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

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.

We thank the iCLOUD team at SDU and the HPC team at QUB for managing the HPC cluster and configuring storage, cluster job-queues, installing software and giving helpful advice. We thank all members of the Tiwari laboratory for their valuable feedback and insightful discussions throughout the study. This study was supported by Max Th.

Harding Larsen’s Fund grants to X.W., Deutsche Forschungsgemeinschaft TI 799/1-3, UKRI’s Innovation to Commercialisation of University Research (ICURe), Novo Nordisk Foundation 3110103 and Danish National Research Foundation DNRF177 grants to V.K.T. These authors contributed equally: Arun Mahesh, Anuj Kumar Dwivedi, Xuan Wang. Institute for Molecular Medicine, University of Southern Denmark, Odense M, Denmark Arun Mahesh, Anuj Kumar Dwivedi, Xuan Wang, Souren Sadhukhan & Vijay K.

Tiwari Graduate School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey Department of Pharmacy Services, Vocational School of Health Services, İstanbul Medipol University, İstanbul, Turkey Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Danish Institute for Advanced Study (DIAS), Odense M, Denmark Department of Clinical Genetics, Odense University Hospital, Odense C, Denmark Mohamed bin Zayed University of Artificial Intelligence, Abu Dhabi, UAE Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry, and Biomedical Science, Queens University Belfast, Belfast, UK The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.