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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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.
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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.
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Romanov, S., Medvedev, A., Gambarian, M. et al. Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nat Methods 5, 253–260 (2008). https://doi.org/10.1038/nmeth.1186
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DOI: https://doi.org/10.1038/nmeth.1186
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