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Quantitative, solution-phase profiling of multiple transcription factors in parallel

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Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Transcription factors are regulatory proteins that bind to specific sites of chromosomal DNA to enact responses to intracellular and extracellular stimuli. Transcription factor signalling networks are branched and interconnected so that any single transcription factor can activate many different genes and one gene can be activated by a combination of different transcription factors. Thus, trying to characterize a cellular response to a stimulus by measuring the level of only one transcription factor potentially ignores important simultaneous events that contribute to the response. Hence, parallel measurements of transcription factors are necessary to capture the breadth of valuable information about cellular responses that would not be obtained by measuring only a single transcription factor. We have sought to develop a new, scalable, flexible, and sensitive approach to analysis of transcription factor levels that complements existing parallel approaches. Here, we describe proof-of-principle analyses of purified human transcription factors and breast cancer nuclear extracts. Our assay can successfully quantify transcription factors in parallel with ∼10-fold better sensitivity than current techniques. Sensitivity of the assay can be further increased by 200-fold through the use of PCR for signal amplification.

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References

  1. Orphanides G, Reinberg D (2002) A unified theory of gene expression. Cell 108(4):439–451

    Article  CAS  Google Scholar 

  2. Babu MM (2010) Structure, evolution and dynamics of transcriptional regulatory networks. Biochem Soc Trans 38:1155–1178

    Article  CAS  Google Scholar 

  3. Emerson BM (2002) Specificity of gene regulation. Cell 109(3):267–270

    Article  CAS  Google Scholar 

  4. Mees C, Nemunaitis J, Senzer N (2009) Transcription factors: their potential as targets for an individualized therapeutic approach to cancer. Cancer Gene Therapy 16(2):103–112

    Article  CAS  Google Scholar 

  5. Nebert DW (2002) Transcription factors and cancer: an overview. Toxicology 181:131–141

    Article  Google Scholar 

  6. Latchman DS (1996) Mechanisms of disease—transcription-factor mutations and disease. N Engl J Med 334(1):28–33

    Article  CAS  Google Scholar 

  7. McCulley DJ, Black BL (2012) Transcription factor pathways and congenital heart disease. Curr Top Dev Biol 100:253–277

    Article  CAS  Google Scholar 

  8. Barnes PJ (2006) Transcription factors in airway diseases. Lab Investig 86(9):867–872

    Article  CAS  Google Scholar 

  9. Johnson DS, Mortazavi A, Myers RM, Wold B (2007) Genome-wide mapping of in vivo protein–DNA interactions. Science 316(5830):1497–1502

    Article  CAS  Google Scholar 

  10. Berger MF, Bulyk ML (2009) Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nat Protoc 4(3):393–411

    Article  CAS  Google Scholar 

  11. Nishiyama A, Xin L, Sharov AA, Thomas M, Mowrer G, Meyers E, Piao YL, Mehta S, Yee S, Nakatake Y, Stagg C, Sharova L, Correa-Cerro LS, Bassey U, Hoang H, Kim E, Tapnio R, Qian Y, Dudekula D, Zalzman M, Li MX, Falco G, Yang HT, Lee SL, Monti M, Stanghellini I, Islam MN, Nagaraja R, Goldberg I, Wang WD, Longo DL, Schlessinger D, Ko MSH (2009) Uncovering early response of gene regulatory networks in ESCs by systematic ınduction of transcription factors. Cell Stem Cell 5(4):420–433

    Article  CAS  Google Scholar 

  12. Matys V, Fricke E, Geffers R, Gossling E, Haubrock M, Hehl R, Hornischer K, Karas D, Kel AE, Kel-Margoulis OV, Kloos DU, Land S, Lewicki-Potapov B, Michael H, Munch R, Reuter I, Rotert S, Saxel H, Scheer M, Thiele S, Wingender E (2003) TRANSFAC (R): transcriptional regulation, from patterns to profiles. Nucleic Acids Res 31(1):374–378

    Article  CAS  Google Scholar 

  13. Matys V, Kel-Margoulis OV, Fricke E, Liebich I, Land S, Barre-Dirrie A, Reuter I, Chekmenev D, Krull M, Hornischer K, Voss N, Stegmaier P, Lewicki-Potapov B, Saxel H, Kel AE, Wingender E (2006) TRANSFAC (R) and its module TRANSCompel (R): transcriptional gene regulation in eukaryotes. Nucleic Acids Res 34:D108–D110

    Article  CAS  Google Scholar 

  14. Ruscher K, Reuter M, Kupper D, Trendelenburg G, Dirnagl U, Meisel A (2000) A fluorescence based non-radioactive electrophoretic mobility shift assay. J Biotechnol 78(2):163–170

    Article  CAS  Google Scholar 

  15. Bronstein I, Fortin J, Stanley PE, Stewart G, Kricka LJ (1994) Chemiluminescent and bioluminescent reporter gene assays. Anal Biochem 219(2):169–181

    Article  CAS  Google Scholar 

  16. Benotmane AM, Hoylaerts MF, Collen D, Belayew A (1997) Nonisotopic quantitative analysis of protein–DNA interactions at equilibrium. Anal Biochem 250(2):181–185

    Article  CAS  Google Scholar 

  17. Shen Z, Peedikayil J, Olson GK, Siebert PD, Fang Y (2002) Multiple transcription factor profiling by enzyme-linked immunoassay. Biotechniques 32(5):1168, -+

    CAS  Google Scholar 

  18. Li XQ, Jiang X, Yaoi T (2006) High throughput assays for analyzing transcription factors. Assay and Drug Development Technologies 4(3):333–341

    Article  CAS  Google Scholar 

  19. Romanov S, Medvedev A, Gambarian M, Poltoratskaya N, Moeser M, Medvedeva L, Diatchenko L, Makarov S (2008) Homogeneous reporter system enables quantitative functional assessment of multiple transcription factors. Nature Methods 5(3):253–260

    Article  CAS  Google Scholar 

  20. Yaoi T, Jiang X, Li XQ (2006) Development of a fluorescent micro sphere-based multiplexed high-throughput assay system for profiling of transcription factor activation. Assay and Drug Development Technologies 4(3):285–292

    Article  CAS  Google Scholar 

  21. Qiao JY, Shao W, Wei HJ, Sun YM, Zhao YC, Xing WL, Zhang L, Mitchelson K, Cheng J (2008) Novel high-throughput profiling of human transcription factors and its use for systematic pathway mapping. Journal of Proteome Research 7(7):2769–2779

    Article  CAS  Google Scholar 

  22. Shao W, Wei HJ, Qiao JY, Zhao YC, Sun YM, Zhou YX, Cheng J (2005) Parallel profiling of active transcription factors using an oligonucleotide array-based transcription factor assay (OATFA). Journal of Proteome Research 4(4):1451–1456

    Article  CAS  Google Scholar 

  23. Jiang X, Roth L, Lai CF, Li XQ (2006) Profiling activities of transcription factors in breast cancer cell lines. Assay and Drug Development Technologies 4(3):293–305

    Article  CAS  Google Scholar 

  24. Wu M, Liu L, Chan C (2011) Identification of novel targets for breast cancer by exploring gene switches on a genome scale. BMC Genomics 12:19

    Article  Google Scholar 

  25. Lee KAW, Bindereif A, Green MR (1988) A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-messenger RNA splicing. Gene Analysis Techniques 5(2):22–31

    Article  CAS  Google Scholar 

  26. Zhang LX, Seitz LC, Abramczyk AM, Liu L, Chan C (2011) cAMP initiates early phase neuron-like morphology changes and late phase neural differentiation in mesenchymal stem cells. Cellular and Molecular Life Sciences 68(5):863–876

    Article  CAS  Google Scholar 

  27. Liu L, Martin R, Chan C (2012) Palmitate-activated astrocytes via serine palmitoyltransferase increase BACE1 in primary neurons by sphingomyelinases. Neurobiol Aging. doi:10.1016/j.neurobiolaging.2012.05.017

  28. Bayer EA, Wilchek M (1990) Protein biotinylation. Methods Enzymol 184:138–160

    Article  CAS  Google Scholar 

  29. Pavletich NP, Pabo CO (1991) Zinc finger DNA recognition—crystal-structure of a ZIF268-DNA complex at 2.1-A. Science 252(5007):809–817

    Article  CAS  Google Scholar 

  30. Kim HJ, Hawke N, Baldwin AS (2006) NF-kappa B and IKK as therapeutic targets in cancer. Cell Death and Differentiation 13(5):738–747

    Article  CAS  Google Scholar 

  31. van Horssen R, ten Hagen TLM, Eggermont AMM (2006) TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist 11(4):397–408

    Article  Google Scholar 

  32. Kielbasa SM, Gonze D, Herzel H (2005) Measuring similarities between transcription factor binding sites. BMC Bioinforma 6:11

    Article  Google Scholar 

Download references

Acknowledgments

We thank all the members of the Cellular and Biomolecular Laboratory (http://www.egr.msu.edu/cbl/) for their advice and support. Financial support for this work was provided in part by Michigan State University, the National Science Foundation (CBET 0941055), the National Institutes of Health (GM079688, RR024439, GM089866, DK081768, DK088251), the Michigan Universities Commercialization Initiative (MUCI), and the Center for Systems Biology.

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Authors and Affiliations

  1. Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, 48824, USA

    Betul Bilgin, Christina Chan & S. Patrick Walton

  2. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, 48824, USA

    Li Liu

  3. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA

    Christina Chan

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  1. Betul Bilgin
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  2. Li Liu
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Correspondence to S. Patrick Walton.

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Bilgin, B., Liu, L., Chan, C. et al. Quantitative, solution-phase profiling of multiple transcription factors in parallel. Anal Bioanal Chem 405, 2461–2468 (2013). https://doi.org/10.1007/s00216-013-6712-9

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  • DOI: https://doi.org/10.1007/s00216-013-6712-9

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