Garcia Grisales, David ADifferent proteins and complexes work together at multiple time scales to orchestrate the activation and silencing of genes in a process called transcription. Understanding transcriptional regulation is of utmost importance to reveal mechanisms behind cell homeostasis and pathologies. The transcription machinery needs to be perfectly tuned in space and time to control the expression of genes to carry out cellular and physiological processes in the noisy and highly heterogeneous nuclear microenvironment. Transcription factors (TF), specialized proteins that bind to specific DNA sequences to regulate mRNA production, are central players in transcriptional regulation. TFs need to navigate the intricate nuclear microenvironment to bind to specific regulatory elements with binding times critically determining their regulatory functions. Recent advances in super-resolution microscopy have allowed us to investigate the dynamics of the transcriptional machinery at the single molecule level, revealing the essential features of transcriptional control. However, how TFs dynamically navigate the nuclear microenvironment and interact with chromatin to activate or silence genes remains poorly understood. I used state of the art microscopy and genomic techniques to show that binding times of TFs to chromatin are power-law distributed. I proposed a new theoretical framework to demonstrate the broad distribution of binding affinity arises from heterogeneity in TF-chromatin interactions and the nuclear microenvironment, contrary to the current paradigm of well-defined and distinguishable TF binding times to specific and non-specific chromatin sites. These studies reconciled discrepancies between genomics, gene expression and TF mobility. I used statistical modeling to show that TFs exhibit two distinguishable low mobility states in the nucleus. One state is related to chromatin binding while the second arises due to protein-protein interactions mediated by intrinsically disordered regions of the TF and potentially controls the initiation rate of transcription. Finally, I studied transcriptional regulation on substrates of different stiffness revealing a connection between the physical properties of the cell microenvironment and TF dynamics. I demonstrated that substrate stiffness activates the estrogen receptor even in the absence of its ligand, with implications for our understanding and treatment of breast cancer. The evidence presented here shows that TF binding times are finely tuned to regulate gene expression.enDissertationBiophysicsPhysicsMolecular biology