Evaluation of the dependency of PAX3-FOXO1 on Cys793 and p300/CBP for transactivation of target genes in alveolar rhabdomyosarcoma

The fusion transcription factor PAX3-FOXO1 (P3F) is the major driver of alveolar rhabdomyosarcoma. We performed a CRISPR/Cas9-based domain screen in P3F-positive rhabdomyosarcoma cells and identified a small, structured and highly important domain in the transactivation domain of P3F. By mutation of individual amino acids in this domain we further demonstrate that cysteine C793 is indispensable for proper function of P3F. The small transactivation domain with C793 mediates the interaction with the transcriptional co-activators p300/CBP. In order to identify P3F target genes that depend on presence of C793, we performed a gene expression profiling in the aRMS cell line Rh4 by RNAseq after exchange of endogenous P3F with either wildtype or C793S mutant P3F. Furthermore, in order to find P3F target genes that are also dependent on p300/CBP, we performed RNAseq analysis with Rh4 cells after double knock-out of p300 and CBP.