Application of crosslinking and mass spectrometry to a challenging target - the case of Dux4

The ectopic expression of Dux4 in myoblasts is the cause of FacioScapuloHumeral muscular Dystrophy (FSHD), a cureless condition affecting 1 person in 8000. Dux4 is a potent transcription factor that binds to several activators and dysregulates the gene expression in myoblasts. The study of these protein interactions has been challenging because the sequence of Dux4 has several regions that present high net electrostatic charges. In addition, the sequence is almost devoid of lysine residues, thus preventing crosslinking and mass spectrometry probing with the standard NHS-ester chemistry. We show how we were able to circumvent these difficulties using alternative crosslinking chemistries. The identified crosslinks mapped the interactions to unexpected regions of Dux4, thus advancing our understanding of its toxicity mechanisms.