Anti-phase clustering of regulatory factors shapes gene bursting

The ability of stem cells to divide and self-renew depends on a complex choreography of molecular events that maintain the transcriptional oscillation of pluripotency genes. Only a handful of transcription factors (TFs) are necessary to preserve pluripotency and reprogram differentiated cells into stem cells. Paradoxically, while the protein players are known, the challenge remains to decipher the series of steps that TFs undertake to modulate "on" and "off" fluctuations of gene transcription. In this study, we employ a trimodal imaging approach that integrates single-molecule tracking of SOX2 mobility, Nanog locus diffusion, and real-time Nanog mRNA synthesis (STEAMING-tag reporter) to observe the coordinated activity of this molecular machinery. This combined system uncovered the temporal clustering dynamics of endogenous SOX2 occupancy at the Nanog locus and reveal how these dynamics relate to both transcriptional activity and the underlying chromatin behaviour in live embryonic stem cells. We link different types of SOX2 binding modes to roles in both transcriptional activation and termination or nucleosome repackaging. This redefines a dual function for SOX2 at the same gene locus during both gene expression and silencing processes. This work exposes a new layer of TF regulation, indicating a priming role rather than direct transcription initiation directly relevant to mammalian stem cell biology.