Epigenetic Analysis in Ewing Sarcoma

Ewing sarcoma is a malignant tumor of bone and soft tissue, primarily driven by the EWSR1::FLI1 fusion oncogene. This chimeric transcription factor reprograms gene regulatory networks by aberrantly activating enhancers and binding to GGAA microsatellite repeats, thereby reshaping the epigenetic landscape of tumor cells. Given that chromatin accessibility governs transcription factor binding and downstream gene expression, high-resolution mapping of protein-DNA interactions is critical for elucidating the epigenetic mechanisms underlying Ewing sarcoma pathogenesis. This chapter presents a comprehensive, end-to-end framework for the analysis of Cleavage Under Targets and Release Using Nuclease (CUT&RUN) data, with a focus on profiling EWSR1::FLI1 binding sites and their associated chromatin features. We detail each step of the analytical pipeline, including raw FASTQ processing, alignment, quality control, peak calling, and downstream analyses. To demonstrate the workflow, both publicly available data and data generated in our laboratory are used. Overall, this chapter serves as a practical and detailed methodological guide for researchers aiming to dissect the epigenetic architecture of Ewing sarcoma using CUT&RUN technology.