Track & trace transcription factor localization and dynamics

Transcription factors (TFs) are key regulators of gene expression. These proteins need to find and bind specific sequences in the DNA to regulate expression of their target genes. How TFs are organized within cells and how they localize their targets is unclear. In this thesis, we use advanced live-cell imaging techniques to study the regulation of transcription factor localization & dynamics in budding yeast. Chapter 2 is a review on how studying dynamics of transcriptional proteins provides insights in the molecular mechanisms underlying gene transcription. Chapter 3 is a protocol on how to perform live-cell single-molecule fluorescence imaging in budding yeast. In Chapter 4, we studied a yeast TF which localizes in clusters inside cells, to identify the regulatory mechanisms of TF clustering and its role in gene regulation in an endogenous context. Finally, in Chapter 5, we present the use of a novel single-molecule imaging technique and apply this to measure both the search time and binding time of this yeast TF at a specific target locus in living cells, combined with genomic perturbations, to study the regulation of TF binding dynamics at their genomic targets.