Life is motion and biological processes are inherently dynamic. When viewed under a microscope, living cells move, change shape, grow and divide. Rapid, seemingly chaotic dynamic movement is also true for many biomolecular processes such as transcriptional regulation: we now know that transcription factors (TFs) bind cognate DNA with residence times of only a few seconds to induce gene expression, and transcription itself occurs in dynamic, stochastic and transient bursts. However, most methods used to probe genome organisation and gene regulation to date rely on endpoint assays that deliver time and ensemble-averaged snapshots based on test tube reactions or dead/fixed cells.
Perhaps not surprisingly, despite decades of study, these traditional experimental strategies have limited our ability to discover the true molecular mechanisms enabling enhancer-promoter (E-P) communication, and it remains unclear how and to what extent 3D genome conformation (DNA-looping) influences transcriptional regulation. Similarly, it remains to be elucidated how the dynamic binding of proteins modulate the exquisite temporal regulation of transcription while navigating and possibly influencing genome organisation. By developing novel methods to study the rapid diffusive behavior and kinetics of TFs in live cells, our lab has recently been able to open a new window into the molecular dynamics of gene regulation. Here we propose to substantially advance the development of live-cell imaging strategies that probe molecular processes dynamically in space and time at single molecule resolution within organized tissues and organoids. A related aspiration is to embrace and foster a culture of “open science” by making our methods, data and unique instruments widely and freely available to the research community.
Within the next decade, great technological advances are expected in the field of microscopy, fluorophore chemistry as well as cellular engineering with new genome editing techniques already in place. These new developments will put us in an ideal situation to monitor several different molecules simultaneously (therefore defining molecular multicomponent complexes) with resolutions close to that of electron microscopy. It will be a unique opportunity to integrate the cellular and biochemical know how of an institute strong of a long experience in gene regulation with advances in biophysics, chemistry and imaging in order to reveal the rules governing transcription regulation within a living cell and eventually living organisms.