Abstract
Over 95% of ependymomas (EPN) that arise in the cortex are characterized by gene fusions commonly involving the zinc finger translocation associated (ZFTA) protein. These fusion oncoproteins (FOs) join ZFTA with either a transcription factor or co-activator, leading to a chimeric protein capable of novel functions. To understand the core mechanism of ZFTA FOs we leveraged super resolution and lattice light sheet microscopy. We show that ZFTA FOs undergo liquid-like phase separation to form dynamic nuclear condensates on chromatin. Nuclear condensate formation is critical for activation of ZFTA FO oncogenic targets and the initiation of brain tumors in mice. Furthermore, we show that DNA binding is necessary for proper condensate distribution in the nucleus and provide the first NMR data of a key DNA binding domain of ZFTA as a resource for drug discovery efforts. Using machine-learning models trained on protein sequences of fusion oncoproteins in pediatric and adult cancers, we identified critical amino acid residues in ZFTA FOs necessary for neoplastic transformation. Critically, insights gained from AI-driven mutagenesis studies enabled the creation of synthetic ZFTA FOs, never seen in biology, and capable of driving oncogenic gene activation and brain tumor development. These findings reveal the molecular building blocks important for the assembly of ZFTA fusion oncoproteins, informing our general understanding of brain tumor initiation and providing opportunities for therapeutic intervention.