PAX3-FOXO1 Contacts BRD4 through Its Acetylated Intrinsically Disordered Region

Intrinsically disordered regions (IDRs) of transcription factors are frequent sites for post-translational modifications (PTMs), which mediate regulation through diverse mechanisms including protein-protein interactions. In the fusion oncoprotein PAX3-FOXO1, which drives alveolar rhabdomyosarcoma, a lysine-rich region of the FOXO1 IDR is subject to acetylation resulting in stabilization and enhanced transcriptional activity. Here, we leveraged ¹³C direct-detect nuclear magnetic resonance (NMR) spectroscopy to characterize acetylation in this system and identified a novel acetylation site corresponding to lysine 233 in endogenous FOXO1. In previous structural characterization of the endogenous FOXO1 DNA binding domain, local structure appears to prevent this site from becoming acetylated, suggesting that it becomes exposed in the context of the fusion protein. In addition, we demonstrate that the first bromodomain of the bromodomain and extraterminal domain-containing protein BRD4 binds to the acetylated region of interest and that this interaction is inhibited through the bromodomain and extraterminal domain inhibitor JQ1. These findings confer molecular mechanistic detail to previous observations that BRD4 and PAX3-FOXO1 colocalize at superenhancers in ARMS, adding to the growing body of literature exploring how BRD4 contacts cancer-relevant transcription factors in ways potentially relevant to the use of bromodomain and extraterminal domain inhibitors in cancer treatment.