR reaction, 1 L of the treated using a SYBR green PCR master mix (Applied Biosystems, Foster City, CA). DNA was used. Primers for bisufite sequencing of genomic targets were All QRT-PCR data were normalized to the expression of the h-tubulin gene. selected using the MethPrimer program (29). Bisulfite PCR products were Microarray-based expression profiling. RNA labeling, microarray cloned into pGEM-T and at least 20 independent clones were sequenced for preparation, hybridization, scanning, and image analysis were done as evaluation of the methylation status of the selected target. described before (26). For each hybridization, 100 Ag of total RNA was fluorescently labeled with Cy3 (HEK-VEC) and Cy5 (HEK-FUS) using direct Results and Discussion dye incorporation by oligo(dT)-primed reverse transcriptase. Annotations of the gene names for each cDNA clone are according to Build 170 of Identification of SS18-SSX2 target genes. For the identifica- the UniGene human sequence collection (http://www.ncbi.nlm.nih.gov/ tion of SS18-SSX2target genes, we generated a stable HEK293-T- UniGene/). The scanned fluorescent images (red and green channels) Rex cell line displaying tetracyclin-inducible SS18-SSX2expression www.aacrjournals.org 9475 Cancer Res 2006; 66: (19). October 1, 2006

(HEK-FUS, see Materials and Methods). At four different time group of genes, two major clusters could be discerned, each points of tetracyclin induction (t = 0, 2, 6, and 24 hours), total RNA encompassing a set of genes displaying specific transcriptional and protein were extracted from the HEK-FUS cells and the responses upon SS18-SSX2induction (Fig. 2 A and B). The first inducible SS18-SSX2expression was monitored on Northern and cluster included 180 genes which were up-regulated or down- Western blots. On Northern blots, using SS18- and SSX2-specific regulated at all time points. A limited subset of this cluster, probes, the SS18-SSX2 mRNA was detected after 2hours of comprising 64 genes, displayed mean log2 ratios meeting more induction (Fig. 1A and B; closed arrowheads). In addition, we stringent criteria at all time points (i.e., threshold levels of À0.8 and detected endogenous SS18 mRNA expression, which does not 0.8). In Fig. 2A, the ratios of all genes in this latter cluster are change during this induction series (Fig. 1A, open arrowhead). depicted in a red/green diagram. Given the low, but detectable, Using real-time QRT-PCR on the same RNA samples, we already SS18-SSX2 mRNA and protein levels already present before detected a low SS18-SSX2 mRNA expression level prior to induction (t = 0; see above), these gene expression changes tetracyclin induction (Fig. 2C), indicating that the HEK293-T-Rex illustrate the sensitivity of this cell system for the expression of cell system is somewhat leaky (confirmed by the manufacturer). On exogenous genes, especially when these genes encode proteins Western blots, using an anti-myc antibody, a 65 kDa protein, involved in transcription regulation. The second cluster included representing the induced myc-tagged SS18-SSX2protein was 72genes, all displaying mean log 2 ratio changes of at least 0.4 detected in the induction series (Fig. 1C; closed arrowhead), between two consecutive time points (corresponding to 30% up- thereby validating the inducible SS18-SSX2 mRNA and protein regulation or down-regulation). These changes were sustained expression in this cell system. Upon longer exposure, the SS18-SSX2 during later time points. In Fig. 2B, the ratios of all genes in this protein was detectable at low levels in the HEK-FUS t = 0 extract as latter cluster are depicted in a red/green diagram. Because this well (data not shown). As expected, HEK-VEC displayed no SS18- cluster, as expected, included the up-regulated SS18 and SSX genes SSX2mRNA expression (Fig. 2 C). As an independent variable of (Fig. 2B, closed arrowheads), an intrinsic validation of the micro- SS18-SSX2expression, we found that the HEK-FUS cell line grows array data was thus obtained. In addition, we did QRT-PCR on six significantly faster than the HEK-VEC cell line (doubling time, 37.7 genes, i.e., the induced SS18-SSX gene, and the LDLR, CD44, EGR1, versus 50.5 hours, respectively). This increase in growth rate is in CXXC5, and IGF2 genes (Fig. 2A and B, arrows). All QRT-PCR data full conformity with the putative role of SS18-SSX in cellular were in accordance with the microarray data, thereby providing a transformation (30). further validation of the microarray data (Fig. 2C). Validation for Total RNAs extracted from the HEK-VEC and HEK-FUS cells the correct annotation of the spotted cDNA clones came from the (both tetracyclin-induced for 0, 2, 6, and 24 hours) were labeled and observation that sequencing of 30 selected clones yielded the used for expression profiling on NHGRI 6K cDNA microarrays (26). expected sequence in 25 of these clones (83%). In a total of nine hybridizations, the Cy5-labeled HEK-FUS and SS18-SSX2 induced deregulation of genes involved in Cy3-labeled HEK-VEC samples were matched for each time point, cholesterol synthesis. Within the cluster of genes that was up- and hybridized in duplicate (t =0AandB,t = 2A and B, t =6AandB) regulated or down-regulated at all time points (first cluster, Fig. 2A), or triplicate (t = 24A, B, and C). Of the 6,548 cDNA clones on the a set of genes that is involved in cholesterol metabolism could read- array, 4,085 (62%) complied with our inclusion criteria (see ily be identified. This set includes the main regulator (low-density Materials and Methods) in at least six out of nine hybridizations lipoprotein receptor, LDLR) and the main rate-limiting enzyme done. The normalized, log2-transformed Cy5/Cy3 ratios of these (3-hydroxy-3-methylglutaryl coenzyme-A reductase, HMGCR), as cDNA clones were combined in one database and the mean ratios well as two other genes which encode enzymes (IDI1 and FDFT1)of from duplicate hybridizations at each time point were used for the de novo cholesterol synthesis pathway (Fig. 3). Based on this clustering purposes. With the preset threshold levels of À0.6 and information, we re-queried the entire data set for additional genes 0.6 (see Materials and Methods), 795 SS18-SSX2–responsive genes involved in cholesterol metabolism and its regulation. By doing so, were identified, each displaying up-regulation or down-regulation we found transcriptional changes that substantiate an up-regulation at one or more time points (Supplementary Table S1). Within this of cholesterol synthesis in response to SS18-SSX2 expression.

Figure 1. Inducible expression of the SS18-SSX2 fusion gene (3) at 2, 6, and 24 hours of tetracyclin induction in the HEK-T-Rex system. SS18-SSX2 mRNA expression was detected on Northern blots using SS18-specific (A) and SSX2-specific (B) probes, respectively. In addition, endogenous SS18 mRNA (A, /), and two unidentified SSX2 signals (B) are visible. SS18-SSX2 protein expression (C) was detected on Western blots using an anti-myc–tagged antibody.

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Figure 2. Two clusters of SS18-SSX2–responsive genes and validation of the microarray data by real-time QRT-PCR. The genes in these clusters display up-regulation or down-regulation at all time points of SS18-SSX2 induction (A) or transcriptional changes between different time points of SS18-SSX2 induction (B). Top, time points (0, 2, 6, and 24 hours) and duplicate experiments, as well as the annotation of accession numbers and gene symbols are indicated. 3,theSS18 and SSX genes, displaying up-regulation within the time series. /, genes exhibiting transcriptional switch responses, as, e.g., the IGF2 and IGFBP5 genes (see text). Expression ratios are displayed in red/green according to the bar scheme (bottom); gray blocks, no data. C, expression analysis of six selected genes (A and B, arrows), including the SS18-SSX2 fusion gene by real-time QRT-PCR. Columns, means of results normalized to h-tubulin expression; bars, SD.

Normally, exogenous cholesterol activates the LDL receptor, by down-regulation of the LDLR and ASNS genes and by up- which induces down-regulation of the HMGCR gene and, thereby, regulation of the HMGCR gene (Fig. 3), which is in conformity with inhibition of de novo cholesterol synthesis (Fig. 3). The LDLR literature data (31, 32). Additionally, we observed the up-regulation gene, in turn, is regulated through association of the early growth of five out of nine genes encoding enzymes of the cholesterol response 1 (EGR1) and CCAAT/enhancer binding protein-h synthesis pathway (Fig. 3). Furthermore, the EGR1 gene displayed (CEBPB) proteins (31). Another, distinct, function of the CEBPB a 3-fold up-regulation at t = 0, but dropped down to threshold protein is the regulation of amino acid synthesis through levels after SS18-SSX2 induction, thereby adding to the effect of asparagine synthetase (ASNS; ref. 32). In the microarray data, we LDLR down-regulation and, hence, HMGCR up-regulation. The observed down-regulation of the CEBPB gene in response to SS18- microarray data were confirmed by QRT-PCR (see above, Fig. 2C). SSX2 induction. This CEBPB down-regulation was accompanied Taken together, our expression profiling data indicate that the www.aacrjournals.org 9477 Cancer Res 2006; 66: (19). October 1, 2006

Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2006 American Association for Cancer Research. Cancer Research regulation of various genes involved in cholesterol metabolism is altered as a direct consequence of SS18-SSX2 fusion gene expression. Because enhanced cholesterol synthesis has been denoted as a hallmark of tumorigenesis (33), these responses underscore a previous notion that SS18-SSX2 fusion gene expression may be directly related to the malignant transformation of cells (30). Interestingly, similar elevated expression of genes involved in cholesterol synthesis has also been associated with elevated IGF2 expression (34). SS18-SSX2-induced deregulation of IGF signaling. Within the cluster of genes displaying up-regulation or down-regulation upon induction of SS18-SSX2 expression (second cluster, Fig. 2B), a set of genes was identified that has previously been implicated in tumor development, i.e., IGF2, NF2, JUNB, CCND1, CAV1, CD44, Figure 4. Transcriptional switch responses of IGF-associated genes between ENPP2, IGFBP5, and IGFBP7. Comparison of our data with data 2 and 6 hours or between 6 and 24 hours after SS18-SSX2 induction in the generated in two independent expression profiling studies of HEK-T-Rex system. Independent clones are indicated by accession numbers primary synovial sarcomas (26, 35) yielded a limited set of genes and UniGene gene symbols. Alternatively regulated IGF2 and IGFBP5 genes are highlighted (brackets). Again, all gene expression ratios are displayed in a that was consistently up-regulated or down-regulated, both in our red/green scheme (see Figs. 2 and 3). inducible cell system and in the primary tumors, and which

included IGF2 and CD44. The up-regulation of the IGF2 gene in our system was accompanied by a down-regulation of the IGF binding protein 5 (IGFBP5) gene. Within the group of IGF-associated genes, this opposite regulation was restricted to the IGF2 and IGFBP5 genes, each represented by two independent cDNA clones on the array (Fig. 4). Complementary expression of these two genes has also been observed during rat pituitary development (36) and in early chicken embryogenesis, where IGFBP5 expression was observed in a pattern similar to Sonic Hedgehog expression (37). Similar transcriptional switch responses, i.e., ratio changes of at least 0.6, either between the 2- and 6-hour time points or between the 6- and 24-hour time points, were observed for 21 genes (Fig. 2B, open arrowheads). These genes included the JUNB gene and two JUN-inducible genes (38), autotaxin (ENPP2), and fatty acid synthase (FASN). Interestingly, we found that this activator- responder combination was uncoupled in response to SS18-SSX2 expression induction (JUNB up-regulated, and ENPP2 and FASN down-regulated). Because a similar effect was observed for the EGR1 gene (down-regulated) and two EGR1-inducible genes (39), IGF2 and CCND1 (both up-regulated), the activatory functions of JUNB and EGR1 seem to be impaired upon SS18-SSX2 induction. An explanation for this phenomenon may be that the transcriptional coactivation machinery, recruited to these transcription factors, functions differently in the presence of the SS18-SSX2 protein. Because the activator protein transcription factor JUNB may recruit members of the SWI/SNF complex for transcriptional coactivation (40), and because the SS18-SSX2fusion protein retains the domain necessary for the interactions with the SWI/SNF complex, this impaired transcriptional activation may be caused by compromised SWI/SNF functions. Epigenetic regulation of SS18-SSX2 target genes. From the genes displaying up-regulation or down-regulation after induction of SS18-SSX2 expression (second cluster, Fig. 2B), we selected the IGF2 and CD44 genes for further analysis. For the IGF2 gene, which displayed the most extreme up-regulation in our microarray data, Figure 3. Transcriptional deregulation of genes involved in cholesterol we confirmed a strong up-regulation at t =24(Fig.2).C For the synthesis. Intermediates of the cholesterol synthesis pathway (inboxes ). Gene symbols of enzymes catalyzing different steps in this pathway or the regulation CD44 gene, we confirmed that it is already down-regulated at t =0, thereof (see text), and which were present on the microarray (inboldface ). and is repressed even more in response to SS18-SSX2 expression Arrows to the left of the gene symbols, up-regulation or down-regulation in induction at t =24(Fig.2C). We also did semiquantitative RT-PCRs response to SS18-SSX2 induction. The expression ratios of these genes are displayed in a red/green scheme (see Fig. 2); time points and duplicate for the CD44 gene on a panel of primary synovial sarcomas, experiments (top), and accession numbers and gene symbols (right). including 11 SS18-SSX1-positive and 11 SS18-SSX2-positive cases.

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By doing so, we found low to undetectable CD44 expression levels antibody, RA2009 (9), the immunoprecipitated SS18-SSX protein in three of the SS18-SSX1-positive tumors (27%) and in six of the was also readily detected (Fig. 5B). In order to establish a direct SS18-SSX2-positive tumors (54%; data not shown). These results binding of the BRG1 protein to the putative IGF2 and CD44 target support the microarray data obtained in our inducible cell system, promoters, we did ChIP on HEK-FUS cells at 0 and 24 hours of and substantiate the notion that the IGF2 and CD44 genes may tetracyclin induction. The recovered ChIP DNAs were analyzed by represent genuine SS18-SSX target genes. Q-PCR for enrichment of IGF2 and CD44 promoter sequences, Previously, it was found that CD44 gene expression is up- expressed as P/I values (see Materials and Methods). By doing so, regulated by the WNT signaling pathway (41) or through DNA we found that all P/I values were >1, indicating that the BRG1 ChIP demethylation after treatment of cells with 5-aza-2-deoxycytidine DNAs were indeed enriched for IGF2 and CD44 promoter (42). Using this information, we again re-queried our microarray sequences (Fig. 5C). In addition, we found that the IGF2 P/I values data set to search for other genes meeting these criteria. WNT decreased significantly upon induction of the SS18-SSX2protein, target genes include Axin, CD44, CCND1, C-MYC, FN1, and TCF1, whereas the CD44 P/I values remained constant after the induction which are activated by h-catenin, in conjunction with TCF of the SS18-SSX2protein (Fig. 5 C). These ChIP data indicate that transcription factors and SWI/SNF proteins such as BRG1 (43). In the presence SS18-SSX2protein leads to dissociation of the BRG1 our data set, the CCND1 gene showed up-regulation, whereas the protein from the IGF2 promoter, but not from the CD44 promoter. CD44, C-MYC, and FN1 genes were either down-regulated, or not Combined with the expression data (IGF2, up-regulation; CD44, affected at all. Additionally, we observed up-regulation of the down-regulation), this implies that the BRG1 protein may, in CXXC5 gene (Fig. 2), of which the encoded protein displays a high conjunction with the SS18-SSX2protein, exert negative regulatory degree of similarity to the WNT-inhibiting protein IDAX, especially effects on the IGF2 and CD44 promoters, respectively. These data around the KTXXXI motif (44, 45). Activation of a putative WNT support our hypothesis that SWI/SNF-related functions may be repressor such as the CXXC5 may be responsible for the observed impaired after SS18-SSX2 expression induction. Through additional SS18-SSX2–induced down-regulation of CD44. However, in line ChIP analyses, using antibodies directed against specifically with the transcriptional uncoupling processes described above, the modified histones, we found that histone modification patterns observed CD44 down-regulation may also result from compro- at the IGF2 promoter change in response to the SS18-SSX2 mised SWI/SNF functions. Previously, CD44 (up)regulation was induction, whereas they remained constant at the CD44 promoter. found to rely specifically on BRG1 protein expression (43). An Specifically, we observed that the levels of H4K16-Ac and H3K4- important premonition for any inhibitory effect exerted by the triMe, which are associated with active genes (46), were increased SS18-SSX2protein on the SWI/SNF protein BRG1 would be that the significantly in the IGF2 promoter (Fig. 5D) upon SS18-SSX2 SS18-SSX2and BRG1 proteins interact directly in our cell system. induction. Together, these data indicate that the SS18-SSX2protein, To establish this, we did an immunoprecipitation of myc-tagged possibly in conjunction with BRG1, affects gene expression in an SS18-SSX2protein from HEK-FUS cells at 24hours of tetracyclin epigenetic fashion. induction. After Western blot analysis with an anti-BRG1 antibody, SS18-SSX2 targets include methylation-sensitive genes. the coimmunoprecipitated BRG1 protein was readily detected Methylation-sensitive genes (http://www.missouri.edu/fhyper- (Fig. 5A), thereby confirming that both proteins interact directly. met/list_of_promoters.htm), including IGF2, SPARC, BTG2, CSPG2, After stripping and reprobing the same blot with the anti-SS18 IGFBP7, and CD44, were also found to be deregulated in response

Figure 5. Immunoprecipitation of the SS18-SSX2 and BRG1 proteins, and ChIP analysis of the IGF2 and CD44 promoters. A, detection of the BRG1 protein in a myc-immunoprecipitate (myc-IP) from the HEK-FUS cell line after 24 hours of tetracyclin induction. The 205 kDa BRG1 protein (/) is visible in both the input and myc-IP lanes, but not in the preimmune lane. B, using an anti-SS18 antibody, the immunoprecipitated SS18-SSX2 protein (3) is likewise detected in the input and myc-IP lanes, but not in the preimmune lane. Molecular weight markers are shown in the second lane with the sizes (in kDa, left). C, Q-PCRof the IGF2 and CD44 promoters (done in triplicate and plotted as P/I values, see text) in an anti-BRG1 ChIP of the HEK-FUS cell line at t = 0 and t = 24 of tetracyclin induction. After SS18-SSX2 induction (t = 24), the enrichment of the IGF2 promoter in the BRG1 ChIP is significantly reduced, whereas that of the CD44 promoter remains the same. D, Q-PCRof the IGF2 and CD44 promoters in H4K16-Ac, H4K20-Me, and H3K4-triMe ChIPs of the HEK-FUS cell line at t = 0 and t = 24 of tetracyclin induction. The levels of H4K16-Ac and H3K4-triMe are elevated in the IGF2, but not in the CD44 promoter. www.aacrjournals.org 9479 Cancer Res 2006; 66: (19). October 1, 2006

Figure 6. Methylation status of the human H19-ICRin the HEK-VEC and HEK-FUS cell lines, before and after SS18-SSX2 induction (t = 0 and t = 24). The ICR is located upstream of the H19 start site (position 0; openbox ) and harbors eight 45 bp consensus CTCF binding sites and six OCT binding sites in repeated domains (light and dark gray blocks). The ICRfragment, assayed by bisulfite sequencing ( BS) is indicated and the results for this fragment in the HEK-VEC and HEK-FUS cell lines at t = 0 and t = 24 of tetracyclin induction are displayed below. In the independently sequenced clones (—), methylated CpG dinucleotides (.), and unmethylated CpGs (o). The percentage of methylated cytosines in the first three CpGs, including the CTCF binding site, are indicated below each set of clones. to SS18-SSX2 expression (Fig. 2). Because the sensitivity of genes with the empty vector control cells (data not shown). Literature for DNA methylation generally relies on the presence of promoter- data indicate that a knockout of the DNA methyltransferase 1 associated CpG islands, we checked for the presence of this (DNMT1) gene induced a 20% decrease in overall mdC levels (50), feature in all genes listed in Fig. 2. Using the UCSC genome whereas DNMT1 overexpression induced a 15% increase in overall browser (http://genome.ucsc.edu/), we found that 112of 136 mdC levels (51). Although the SS18-SSX2–induced effect on overall (82%) of these genes indeed contain CpG islands in their DNA methylation was limited, our data provides support for the promoter regions, thereby turning them into putative targets for proposed DNA methylation hypothesis. transcriptional (de)regulation through DNA methylation. A In addition, we selected the IGF2 gene for a locus-specific DNA genome-wide survey of the UCSC data indicated that the methylation analysis. The IGF2 gene, which we found to be the promoter regions of 16,983 of 96,377 (18%) singly mapped most extremely up-regulated gene in response to SS18-SSX2 UniGene clusters are associated with CpG islands. Based on the expression, and which was found to be consistently up-regulated striking enrichment of CpG island–positive genes within the SS18- in primary synovial sarcomas (26, 35), is equipped with both a CpG SSX2–responsive genes, we hypothesize that the SS18-SSX2– island and a differentially methylated region, also referred to as mediated transcriptional effects may, in part, be induced by H19 imprinting control region (H19-ICR). In its unmethylated (epigenetic) changes in DNA methylation. Either through, or in state, this ICR forms a chromatin boundary that prevents IGF2 parallel with, defects in the SWI/SNF complex, this may lead to activation by distant enhancers. Binding of the zinc finger protein changes in DNA (cytosine) methylation. These methylation CTCF to the ICR is essential for this chromatin boundary function changes, in turn, may induce changes in expression of down- (52). Methylation of the CTCF binding sites within the ICR stream target genes. The observations that the SS18-SSX2and abolishes CTCF binding, thereby leading to IGF2 activation. BRG1 proteins interact physically, that the SWI/SNF subunits are Because we observed genome-wide DNA hypermethylation, as well associated with transcriptional repression through DNA methyl- as IGF2 up-regulation (see above), we decided to analyze the DNA ation (47), and that BRG1-induced DNA methylation changes methylation status of the H19-ICR in detail. To this end, we did affect CD44 expression (48), are in full support of this hypothesis. bisulfite sequencing on genomic DNAs extracted from HEK-VEC SS18-SSX2 expression affects DNA methylation at the IGF2 and the HEK-FUS cells after 0 and 24 hours of tetracyclin induction. locus. The above hypothesis prompted us to measure genome- As a target, we selected one of the eight consensus CTCF binding wide DNA methylation levels in the SS18-SSX2–expressing cells. To sites located upstream of the H19 gene (Fig. 6). After sequencing of this end, genomic DNAs extracted from the HEK-VEC and HEK- 20 to 22 independently cloned PCR products from each time point FUS cell lines (t = 0 and t = 24), were digested to mononucleotides, of both cell lines, we observed a significant increase of methylated and the total methyl-deoxycytosine (mdC) content in these cytosine residues in the HEK-FUS cell line as compared with the samples was measured by high-performance liquid chromatogra- HEK-VEC cell line (Fig. 6). The most striking differences were phy. In the past, this method has been used to detect genome-wide observed in the first three CpG dinucleotides (including the CTCF mdC levels in tumor samples, normal tissues, and knockout models binding site, Fig. 6). This enhanced ICR methylation is fully (49, 50). Through this approach, we detected an increase (5%) in compatible with the observed IGF2 up-regulation and supports our the overall mdC level in SS18-SSX2–expressing cells as compared hypothesis that SS18-SSX2 expression affects DNA methylation,

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2006 American Association for Cancer Research. Synovial Sarcoma–Associated SS18-SSX Fusion Proteins thereby leading to transcriptional deregulation of downstream acetylation and methylation modifications within the IGF2 target genes. promoter in response to SS18-SSX2protein expression, which are In conclusion, we did cDNA microarray-based expression fully compatible with the enhanced activity of this gene. Finally, we profiling in conjunction with a SS18-SSX2 inducible cell system. observed SS18-SSX2–induced changes in DNA methylation, both at By doing so, we identified various SS18-SSX2–responsive genes, the genome-wide level and at the single-locus level in the IGF2 including a group of genes involved in cholesterol synthesis, a imprinting control region. Taken together, our data indicate that hallmark of malignancy. Integration of our data with literature data the SS18-SSX2–induced transcriptional responses may represent on primary synovial sarcomas led to the identification of a group of epigenetic phenomena hampering normal SWI/SNF functions. The genes, including IGF2 and CD44, that seems to be controlled recent finding that SWI/SNF subunits may act as tumor directly by the SS18-SSX2fusion protein. The opposite transcrip- suppressors (53), and that the histone deacetylase–inhibitory drug tional regulation observed for the IGF2 and IGFBP5 genes indicates FK228 exerts a positive effect on the expression of the SWI/SNF that SS18-SSX2 expression may affect IGF signaling. In addition, protein BRM (54) and a concomitant negative effect on the growth SS18-SSX expression may lead to an uncoupling of JUN and EGR1- of synovial sarcoma cells (55), fully supports our hypothesis. mediated transcription regulatory processes. The deregulation of the SWI/SNF-responsive gene CD44, prompted us to hypothesize that this uncoupling of transcription regulatory processes may be Acknowledgments caused by an SS18-SSX–induced abrogation of the SWI/SNF Received 10/20/2005; revised 7/7/2006; accepted 8/9/2006. complex. This hypothesis is in line with a proposed function of Grant support: Dutch Cancer Society (Koningin Wilhelmina Fonds). the SS18-SSX fusion protein as an ‘‘activator-repressor,’’ and is fully The costs of publication of this article were defrayed in part by the payment of page supported by our observations that the SS18-SSX and BRG1 charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. proteins interact directly and that BRG1 binds directly to the IGF2 The authors thank Paul Joosten (Department of Cell Biology, Radboud University and CD44 promoters. In addition, we observed specific histone Nijmegen) for providing the anti-BRG1 antibody.

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Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 2006 American Association for Cancer Research. The Synovial Sarcoma−Associated SS18-SSX2 Fusion Protein Induces Epigenetic Gene (De)Regulation

Diederik R.H. de Bruijn, Susanne V. Allander, Anke H.A. van Dijk, et al.

Cancer Res 2006;66:9474-9482.

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