Reprogramming of mesenchymal stem cells by oncogenes.

Mesenchymal stem cells (MSCs) originate from embryonic mesoderm and give rise to the multiple lineages of connective tissues. Transformed MSCs develop into aggressive sarcomas, some of which are initiated by specific chromosomal translocations that generate fusion proteins with potent oncogenic properties. The sarcoma oncogenes typically prime MSCs through aberrant reprogramming. They dictate commitment to a specific lineage but prevent mature differentiation, thus locking the cells in a state of proliferative precursors. Deregulated expression of lineage-specific transcription factors and controllers of chromatin structure play a central role in MSC reprogramming and sarcoma pathogenesis. This suggests that reversing the epigenetic aberrancies created by the sarcoma oncogenes with differentiation-related reagents holds great promise as a beneficial addition to sarcoma therapies.

Genome-wide recruitment to Polycomb-modified chromatin and activity regulation of the synovial sarcoma oncogene SYT-SSX2.

BACKGROUND: SYT-SSX is the oncogene associated with synovial sarcoma (SS), a stem cell disease. SYT-SSX is thought to be responsible for sarcoma initiation and development. It interacts with components of Polycomb and SWI/SNF complexes, the two epigenetic controllers that maintain the heritable status of differentiation-specific genes in the stem/progenitor cell. Through these associations SYT-SSX is thought to alter gene expression programs by epigenetic mechanisms. Recently, we reported that SYT-SSX2 reprograms mesenchymal stem cells and myoblasts by dictating their commitment to the neural lineage while disrupting their normal differentiation. This reprogramming was due to the direct occupancy of proneural genes by the SYT-SSX2 nuclear complex. To gain a clear understanding of SYT-SSX2 control of gene expression networks, we conducted a thorough genome-wide analysis to determine the mechanism of its recruitment and identify signature sets of epigenetic markers that would predict its targeting and transcriptional activity. RESULTS: SYT-SSX2 was recruited to distinct loci across all chromosomes, and an overwhelming number of Polycomb-modified sites enriched with the trimethylated histone H3 on lysine 27 (H3K27me3) formed the main recruiting module for SYT-SSX2. Not all SYT-SSX2/H3K27me3-occupied genes had altered expression, denoting the requirement for additional signals upon oncogene binding. Differential binding and epigenetic patterns distinguished upregulated and downregulated genes. Most activated genes had SYT-SSX2 sites enriched with H3K27me3 within their body or near their transcription start site (TSS) whereas a majority of downregulated genes were characterized by SYT-SSX2/H3K27me3-rich regions at long-range, or by modifications associated with transcription activation within the gene body or near the TSS. Hierarchical and functional clustering identified H3K27me3 as the dominant epigenetic marker associated with SYT-SSX2 binding and gene expression. Notably, this analysis revealed a cluster of upregulated neuronal genes densely covered by H3K27me3, consistent with programming toward the neural lineage by SYT-SSX2 observed previously. CONCLUSIONS: The data analysis revealed that Polycomb complexes or their modified chromatin and their stably silenced differentiation programs seem to be the main target for SYT-SSX2, suggesting that their perturbation is at the center of tumorigenesis driven by the oncogene. Further research into this mechanism is crucial to the full understanding of SS biology.

Integrating research into a molecular cloning course to address the evolving biotechnology landscape.

The rapid development of molecular biotechnology presents a curricular challenge for educators trying to provide students with relevant coursework. A comprehensive biology education should also include opportunities for students to develop intellectual and technical skills through authentic research experiences. Integrating relevant and interesting research projects into their classes, however, can be a challenging task for instructors. To address these varied demands, we redesigned our existing molecular cloning course to incorporate an independent research project assessing calcium signaling. In the revised course, students use traditional and recombination-based cloning strategies to generate bacterial and mammalian expression vectors encoding CaMPARI, a novel fluorescent calcium indicator. Bacterially-expressed CaMPARI is used in protein quantification and purification assays. Students must also design their own research project evaluating the effect of chemotherapeutic agents on calcium signaling in a mammalian system. Revised and novel labs were designed to be modular, facilitating their integration into the course over 2 years. End-of-semester student evaluations were compared between years revealing a significant difference in students' perception of the course's difficulty between years. This change in attitude highlights potential pedagogical considerations that must be examined when introducing new material and activities into existing courses. Since calcium signaling is important for cellular process across diverse species, instructors may be able to develop research projects within their respective areas of interest. Integration of authentic research experiences into the curriculum is challenging; however, the framework described here provides a versatile structure that can be adapted to merge diverse instructor interests with evolving educational needs.

The synovial sarcoma SYT-SSX2 oncogene remodels the cytoskeleton through activation of the ephrin pathway.

Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. In this study, we demonstrate that SYT-SSX2 is a unique oncogene. Rather than confer enhanced proliferation on its target cells, SYT-SSX2 instead causes a profound alteration of their architecture. This aberrant morphology included elongation of the cell body and formation of neurite-like extensions. We also observed that cells transduced with SYT-SSX2 often repulsed one another. Notably, cell repulsion is a known component of ephrin signaling. Further analysis of SYT-SSX2-infected cells revealed significant increases in the expression and activation of Eph/ephrin pathway components. On blockade of EphB2 signaling SYT-SSX2 infectants demonstrated significant reversion of the aberrant cytoskeletal phenotype. In addition, we discovered, in parallel, that SYT-SSX2 induced stabilization of the microtubule network accompanied by accumulation of detyrosinated Glu tubulin and nocodazole resistance. Glu tubulin regulation was independent of ephrin signaling. The clinical relevance of these studies was confirmed by abundant expression of both EphB2 and Glu tubulin in SYT-SSX2-positive synovial sarcoma tissues. These results indicate that SYT-SSX2 exerts part of its oncogenic effect by altering cytoskeletal architecture in an Eph-dependent manner and cytoskeletal stability through a concurrent and distinct pathway.

Mineral accumulation in vegetative and reproductive tissues during seed development in Medicago truncatula.

Enhancing nutrient density in legume seeds is one of several strategies being explored to improve the nutritional quality of the food supply. In order to develop crop varieties with increased seed mineral concentration, a more detailed understanding of mineral translocation within the plant is required. By studying mineral accumulation in different organs within genetically diverse members of the same species, it may be possible to identify variable traits that modulate seed mineral concentration. We utilized two ecotypes (A17 and DZA315.16) of the model legume, Medicago truncatula, to study dry mass and mineral accumulation in the leaves, pod walls, and seeds during reproductive development. The pod wall dry mass was significantly different between the two ecotypes beginning at 12 days after pollination, whereas there was no significant difference in the average dry mass of individual seeds between the two ecotypes at any time point. There were also no significant differences in leaf dry mass between ecotypes; however, we observed expansion of A17 leaves during the first 21 days of pod development, while DZA315.16 leaves did not display a significant increase in leaf area. Mineral profiling of the leaves, pod walls, and seeds highlighted differences in accumulation patterns among minerals within each tissue as well as genotypic differences with respect to individual minerals. Because there were differences in the average seed number per pod, the total seed mineral content per pod was generally higher in A17 than DZA315.16. In addition, mineral partitioning to the seeds tended to be higher in A17 pods. These data revealed that mineral retention within leaves and/or pod walls might attenuate mineral accumulation within the seeds. As a result, strategies to increase seed mineral content should include approaches that will enhance export from these tissues.

Targeting the Wnt pathway in synovial sarcoma models.

UNLABELLED: Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In an SYT-SSX2 transgenic model, we show that a constitutive Wnt/ß-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of ß-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/ß-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/ß-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents. SIGNIFICANCE: Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT­SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans.

The synovial sarcoma-associated SYT-SSX2 oncogene antagonizes the polycomb complex protein Bmi1.

This study demonstrates deregulation of polycomb activity by the synovial sarcoma-associated SYT-SSX2 oncogene, also known as SS18-SSX2. Synovial sarcoma is a soft tissue cancer associated with a recurrent t(X:18) translocation event that generates one of two fusion proteins, SYT-SSX1 or SYT-SSX2. The role of the translocation products in this disease is poorly understood. We present evidence that the SYT-SSX2 fusion protein interacts with the polycomb repressive complex and modulates its gene silencing activity. SYT-SSX2 causes destabilization of the polycomb subunit Bmi1, resulting in impairment of polycomb-associated histone H2A ubiquitination and reactivation of polycomb target genes. Silencing by polycomb complexes plays a vital role in numerous physiological processes. In recent years, numerous reports have implicated gain of polycomb silencing function in several cancers. This study provides evidence that, in the appropriate context, expression of the SYT-SSX2 oncogene leads to loss of polycomb function. It challenges the notion that cancer is solely associated with an increase in polycomb function and suggests that any imbalance in polycomb activity could drive the cell toward oncogenesis. These findings provide a mechanism by which the SYT-SSX2 chimera may contribute to synovial sarcoma pathogenesis.