Phosphorylation-dependent control of transcription factor activity regulates temporal patterning of cortical cell fate specification

Abstract The development of the cerebral cortex requires precise temporal control of transcription factor (TF) activity to coordinate neuronal and glial lineage transitions. However, the molecular mechanisms coupling signaling dynamics to TF function remain unclear. Here, we combine proteomic, phosphoproteomic, and multi-omic analyses of the developing mouse cortex to define phosphorylation as a key post translational mechanism governing cortical cell fate decisions. Across the neurogenic to gliogenic transition, we identify a global remodeling of the phosphoproteome, including extensive phosphorylation of distinct TFs that is linked to their developmental-stage and cell-type-specific gene regulatory activity. Among these, Hmgn3 and Nfib emerge as pivotal regulators acting in sequential developmental phases. Phosphorylation of Hmgn3 at S78 enhances its chromatin association and activation of progenitor related genes, promoting progenitor proliferation. In contrast, phosphorylation of Nfib at S265 strengthens DNA binding at distal enhancers, driving axonogenesis and neuronal maturation. In utero perturbations with wild-type and phospho-mutant TFs combined with single-cell transcriptomic analyses reveal corresponding shifts in cell-type proportions. Our findings establish site-specific phosphorylation as a molecular timer that orchestrates TF activity to control the temporal patterning of cortical fate specification and ensure the precise assembly of cortical circuits.