PAX3-FOXO1 directs CBP/P300 via its activation domain to build RNA Pol2 clusters

Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of PAX3-FOXO1 forms a small alpha helical hook that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine, and also CBP/p300. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription. RNA-seq and Pol2 HiChIP in RMS cells treated with HAT inhibitors and degraders. Pol2 ChIP-seq in ABE RMS cells with wt P3F or C793R mutant P3F and H3K27ac ChIP-seq in RMS cells treated with HAT degraders. Pol2 HiChIP in RMS cells with P3F-FKBP12 treated with DMSO or dTAG.