Phase separation control of transcription in Ewing sarcoma Eighty-five percent of Ewing sarcomas are caused by translocations between the genes EWS and FLI1, creating a powerful oncogene – EWS-FLI1. The remainder are caused by translocations involving an EWS homologue, FUS. This sarcoma is an aggressive primary bone tumor affecting 1 in 300,000 people annually with more than 80% of tumors occurring in adolescents, making it the second most common pediatric bone cancer. Half of known translocations identified in sarcomas involve one of a family of three proteins – FUS, EWS, and TAF15, known as the FET family of proteins. Our work over the last few years has contributed to expanding the model for FET protein regulation of transcription. FET proteins control transcription in an organizational capacity. FET proteins undergo an assembly process called phase separation, which is the process thought to drive formation of non-membrane bound organelles, also called granules. In this proposal, we highlight our accomplishments isolating an EWS-FLI1 associated granule from cells. These granules have considerable similarity to those we have discovered to associate with RNA polymerase II (RNA Pol II) during transcription, which we refer to as transcription granules. This proposal aims to establish a model that EWS- FLI1 fusion protein seed aberrant granules that incorporate EWSR1 and other FET proteins to alter transcription and promote tumorigenesis. Our proposed work will investigate basic molecular mechanisms by which EWS-FLI1 can contribute and drive pediatric sarcoma pathology. Our proposal involves three aims. (1) We will determine the binding partners that comprise fusion protein granules for EWS-FLI1. (2) We will investigate EWS-FLI1 and EWSR1 activities to bind nucleic acids, phase separate, and alter transcription through effects on nucleic structures. Finally, (3) we will investigate how changes to DNA structure and chromatin are linked to aberrant phase separation b