Deciphering the Role of Transcription Factor Mobility and RNA Interplay in Embryonic Stem Cell Pluripotency

Embryonic stem cells require precise regulation of gene expression to maintain pluripotency, which enables sustained cell division and self-renewal. This process involves coordinated molecular events, driven by key transcription factors (TFs) such as NANOG, SOX2, and OCT4, that sustain the transcriptional oscillation of pluripotency genes. Despite well-characterised static binding profiles and mutual regulatory roles, the temporal coordination of these regulators and their impact on gene expression remains poorly understood. In my PhD, I developed a multimodal imaging method to quantify molecular events during gene transcription, using an advanced Marianas microscope equipped with integrated confocal and TIRF modules. This setup enables simultaneous imaging of the STREAMING-tag transcriptional reporter system and single-molecule tracking (SMT). This allows real-time visualisation of a single gene locus, its nascent mRNA, and TF mobility at the same site. This approach provides the first direct measurement of TF dynamics in relation to transcriptional progression at a target gene locus in live cells. Using this system, I identified three distinct SOX2 clustering patterns, each associated with different stages of the Nanog transcription cycle. These patterns differed in visiting frequency and dwell time: Type I showed low visiting frequency with short-lived binding; Type II showed medium to high visiting frequency with short-lived binding; and Type III displayed high visiting frequency with long-lived binding. Long-term multimodal imaging revealed that Type I and II clustering primarily occur before or during elongation, whereas Type III events are out of phase with elongation. In parallel, to assess the role of RNA in TF mobility, I investigated Nanog enhancer RNA (eRNA) using large-scale fixed-cell imaging of DNA/RNA-FISH-labelled Nanog loci, mRNA and eRNAs. Colocalisation analysis revealed that eRNA transcription follows a cycle temporally independent of gene body transcription. Given that both the densest SOX2 clustering and eRNA transcription were out of phase with the time window of most active Nanog mRNA synthesis, I further examined their physical coordination using pulldown and downstream detection assays. Through this work, I established a framework to dissect dynamic transcriptional regulation at single-gene resolution in live cells, advancing our understanding of TF mode of action from a temporal perspective in the context of pluripotency.