Institut de biologie de l’école normale supérieure

INSERM 1024 - CNRS 8197
Director: Antoine Triller

       

Functional imaging of transcription | Xavier Darzacq

  • Goal. Understanding gene expression regulation mechanisms.
  • Background. Our group focuses on enhancing understanding of how the cellular machinery reads and uses our genome. Our genome cannot be reduced to a simple linear structure and what we refer to as cis or trans effects reflect nuclear spatial organization of genes and biophysical rules of nuclear factors dif- fusing and binding to their targets. We study general transcription mechanisms in model cell cultures ob- tained from tumors. Another model in our lab is human primary skin fibroblasts differentiating into myofibroblasts, in which we can study genetic reprogramming occurring during differentiation. Besides general molecular biology approaches, we use imaging to address questions that can only be solved at the single cell or single molecule scale. Photo- activated light microscopy is used to gain spatial resolution in the nucleus and to monitor the mobility of nuclear factors by single-protein tracking. We use methodologies involving the engineering of human cell lines where traceable loci are integrated and serve as live cell–specific transcriptional markers (reviewed in Darzacq et al 2006, Curr. Opin. Cell. Biol., Shav-Tal et al 2004 Nat Rev Mol Cell Biol.). We use these cellular systems in order to measure the temporal relations that govern the ordered co-transcriptional re- cruitment of specific factors involved in gene activity regulation. We interpret time-resolved fluorescence photo-attenuation and photoactivation data, comparing them with mathematical mechanistic models in order to extract kinetic parameters from enzymes working in their cellular environment
  • Research highlights. The transcriptional molecular machine differs in nature depending on the gene on which it is recruited or the state of the cell. Our goal is to quantify in live cells how RNA Pol II has different kinetics when recruited in different situations. This work starts from the quantification of the target search process of transcription factors finding a particular gene in the nucleus to the precise measurement of mRNAs produced from a single locus. We use live cell assays allowing the resolution of transcriptional kinetics that involve the simultaneous following of particular loci to measure their RNA production activity in real time, using three- dimensional live microscopy and the monitoring of the site-specific interaction of transcription fac- tors. We are also investigating the regulation of the intranuclear mobility of transcriptional machinery factors, with a view to understanding how their different interactions influence their mobility and distribution.

Selected Publications

Darzacq X., Yao J., Larson D., Causse S.Z., Bosanac L., de Turris V., Ruda V.M., Lionnet T.,Zenklusen D., Guglielmi B., Tjian R. and Singer R.H., Imaging Transcription in Living Cells. Annual Review of Biophysics (2009), 38 — 173-196.

Darzacq X., Shav-Tal Y, de Turis, V. Brody, Y., Shenoy, S.M., Phair, R.D., Singer R.H., In Vivo Dynamics of Polymerase II Transcription. Nat Struct Mol Biol (2007), 14 — 796-806.

Darzacq X., Singer R.H., Shav-Tal Y., Dynamics of transcription and mRNA export. Curr. Opin. Cell. Biol. (2005), 17 — 332-9.

Shav-Tal Y, Singer R.H., Darzacq X., Imaging gene expression in single living cells. Nat Rev Mol Cell Biol. (2004), 5 — 5855-62.

Shav-Tal Y, Darzacq X., Shenoy, S.M., Fusco D., Janicki S.M., Spector D.L., Singer R.H., Dynamics of Single mRNPs in Nuclei of Living Cells. Science (2004), 304 — 1797-80.

Team Members