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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.


  1. Babu, M.M., Luscombe, N.M., Aravind, L., Gerstein, M. & Teichmann, S.A. Structure and evolution of transcriptional regulatory networks. Curr. Opin. Struct. Biol. 14, 283–291 (2004).

    Article  CAS  Google Scholar 

  2. Stegmaier, P., Kel, A.E. & Wingender, E. Systematic DNA-binding domain classification of transcription factors. Genome Inform. 15, 276–286 (2004).

    CAS  PubMed  Google Scholar 

  3. Matys, V. et al. TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 34 Database issue, D108–D110 (2006).

    Article  CAS  Google Scholar 

  4. Benotmane, A.M., Hoylaerts, M.F., Collen, D. & Belayew, A. Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium. Anal. Biochem. 250, 181–185 (1997).

    Article  CAS  Google Scholar 

  5. Rosenau, C., Emery, D., Kaboord, B. & Qoronfleh, M.W. Development of a high-throughput plate-based chemiluminescent transcription factor assay. J. Biomol. Screen. 9, 334–342 (2004).

    Article  CAS  Google Scholar 

  6. Hermanson, O., Glass, C.K. & Rosenfeld, M.G. Nuclear receptor coregulators: multiple modes of modification. Trends Endocrinol. Metab. 13, 55–60 (2002).

    Article  CAS  Google Scholar 

  7. Bronstein, I., Fortin, J., Stanley, P.E., Stewart, G.S. & Kricka, L.J. Chemiluminescent and bioluminescent reporter gene assays. Anal. Biochem. 219, 169–181 (1994).

    Article  CAS  Google Scholar 

  8. Kovacs, D.M. & Kaplan, B.B. Discordant estimates of heterologous promoter activity as determined by reporter gene mRNA levels and enzyme activity. Biochem. Biophys. Res. Commun. 189, 912–918 (1992).

    Article  CAS  Google Scholar 

  9. Li, X., Jiang, X. & Yaoi, T. High throughput assays for analyzing transcription factors. Assay Drug Dev. Technol. 4, 333–341 (2006).

    Article  CAS  Google Scholar 

  10. Guhaniyogi, J. & Brewer, G. Regulation of mRNA stability in mammalian cells. Gene 265, 11–23 (2001).

    Article  CAS  Google Scholar 

  11. Shim, J. & Karin, M. The control of mRNA stability in response to extracellular stimuli. Mol. Cells 14, 323–331 (2002).

    CAS  PubMed  Google Scholar 

  12. Levitt, N., Briggs, D., Gil, A. & Proudfoot, N.J. Definition of an efficient synthetic poly(A) site. Genes Dev. 3, 1019–1025 (1989).

    Article  CAS  Google Scholar 

  13. Enriquez-Harris, P., Levitt, N., Briggs, D. & Proudfoot, N.J. A pause site for RNA polymerase II is associated with termination of transcription. EMBO J. 10, 1833–1842 (1991).

    Article  CAS  Google Scholar 

  14. MAQC Consortium. The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat. Biotechnol. 24, 1151–1161 (2006).

  15. Oberg, M., Bergander, L., Hakansson, H., Rannug, U. & Rannug A. Identification of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole, in cell culture medium, as a factor that controls the background aryl hydrocarbon receptor activity. Toxicol. Sci. 85, 935–943 (2005).

    Article  Google Scholar 

  16. Montminy, M.R. & Bilezikjian, L.M. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature 328, 175–178 (1987).

    Article  CAS  Google Scholar 

  17. Dennler, S. et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 17, 3091–3100 (1998).

    Article  CAS  Google Scholar 

  18. Eisen, M.B., Spellman, P.T., Brown, P.O. & Botstein, D. Cluster analysis and display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA 95, 14863–14868 (1998).

    Article  CAS  Google Scholar 

  19. Zhang, L. et al. Gene expression profiles in normal and cancer cells. Science 276, 1268–1272 (1997).

    Article  CAS  Google Scholar 

  20. Perou, C.M. et al. Molecular portraits of human breast tumours. Nature 406, 747–752 (2000).

    Article  CAS  Google Scholar 

  21. Okabe, H. et al. Genome-wide analysis of gene expression in human hepatocellular carcinomas using cDNA microarray: identification of genes involved in viral carcinogenesis and tumor progression. Cancer Res. 61, 2129–2137 (2001).

    CAS  PubMed  Google Scholar 

  22. Ross, D.T. et al. Systematic variation in gene expression patterns in human cancer cell lines. Nat. Genet. 24, 227–235 (2000).

    Article  CAS  Google Scholar 

  23. Korinek, V. et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC−/− colon carcinoma. Science 275, 1784–1787 (1997).

    Article  CAS  Google Scholar 

  24. Morin, P.J. et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275, 1787–1790 (1997).

    Article  CAS  Google Scholar 

  25. Nakshatri, H., Bhat-Nakshatri, P., Martin, D.A., Goulet, R.J. Jr. & Sledge, G.W. Jr. Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol. Cell. Biol. 17, 3629–3639 (1997).

    Article  CAS  Google Scholar 

  26. Troester, M.A. et al. Cell-type-specific responses to chemotherapeutics in breast cancer. Cancer Res. 64, 4218–4226 (2004).

    Article  CAS  Google Scholar 

  27. Waldman, T., Lengauer, C., Kinzler, K.W. & Vogelstein, B. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 381, 713–716 (1996).

    Article  CAS  Google Scholar 

  28. Goyette, M.C. et al. Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol. Cell. Biol. 12, 1387–1395 (1992).

    Article  CAS  Google Scholar 

  29. Shao, Z.M. et al. p53 independent G0/G1 arrest and apoptosis induced by a novel retinoid in human breast cancer cells. Oncogene 11, 493–504 (1995).

    CAS  Google Scholar 

  30. Sugikawa, E. et al. Mutant p53 mediated induction of cell cycle arrest and apoptosis at G1 phase by 9-hydroxyellipticine. Anticancer Res. 19, 3099–3108 (1999).

    CAS  PubMed  Google Scholar 

  31. Tamaru, N. et al. Estrogen receptor-associated expression of keratinocyte growth factor and its possible role in the inhibition of apoptosis in human breast cancer. Lab. Invest. 84, 1460–1471 (2004).

    Article  CAS  Google Scholar 

  32. Nuwaysir, E.F., Bittner, M., Trent, J., Barrett, J.C. & Afshari, C.A. Microarrays and toxicology: the advent of toxicogenomics. Mol. Carcinog. 24, 153–159 (1999).

    Article  CAS  Google Scholar 

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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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