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Combining protein and mRNA quantification to decipher transcriptional regulation

Nature Methods volume 12, pages 739–742 (2015)Cite this article

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Abstract

We combine immunofluorescence and single-molecule fluorescence in situ hybridization (smFISH), followed by automated image analysis, to quantify the concentration of nuclear transcription factors, number of transcription factors bound, and number of nascent mRNAs synthesized at individual gene loci. A theoretical model is used to decipher how transcription factor binding modulates the stochastic kinetics of mRNA production. We demonstrate this approach by examining the regulation of hunchback in the early Drosophila embryo.

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Figure 1: Simultaneous quantification of Bcd protein and hb mRNA in a single embryo.
Figure 2: The regulatory relationship between Bcd concentration and hb transcription.
Figure 3: Transcription factor binding at the hb locus.

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Acknowledgements

We thank J. Reinitz (University of Chicago) for the gift of anti-Hb antibody, and the following people for their generous advice: A. Boettiger, J. Elf, H. Garcia, D. Larson, S. Little, J. Ma, A. Raj, A. Sanchez, E. Segal, D. van Dijk and all members of the Golding and Sokac labs. Work in the Golding lab was supported by grants from the US National Institutes of Health (NIH R01 GM082837), US National Science Foundation (PHY 1147498, PHY 1430124 and PHY 1427654) and Welch Foundation (Q-1759). H.X. is supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface. A.M.S. and L.F. are supported by a grant from the NIH (R01 GM115111), a Computational and Integrative Biomedical Research (CIBR) Center Seed Award and the Curtis Hankamer Basic Research Fund Award from Baylor College of Medicine. We gratefully acknowledge the computing resources provided by the CIBR Center of Baylor College of Medicine.

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Authors and Affiliations

  1. Verna & Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA

    Heng Xu, Leonardo A Sepúlveda, Anna Marie Sokac & Ido Golding

  2. Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Heng Xu & Ido Golding

  3. Center for Theoretical Biological Physics, Rice University, Houston, Texas, USA

    Heng Xu, Leonardo A Sepúlveda & Ido Golding

  4. Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas, USA

    Lauren Figard

Authors
  1. Heng Xu
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  2. Leonardo A Sepúlveda
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  3. Lauren Figard
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  4. Anna Marie Sokac
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  5. Ido Golding
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Contributions

H.X., L.A.S. and I.G. conceived the experimental and analysis methods. H.X. and L.A.S. developed image and data analysis algorithms. H.X. performed the experiments, developed algorithms and theoretical models, and analyzed the data. L.F. performed fly-injection experiments. A.M.S. provided guidance on fly biology and microscopy. I.G. supervised the project. H.X., A.M.S. and I.G. wrote the manuscript.

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Correspondence to Ido Golding.

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The authors declare no competing financial interests.

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Supplementary Figures 1–18, Supplementary Table 1 and Supplementary Note (PDF 8355 kb)

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MATLAB code used for image and data analysis. (ZIP 860 kb)

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Xu, H., Sepúlveda, L., Figard, L. et al. Combining protein and mRNA quantification to decipher transcriptional regulation. Nat Methods 12, 739–742 (2015). https://doi.org/10.1038/nmeth.3446

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