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

Abstract Maintenance of naïve 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, NANOG’s 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 naïve pluripotency while providing quantitative pipelines for dissecting spatiotemporal TF dynamics. Competing Interest Statement The authors have declared no competing interest.