PCV - Programme interdiciplinaire en physique et chimie du vivant

Dynamique de facteurs de transcriptions individuels dans une cellule eukaryote – DynaFT

Submission summary

Over the ~23k genes present in a human cell, only a few thousands are expressed at a given time in order to specify a cellular function integrated in an organism. The regulation of gene expression is ensured by specific transcription factors (TFs). TFs control the genes that are silenced or activated by transiently associating to promoter sequences on the genome. Therefore, understanding the dynamics of gene regulation requires elucidating the mechanisms underlying TF target search and the kinetics of DNA-binding and release. Within the nucleus, TFs are often present in small amount compared to the number of genes they can regulate and, therefore, their activity should ideally be addressed at the single molecule level. Here, we propose to develop a combination of biological and physical tools that enables the visualization of individual specific TFs, labeled with a dye or a quantum dot and microinjected in the nucleus. We will use engineered human cells in which these TFs can bind to a single locus controlling a single gene. In these conditions, tracking individual TFs in the nucleus of live cells will allow us to directly address a variety of important and largely unexplored questions : (1) How does a TF find its target and how long does it take for a TF to reach its binding site?; (2) What are the parameters (non specific interaction with DNA, molecular crowding, nuclear structure, association rate…) that primarily determine the search kinetics?; (3) In the presence of multiple binding sites, is there competition or cooperation in the TF recruitment and how does it depend on their relative distance?; (4) Once a TF has reached its target site, how long does it stay at the locus and what is the relation between the production of transcripts and the binding time? The in vivo single-molecule (SM) measurements will be complemented by in vitro experiments to investigate TF-DNA interactions and by analytical tools to dissect the TF search mechanisms within the nucleus. The project is based on the close collaboration of three groups with complementary expertise in single molecule biophysics, cell biology of gene transcription and statistical modeling of stochastic processes. We expect that this combination of experimental tools and experiments will provide a novel and quantitative view of the processes governing the nuclear dynamics of TFs and their implication in gene regulation.

Project coordination

Maxime DAHAN (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Help of the ANR 450,000 euros
Beginning and duration of the scientific project: - 36 Months

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