jats:titleAbstract</jats:title>jats:sec jats:titleBackground</jats:title> jats:pThe pro-neural transcription factor ASCL1 is a master regulator of neurogenesis and a key factor necessary for the reprogramming of permissive cell types to neurons. Endogenously, ASCL1 expression is often associated with neuroblast stem-ness. Moreover, ASCL1-mediated reprogramming of fibroblasts to differentiated neurons is commonly achieved using artificially high levels of ASCL1 protein, where ASCL1 acts as an “on-target” pioneer factor. However, the genome-wide effects of enhancing ASCL1 activity in a permissive neurogenic environment has not been thoroughly investigated. Here, we overexpressed ASCL1 in the neuronally-permissive context of neuroblastoma (NB) cells where modest endogenous ASCL1 supports the neuroblast programme.</jats:p> </jats:sec>jats:sec jats:titleResults</jats:title> jats:pIncreasing ASCL1 in neuroblastoma cells both enhances binding at existing ASCL1 sites and also leads to creation of numerous additional, lower affinity binding sites. These extensive genome-wide changes in ASCL1 binding result in significant reprogramming of the NB transcriptome, redirecting it from a proliferative neuroblastic state towards one favouring neuronal differentiation. Mechanistically, ASCL1-mediated cell cycle exit and differentiation can be increased further by preventing its multi-site phosphorylation, which is associated with additional changes in genome-wide binding and gene activation profiles.</jats:p> </jats:sec>jats:sec jats:titleConclusions</jats:title> jats:pOur findings show that enhancing ASCL1 activity in a neurogenic environment both increases binding at endogenous ASCL1 sites and also results in additional binding to new low affinity sites that favours neuronal differentiation over the proliferating neuroblast programme supported by the endogenous protein. These findings have important implications for controlling processes of neurogenesis in cancer and cellular reprogramming.</jats:p> </jats:sec>