Modeling initiation of Ewing sarcoma in human neural crest cells

Ewing sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that express EWS-ETS fusion genes as driver mutations. Although the histogenesis of ESFT is controversial, mesenchymal (MSC) and/or neural crest (NCSC) stem cells have been implicated as cells of origin. For the current study we evaluated the consequences of EWS-FLI1 expression in human embryonic stem cell-derived NCSC (hNCSC). Ectopic expression of EWS-FLI1 in undifferentiated hNCSC and their neuro-mesenchymal stem cell (hNC-MSC) progeny was readily tolerated and led to altered expression of both well established as well as novel EWS-FLI1 target genes. Importantly, whole genome expression profiling studies revealed that the molecular signature of established ESFT is more similar to hNCSC than any other normal tissue, including MSC, indicating that maintenance or reactivation of the NCSC program is a feature of ESFT pathogenesis. Consistent with this hypothesis, EWS-FLI1 induced hNCSC genes as well as the polycomb proteins BMI-1 and EZH2 in hNC-MSC. In addition, up-regulation of BMI-1 was associated with avoidance of cellular senescence and reversible silencing of p16. Together these studies confirm that, unlike terminally differentiated cells but consistent with bone marrow-derived MSC, NCSC tolerate expression of EWS-FLI1 and ectopic expression of the oncogene initiates transition to an ESFT-like state. In addition, to our knowledge this is the first demonstration that EWS-FLI1-mediated induction of BMI-1 and epigenetic silencing of p16 might be critical early initiating events in ESFT tumorigenesis.     

A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells

The Rb/E2F complex represses S-phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S-phase gene repression, which involves histone-modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock-down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S-phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h-depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.