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Quantifying transcription factor–DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy

Nature Protocols volume 12, pages 1458–1471 (2017)Cite this article

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Abstract

Probing transcription factor (TF)–DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF–DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.

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Figure 1: paFCS workflow.
Figure 2: Selective photoactivation of a TF subpopulation and optimization of photoactivation and paFCS acquisition parameters.
Figure 3: Acquisition of fluorescence intensity fluctuation traces and ACF curves.
Figure 4: Model selection for fitting paFCS data.
Figure 5: Probing the impact of mutation and drug treatment on TF-binding dynamics with paFCS.

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Acknowledgements

We thank J. Silva for help with embryo isolation and microinjection, and V. Levi for advice on modeling. This work was supported by an A*STAR National Science Scholarship (to Z.W.Z.), a Human Frontiers Science Program Fellowship (to J.Z.), and an A*STAR Investigatorship and EMBO Young Investigator grants (to N.P.).

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Author notes

  1. Ziqing Winston Zhao, Melanie D White, Yanina D Alvarez and Jennifer Zenker: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Ziqing Winston Zhao, Melanie D White, Yanina D Alvarez, Jennifer Zenker, Stephanie Bissiere & Nicolas Plachta

  2. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina

    Yanina D Alvarez

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Contributions

N.P. conceived the project; Z.W.Z. and Y.D.A. performed the microscope calibration, optimization of photoactivation/acquisition parameters, and paFCS measurements; M.D.W. and J.Z. performed drug treatment and mutant TF experiments. Y.D.A. performed data analysis and modeling. S.B. performed embryo isolation, culture, and mRNA microinjections. Z.W.Z., M.D.W., Y.D.A., J.Z., S.B. and N.P. wrote the manuscript.

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Correspondence to Nicolas Plachta.

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Zhao, Z., White, M., Alvarez, Y. et al. Quantifying transcription factor–DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy. Nat Protoc 12, 1458–1471 (2017). https://doi.org/10.1038/nprot.2017.051

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