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Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors

Nature Methods volume 5, pages 253260 (2008) | Download Citation

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Abstract

We developed a high-content reporter system that allows quantitative assessment of activities of multiple transcription factors (TFs) in a eukaryotic cell. The system comprises a library of reporter constructs that are evaluated according to their transcription rates. All reporters produce essentially identical messages that are subjected to 'processing', which generates a spectrum of distinguishable fragments that are analyzed quantitatively. The homogeneity of the reporter library afforded inherently uniform detection conditions for all reporters and provided repeatability, accuracy and robustness of assessment. We showed that this technology can be used to identify pathways transmitting cell responses to inducers, and that the profile of TF activities generated using this system represents a stable and sustained cell signature that clearly distinguishes different cell types and pathological conditions. This technology provides a framework for functional characterization of signal transduction networks through profiling activities of multiple TFs.

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Acknowledgements

We thank S. Langdon for invaluable help with fragment analysis, and A.S. Baldwin and R. Gaynor for discussions. The work was supported by US National Institutes of Health grants 1R43CA101636, 1R43CA101271 and UO1 AI061360.

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Affiliations

  1. Attagene Inc., P.O. Box 12054, Research Triangle Park, North Carolina 27709, USA.

    • Sergei Romanov
    • , Alexander Medvedev
    • , Maria Gambarian
    • , Natalia Poltoratskaya
    • , Matt Moeser
    • , Liubov Medvedeva
    •  & Sergei Makarov
  2. MishaGam Software, 525 Riverside Ln., Cayce, South Carolina 29033, USA.

    • Mikhail Gambarian
  3. Center for Neurosensory Disorders, 2120 Old Dental Building, CB 7450, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    • Luda Diatchenko

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Contributions

S.R., A.M. and S.M. are authors of the concept. A.M. and M.M. constructed the RTU library; A.M. and S.R. designed the experiments; Ma.G., M.M., N.P., L.M. and L.D. performed the experiments; Mi.G. designed the software; S.R. and S.M. wrote the manuscript.

Competing interests

S.R., A.M., Ma.G., N.P., M.M., L.M. and S.M. are current employees of Attagene Inc. S.R., L.D. and S.M. have significant financial interest in company.

Corresponding author

Correspondence to Sergei Makarov.

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    Supplementary Methods, Supplementary Figures 1–4, Supplementary Tables 1–5

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https://doi.org/10.1038/nmeth.1186

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