PolyA/polyQ-mediated conformational rewiring regulates DNA engagement and drives aggregation in the neuronal transcription factor Ascl1

Ascl1 is a pioneer transcription factor that drives neuronal fate decisions, yet the structural basis of its activity remains elusive. Besides the basic helix-loop-helix (bHLH) domain which dimerizes with other transcription factors and binds DNA, Ascl1 contains long low-complexity intrinsically disordered regions (IDRs), including a polyA/polyQ tract of unknown function. Here we use single-molecule FRET to map the conformational ensemble of full-length Ascl1 across monomeric, heterodimeric, and DNA-bound states. Monomeric Ascl1 is largely disordered but displays multivalent coupling between the N-terminal IDR and the bHLH domain, with the polyA/polyQ tract opposing bHLH compaction. E12 binding folds the bHLH domain and remodels the N-IDR, increasing dynamics across the N-IDR while suppressing them locally within the polyA/polyQ tract. Deleting the tract weakens nonspecific DNA binding by the Ascl1/E12 heterodimer without disrupting heterodimerization or high-affinity E-box binding, abolishes aggregation in vitro, and increases Ascl1 abundance in HEK293T cells. Thus, the polyA/polyQ tract acts as a regulatory module that promotes nonspecific DNA engagement while imposing a cost in solubility and cellular abundance.