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Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

Author

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  • Davide Normanno

    (Laboratoire Kastler Brossel, CNRS UMR 8552, École normale supérieure, Université Pierre et Marie Curie
    Functional Imaging of Transcription, CNRS UMR 8197, École normale supérieure, Institut de Biologie de l’ENS, IBENS
    Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute
    Physico-Chimie Curie, Institut Curie, CNRS UMR 168, Université Pierre et Marie Curie, Paris 6)

  • Lydia Boudarène

    (Laboratoire Kastler Brossel, CNRS UMR 8552, École normale supérieure, Université Pierre et Marie Curie
    Functional Imaging of Transcription, CNRS UMR 8197, École normale supérieure, Institut de Biologie de l’ENS, IBENS)

  • Claire Dugast-Darzacq

    (Functional Imaging of Transcription, CNRS UMR 8197, École normale supérieure, Institut de Biologie de l’ENS, IBENS
    Université Paris-Diderot
    Present address: Li Ka Shing Center, Molecular and Cell Biology Department, University of California, #3370, Berkeley, California 94720-3370, USA)

  • Jiji Chen

    (Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute)

  • Christian Richter

    (Laboratoire Kastler Brossel, CNRS UMR 8552, École normale supérieure, Université Pierre et Marie Curie
    Present address: Universität Osnabrück, Fachbereich Biologie, Barbarastraße 11, 49076 Osnabrück, Germany)

  • Florence Proux

    (Functional Imaging of Transcription, CNRS UMR 8197, École normale supérieure, Institut de Biologie de l’ENS, IBENS)

  • Olivier Bénichou

    (Laboratoire de Physique Théorique de la Matière Condensée, CNRS UMR 7600, Université Pierre et Marie Curie)

  • Raphaël Voituriez

    (Laboratoire de Physique Théorique de la Matière Condensée, CNRS UMR 7600, Université Pierre et Marie Curie)

  • Xavier Darzacq

    (Functional Imaging of Transcription, CNRS UMR 8197, École normale supérieure, Institut de Biologie de l’ENS, IBENS
    Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute
    Present address: Li Ka Shing Center, Molecular and Cell Biology Department, University of California, #3370, Berkeley, California 94720-3370, USA)

  • Maxime Dahan

    (Laboratoire Kastler Brossel, CNRS UMR 8552, École normale supérieure, Université Pierre et Marie Curie
    Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute
    Physico-Chimie Curie, Institut Curie, CNRS UMR 168, Université Pierre et Marie Curie, Paris 6)

Abstract

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Suggested Citation

  • Davide Normanno & Lydia Boudarène & Claire Dugast-Darzacq & Jiji Chen & Christian Richter & Florence Proux & Olivier Bénichou & Raphaël Voituriez & Xavier Darzacq & Maxime Dahan, 2015. "Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher," Nature Communications, Nature, vol. 6(1), pages 1-10, November.
    • Handle: RePEc:nat:natcom:v:6:y:2015:i:1:d:10.1038_ncomms8357
    DOI: 10.1038/ncomms8357
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    Cited by:

    1. Matteo Mazzocca & Alessia Loffreda & Emanuele Colombo & Tom Fillot & Daniela Gnani & Paola Falletta & Emanuele Monteleone & Serena Capozi & Edouard Bertrand & Gaelle Legube & Zeno Lavagnino & Carlo Ta, 2023. "Chromatin organization drives the search mechanism of nuclear factors," Nature Communications, Nature, vol. 14(1), pages 1-17, December.

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