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Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors


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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Figure 1: Reporter system for functional assessment of multiple TFs.
Figure 2: TF activity profile in HepG2 cells transiently transfected with the RTU library.
Figure 3: Robustness of the MRTU assay.
Figure 4: Evaluation of biologically active compounds using MRTU assay.
Figure 5: Profile of TF activities provides a distinct cell signature that is stable over time.
Figure 6: Anatomically related cancer cells have distinct TF activity profiles.


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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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Authors and Affiliations



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.

Corresponding author

Correspondence to Sergei Makarov.

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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.

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Supplementary Methods, Supplementary Figures 1–4, Supplementary Tables 1–5 (PDF 338 kb)

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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).

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