Reprogramming of mesenchymal stem cells by the synovial sarcoma-associated oncogene SYT-SSX2.

Cell identity is determined by its gene expression programs. The ability of a cell to change its identity and produce cell types outside its lineage is achieved by the activity of transcription controllers capable of reprogramming differentiation gene networks. The synovial sarcoma (SS)-associated protein, SYT-SSX2, reprograms myogenic progenitors and human bone marrow-derived mesenchymal stem cells (BMMSCs) by dictating their commitment to a pro-neural lineage. It fulfills this function by directly targeting an extensive array of neural-specific genes as well as genes of developmental pathway mediators. Concomitantly, the ability of both myoblasts and BMMSCs to differentiate into their normal myogenic and adipogenic lineages was compromised. SS is believed to arise in mesenchymal stem cells where formation of the t(X/18) translocation product, SYT-SSX, constitutes the primary event in the cancer. SYT-SSX is therefore believed to initiate tumorigenesis in its target stem cell. The data presented here allow a glimpse at the initial events that likely occur when SYT-SSX2 is first expressed, and its dominant function in subverting the nuclear program of the stem cell, leading to its aberrant differentiation, as a first step toward transformation. In addition, we identified the fibroblast growth factor receptor gene, Fgfr2, as one occupied and upregulated by SYT-SSX2. Knockdown of FGFR2 in both BMMSCs and SS cells abrogated their growth and attenuated their neural phenotype. These results support the notion that the SYT-SSX2 nuclear function and differentiation effects are conserved throughout sarcoma development and are required for its maintenance beyond the initial phase. They also provide the stem cell regulator, FGFR2, as a promising candidate target for future SS therapy.

The synovial sarcoma translocation protein SYT-SSX2 recruits beta-catenin to the nucleus and associates with it in an active complex.

Localization of beta-catenin in the cell is a key determinant in its decision to function as a critical mediator of cell adhesion at the surface or a transcription activator in the nucleus. SYT-SSX2 is the fusion product of the chromosomal translocation, t(X;18)(p11.2;q11.2), which occurs in synovial sarcoma, a soft tissue tumor. SYT-SSX2 is known to associate with chromatin remodeling complexes and is proposed to be involved in controlling gene expression. We report that SYT-SSX2 plays a direct role in beta-catenin regulation. When expressed in mammalian cells, SYT-SSX2-induced beta-catenin recruitment to the nucleus. Interestingly, known target genes of canonical Wnt were not activated as a result of SYT-SSX2 expression, nor was the nuclear localization of beta-catenin due to one of the signaling pathways normally implicated in this event. beta-Catenin accumulation in the nucleus led to the formation of a transcriptionally active nuclear complex that contained SYT-SSX2 and beta-catenin. More importantly, depletion of SYT-SSX2 in primary synovial sarcoma cells resulted in loss of nuclear beta-catenin signal and a significant decrease in its signaling activity. These results unravel a novel pathway in the control of beta-catenin cellular transport and strongly suggest that SYT-SSX2 contributes to tumor development, in part through beta-catenin signaling.

p300 interacts with the nuclear proto-oncoprotein SYT as part of the active control of cell adhesion.

Complexes containing p300, but not CBP, and the nuclear proto-oncoprotein SYT were detected in confluent cultures of G1-arrested cells but not in sparse cells or during S or G2. SYT sequences constitute the N-terminal segment of a fusion oncogene product, SYT-SSX, routinely detected in synovial sarcoma, an aggressive human tumor. SYT/p300 complex formation promotes cell adhesion to a fibronectin matrix, as reflected by compromise of this process in cells expressing SYT dl mutants that retain p300 binding activity and in the primary fibroblasts of p300 but not CBP heterozygous null mice. The mechanism linking the action of SYT/p300 complexes to adhesion function is, at least in part, transcription activation-independent and results in proper activation of beta1 integrin, a major adhesion receptor.

ST-2, a telomere and subtelomere duplex and G-strand binding protein activity in Trypanosoma brucei.

From Trypanosoma brucei, we identified ST-2, a protein complex that interacts with telomeric DNA and exhibits novel features. It binds specifically to the double-stranded telomere repeats (TTAGGG) and more tightly to the subtelomere 29-base pair elements that separate the telomere repeats from their proximal telomere-associated sequences. Interestingly, ST-2 showed still greater affinity for the G-rich strand of the telomere present either as an overhang or in a single-stranded form, but it exhibited the highest affinity for the G-rich strand of the subtelomere repeats. The binding characteristics of ST-2 are complementary to those of ST-1, a 39-kDa polypeptide we previously identified in T. brucei (Eid, J., and Sollner-Webb, B. (1995) Mol. Cell. Biol. 15, 389-397) that binds preferentially to the C-rich strands of the subtelomere and telomere repeats. UV cross-linking revealed five polypeptides of ST-2 that bind directly to the G-rich strand of the DNA, one of which is phosphorylated. Furthermore, the presence of ST-1 is critical for ST-2 complex binding both to the G-rich strand and to the duplex DNA, evidently as part of the ST-2 complex. This indicates that when binding to the duplex subtelomere and telomere repeats, ST-2 may act as a protein bridge with its ST-1 subunit binding to the C-rich strand and its five other cross-linkable polypeptides binding to the G-rich strand. Such an association could serve to hold the genomic subtelomeric and telomeric sequences in a partially single-stranded configuration to facilitate the recombinational events in this region that are crucial to the parasite.

ST-1, a 39-kilodalton protein in Trypanosoma brucei, exhibits a dual affinity for the duplex form of the 29-base-pair subtelomeric repeat and its C-rich strand.

In our attempt to identify telomere region-binding proteins in Trypanosoma brucei, we identified ST-1, a polypeptide with novel features. ST-1 was chromatographically purified from S-100 cell extracts and was renatured from a sodium dodecyl sulfate-protein gel as a 39-kDa polypeptide. It forms a specific complex with the trypanosome telomere repeats of TTAGGG, but more significantly, it shows a higher affinity for the 29-bp subtelomere repeats of T. brucei. These 29-mer boxes are a large tandem series of telomere-derived repeats which separate the simple telomere DNA from middle-repetitive telomere-associated sequences on many chromosomes. ST-1 is the first example of a protein binding within such large repetitive subtelomere elements in trypanosomes or other organisms. ST-1 is also novel in that it has a selective affinity for the C-rich strands of both the subtelomeric 29-mer and the telomere repeats, comparable to that for the duplex form of the respective repeats. All previously described telomere-binding proteins have affinity for only the duplex form or for the G-rich strand. This C-rich strand binding specificity of ST-1 may provide insight into this protein's mechanism of binding in vivo.

Homologous recombination in the tandem calmodulin genes of Trypanosoma brucei yields multiple products: compensation for deleterious deletions by gene amplification.

Homologous recombination between a calmodulin-neomycin-resistance fusion gene and the Trypanosoma brucei chromosome takes place not only in the large 5'- and 3'-flanking segments of the calmodulin locus but also in any of the four tandem genomic calmodulin genes. This results in a recombined locus consisting of the chimeric neor gene and four, three, two, one, or zero functional calmodulin genes. Cells bearing this latter event have half of their normal number of intact calmodulin genes and an accompanying phenotype of slow growth. Over months of propagation, these lines acquire additional calmodulin genes, frequently by amplifying a calmodulin gene at the untargeted locus, and concomitantly revert to normal growth rate. This response could be related to the property of the trypanosome of maintaining most housekeeping genes in tandem chromosomal arrays. Recombination appears to be initiated by a crossover event between the linearized end of the transfecting plasmid and a homologous region in the host genome; the second crossover generally occurs internally and in that region requires no more than 87 bp of homology.