SUMMARY Ewing sarcoma, a cancer of the bone and soft tissue of children and young adults, remains a highly lethal cancer despite the use of aggressive chemotherapy, radiation, and surgery. The tumor is dependent on the development of a hybrid gene that brings together parts of two different genes, EWSR1 and FLI1, through chromosomal translocation or chromoplexy. The resulting EWS-FLI1 fusion oncoprotein acts as a transcriptional and chromatin regulator. Building from the work of our lab and others demonstrating that EWS-FLI1 gains neomorphic activity to regulates chromatin state at microsatellite coopted to become enhancers in tumor cells. However, we have shown that the activity and genomic targeting of EWS-FLI1 is influenced by the underlying epigenomic state of the cell. We demonstrated that primary and in vitro differentiated mesenchymal stem cells offer a chromatin state similar to that of Ewing sarcoma. We hypothesize that during stem cell differentiation a unique permissive chromatin state develops that enables EWS-FLI1. Further, we hypothesize that this permissive state is made up of chromatin regulators, characteristically modified histones and specific RNAs. In this project we will employ cancer cell (Aim 1) and stem cell developmental approaches (Aim 2) to identify the protein and RNA interactors and the posttranslational modifications of histones that create a favorable environment. We predict that critical features will be shared between both model systems. We will test the impact of these factors by evaluating the activity of EWS-FLI1 on chromatin states and transcription when these factors are modulated. The generation of Ewing sarcoma patient derived stem cells will enable us to evaluate the impact of EWS-FLI1 across the process of cellular differentiation. Integrated single cell analytics of chromatin accessibility and the transcriptome will enable direct evaluation of the impact of EWS-FLI1 on chromatin and, reciprocally, chromatin on EWS-FLI1. We will specifically study the biochemical interaction and functional relationship between EWS-FLI1 and the transcriptional regulator PAX7, one of the interactors identified during pilot experimentation.