Highly connected 3D chromatin networks established by an oncogenic fusion protein shape tumor cell identity

Tumor cell identity is the product of complex interactions between oncogenic drivers and mechanisms regulating normal differentiation pathways. Cell fate transitions observed in embryonic development involve changes in 3D genomic organization that provide proper lineage specification, however, whether similar events also occur within tumor cells and contribute to cancer evolution remains largely unexplored. Here we modeled this process in the pediatric bone cancer Ewing sarcoma and investigated high resolution looping and large-scale 3D nuclear conformation changes associated with EWS-FLI1, the oncogenic fusion protein that drives this tumor. We show that chromatin interactions in Ewing sarcoma cells are dominated by highly connected looping hubs centered on EWS-FLI1 binding sites, which directly control the activity of linked enhancers and promoters to establish oncogenic expression programs. Depletion of EWS-FLI1 led to the loss of looping networks associated with the oncoprotein and, strikingly, also resulted in widespread nuclear reorganization through the establishment of new patterns of looping and large-scale inter-compartment connectivity characteristic of mesenchymal stem cells, a candidate cell of origin for this tumor. Our data thus demonstrate that major architectural features of nuclear organization in cancer cells can be dependent on a single oncogenic event and readily reversed to re-establish latent differentiation programs.