Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcriptional activities. The SS18 protein functions as a transcriptional coactivator and is associated with the SWI/SNF complex, whereas the SSX proteins function as transcriptional corepressors and are associated with the polycomb complex. The domains involved in these opposite transcriptional activities are retained in the SS18-SSX fusion proteins. Here, we set out to determine the direct transcriptional consequences of conditional SS18-SSX2 fusion protein expression using complementary DNA microarray-based profiling. By doing so, we identified several clusters of SS18-SSX2-responsive genes, including a group of genes involved in cholesterol synthesis, which is a general characteristic of malignancy. In addition, we identified a group of SS18-SSX2-responsive genes known to be specifically deregulated in primary synovial sarcomas, including IGF2 and CD44. Furthermore, we observed an uncoupling of EGR1, JUNB, and WNT signaling in response to SS18-SSX2 expression, suggesting that the SWI/ SNF-associated coactivation functions of the SS18 moiety are impaired. Finally, we found that SS18-SSX2 expression affects histone modifications in the CD44 and IGF2 promoters and DNA methylation levels in the IGF2 imprinting control region. Together, we conclude that the SS18-SSX2 fusion protein may act as a so-called transcriptional ''activator-repressor,'' which induces downstream target gene deregulation through epigenetic mechanisms. Our results may have implications for both the development and clinical management of synovial sarcomas. (Cancer Res 2006; 66(19): 9474-82)

The papillary renal cell carcinoma (RCC)-associated (X;1)(p11;q21) translocation fuses the genes PRCC and TFE3 and leads to cancer by an unknown molecular mechanism. We here demonstrate that the mitotic checkpoint protein MAD2B interacts with PRCC. The PRCC-TFE3 fusion protein retains the MAD2B interaction domain, but this interaction is impaired. In addition, we show that two t(X;1)-positive RCC tumor cell lines are defective in their mitotic checkpoint. Transfection of PRCCTFE3, but not the reciprocal product TFE3PRCC, disrupts the mitotic checkpoint in human embryonic kidney cells. Our results suggest a dominant-negative effect of the PRCCTFE3 fusion gene leading to a mitotic checkpoint defect as an early event in papillary RCCs. Chromosomal translocations often occur as tumor-specific abnormalities, suggesting that the underlying molecular alterations are crucial for tumor development (1, 2). In a subset of renal cell carcinomas (RCCs) with chromophilic histology and a mainly papillary growth pattern, referred to as papillary RCCs, chromosomal translocations involving the Xp11 region, usually t(X;1)(p11;q21), are recurrently encountered (3-11). Positional cloning of the Xp11 breakpoint by us and others revealed that the t(X;1)(p11;q21) translocation results in an in-frame fusion of the transcription factor TFE3 gene on the X-chromosome to the PRCC gene on chromosome 1 (12-14). Consequently, two fusion genes are formed, TFE3PRCC and PRCCTFE3, both of which are expressed in t(X;1)-positive tumor cells (13).TFE3 is a ubiquitously expressed transcription factor characterized by the presence of a basic region followed by helix-loophelix and leucine zipper domains, both of which are needed for dimerization and DNA binding of the transcription factor (15-17). The fusion protein PRCCTFE3 retains all these domains. PRCC is also ubiquitously expressed and characterized by a relatively high proline content. We have shown that the Nterminal 156 amino acids of PRCC, when fused to TFE3, significantly elevate the transactivating capacity of this fusion protein as compared with wild-type TFE3 (18). Moreover, transfection studies with conditionally immortalized mouse renal proximal epithelial cells, from which chromophilic tumors are thought to arise, showed that PRCCTFE3 could bypass temperature-induced growth arrest and differentiation (19). On the basis of the limited functional information available, we chose to further characterize PRCC via the identification of interacting proteins through yeast two-hybrid screening. This resulted in the identification of MAD2B, member of a family of genes involved in processes of mitotic checkpoint control mechanisms (20-22). Our results indicate that PRCCTFE3 expression may contribute to RCC development through a mechanism that affects the PRCC-MAD2B interaction. Materials and MethodsYeast Two-Hybrid Analysis. Yeast two-hybrid analysis and filter lift assays were basically performed as described by the manufacturer (Stratagene). In short, yeast cells (pJ69-4A), kindly provided by Phi...

The papillary renal cell carcinoma-associated t(X;1)(p11;q21) leads to fusion of the transcription factor TFE3 gene on the X-chromosome to a novel gene, PRCC, on chromosome 1. As a result, two putative fusion proteins are formed: PRCCTFE3, which contains all known domains for DNA binding, dimerization, and transactivation of the TFE3 protein, and the reciprocal product TFE3PRCC. Upon transfection into COS cells, both wild type and fusion proteins were found to be located in the nucleus. When comparing the transactivating capacities of these (fusion) proteins, signi®cant dierences were noted. PRCCTFE3 acted as a threefold better transactivator than wild type TFE3 both in a TFE3-speci®c and in a general (Zebra) reporter assay. In addition, PRCC and the two fusion proteins were found to be potent transactivators in the Zebra reporter assay. We propose that, as a result of the (X;1) translocation, fusion of the N-terminal PRCC sequences to TFE3 alters the transactivation capacity of the transcription factor thus leading to aberrant gene regulation and, ultimately, tumor formation. Oncogene (2000) 19, 69 ± 74.

BackgroundPreviously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established.Methodology/Principal FindingsUsing a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed.Conclusions/SignificanceThe small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.

nma, a novel gene, was isolated by using a subtractive hybridization technique in which the gene expression was compared in a panel of human melanoma cell lines with different metastatic potential. nma mRNA expression (I .5 kb) is high in poorly metastatic human melanoma cell lines and xenografts and completely absent in highly metastatic human melanoma cell lines. Fluorescence in situ hybridization combined with the analysis of a panel of human-rodent somatic cell hybrids indicated that the nma gene is located on human chromosome 10, in the region p I I .2-p 12.3. Sequence analysis of nma showed no homologies with other known genes or proteins, except for several partially sequenced cDNAs. The predicted amino acid sequence suggests that the protein encoded by nma contains a transmembrane domain. Expression of nma is high in human kidney medulla, placenta and spleen, low in kidney cortex, liver, prostate and gut and absent in lung and muscle. Whereas nma is not expressed in normal skin tissue, expression is high in melanocytes and in 3 out of I I melanoma metastases tested.