Multi-layered regulatory networks driving human dopaminergic neuron differentiation

Neuronal differentiation requires coordinated regulation across chromatin organization, gene expression, protein abundance, and post-translational modifications. Using the LUHMES human dopaminergic neuronal differentiation model, we integrated proteome and phosphoproteome profiling with previously generated enhancer–promoter interaction maps from NET-CAGE and HiCap and transcriptomic analysis across three consecutive differentiation stages. Differentiation was accompanied by increased abundance and phosphorylation of proteins involved in axon guidance, cytoskeletal organization, and synaptic signaling, alongside repression of the cell cycle, DNA replication, and chromatin-associated programs. Phosphoproteome analysis further revealed extensive remodeling of signaling networks associated with neuronal maturation. Enhancer–promoter interaction analysis revealed substantially greater rewiring at enhancers than promoters and identified master and relay transcription factors regulated across multiple molecular layers. siRNA-mediated knockdown showed that transcription factors MYT1, ISL2, and NHLH2 are crucial for proper neuronal maturation, whereas LCOR acts as a negative regulator of differentiation. Integrative network reconstruction further nominated MEOX2 as a candidate enhancer-associated regulator of late dopaminergic maturation. Together, these findings provide a multi-layered view of regulatory networks governing human neuronal differentiation.