In this work, Bunina et al. profiled transcriptome, proteome, and chromatin accessibility dynamics during differentiation of mouse embryonic stem cells to postmitotic neurons. They revealed extensive associations between molecular layers within and across differentiation time points and uncovered an unexpected interaction of two chromatin-bound proteins, SOX2 and ATRX, in neurons. Their role in regulating neuronal genes was validated by CRISPR. The study highlights the significance of multi-omic approaches followed by specific functional experiments in understanding gene regulation in complex systems.