Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells

Agsu, G. G.; Cao, Y.; Jain, D.; Strawbridge, S.; Daly, S.; Zhang, Z.; Wurmser, A.; Bowers, K.; Orsine de Almeida, I.; Nikolenyi, G.; Berger, S.; Jonaitis, M.; Guma'a, K.; Pauzaite, T.; Maretvadakethope, S.; Bates, L.; Nathan, J. A.; Hendrich, B.; Perez-Carrasco, R.; Chalut, K.; Klenerman, D.; Boroviak, T.; Lee, S. F.; Holcman, D.; Basu, S.

doi:10.64898/2025.12.03.691924

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bioRxiv · 10.64898/2025.12.03.691924

Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells

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Abstract

Maintenance of naive pluripotency requires core transcription factors (TFs) like SOX2 and auxiliary TFs like NANOG, yet molecular mechanisms governing their intra-nuclear dynamics and DNA binding interactions remain unclear. Here, using high-density 3D single-molecule light-field microscopy combined with novel spatiotemporal analysis pipelines, we track SOX2 and NANOG dynamics in live cells. Despite lower protein abundance, NANOG displays a similar chromatin-bound fraction to SOX2. This arises partially because, while both TFs undergo frequent transient non-specific binding interactions ([~]0.5-0.7s), NANOG exhibits more stable specific binding ([~]25s vs [~]16s). Both TFs also assemble into phase-separated domains of [~]400 nm containing both freely diffusing and chromatin-bound proteins, which further influences their dynamics. Strikingly, NANOGs protein-protein interaction domain markedly increases chromatin residence time (>5-fold) and the size of these phase-separated domains. Our work uncovers how NANOG and SOX2 stabilise gene regulatory networks that maintain naive pluripotency while providing quantitative pipelines for dissecting spatiotemporal TF dynamics.

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Agsu, G. G., Cao, Y., Jain, D., Strawbridge, S., Daly, S., Zhang, Z., Wurmser, A., Bowers, K., Orsine de Almeida, I., Nikolenyi, G., Berger, S., Jonaitis, M., Guma'a, K., Pauzaite, T., Maretvadakethope, S., Bates, L., Nathan, J. A., Hendrich, B., Perez-Carrasco, R., Chalut, K., Klenerman, D., Boroviak, T., Lee, S. F., Holcman, D., Basu, S.. 2025-12-03. Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells. https://doi.org/10.64898/2025.12.03.691924

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