Introduction In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. Methods Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. Findings We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. Conclusions We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters.
References
[1]
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, et al. (2001) The sequence of the human genome. Science 291: 1304–1352.
[2]
Elkon R, Linhart C, Sharan R, Shamir R, Shilon Y (2003) Genome-wide in silico identification of transcriptional regulators controlling the cell cycle in human cells. Genome Res 13: 773–780.
[3]
Calkhoven CF, Geert AB (1996) Multiple steps in the regulation of transcription-factor level and activity. Biochem J 317: 329–342.
[4]
McCoy C, Smith DE, Cornwell MM (1995) 12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1. Mol Cell Biol 15: 6100–6108.
[5]
Wasserman WW, Fickett JW (1998) Identification of regulatory regions which confer muscle-specific gene expression. J Mol Biol 278: 167–181.
[6]
Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, et al. (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445: 168–176.
[7]
Qiao JY, Shao W, Wei H-J, Sun YM, Zhao Y-C, et al. (2008) Novel High-Throughput profiling of human transcription factors and its use for systematic pathway mapping. J Proteome Res 7: 2769–2779.
[8]
Ho Sui SJ, Mortimer JR, Arenillas DJ, Brumm J, Walsh CJ, et al. (2005) oPOSSUM, identification of over-represented transcription factor binding sites in coexpressed genes. Nucleic Acids Res 33: 3154–3164.
[9]
Yu X, Lin J, Zack DJ, Qian J (2006) Computational analysis of tissue-specific combinatorial gene regulation, predicting interaction between transcription factors in human tissues. Nucleic Acids Res 34: 4925–4936.
[10]
Smith AD, Sumazin P, Zhang MQ (2007) Tissue-specific regulatory elements in mammalian promoters. Mol Syst Biol 3: 73.
[11]
Barrera LO, Li Z, Smith AD, Arden KC, Cavenee WK, et al. (2008) Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs. Genome Res 18: 46–59.
[12]
Romanov S, Medvedev A, Gambarian M, Poltoratskaya N, Moeser M, et al. (2008) Homogenous reporter system enables quantitative functional assessment of multiple transcription factors. Nature Methods 5: 253–260.
[13]
Botvinnik A, Wichert SP, Fischer TM, Rossner MJ (2010) Integrated analysis of receptor activation and downstream signalling with EXTassays. Nature Methods 7: 74–80.
[14]
Heid CA, Stevens J, Lival KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6: 986–994.
[15]
Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11: 1026–1030.
[16]
Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nature Protocols 1: 1559–1582.
[17]
Storhoff JJ, Marla SS, Garimella V, Mirkin CA (2005) Labels and Detection Methods. In Müller UR, Nicolau DV, editors. Microarray technology and its applications. Berlin: Springer. 147–180.
[18]
Yoo SM, Choi JH, Lee SY, Yoo NC (2009) Applications of DNA microarray in disease diagnostics. J Microbiol Biotechnol 19: 635–646.
[19]
Ramaswamy S, Golub TR (2002) DNA microarrays in clinical oncology. J Clinical Oncology 20: 1932–1941.
[20]
Jiwaji M, Daly R, Pansare K, McMcLean P, Yang J, et al. (2010) The Renilla luciferase gene as a reference gene for normalization of gene expression in transiently transfected cells. BMC Mol Biol 11: 103.
[21]
Whelan JA, Russell NB, Whelan MA (2003) A method for using absolute quantification of cDNA using real-time PCR. J Immunological Methods 278: 261–269.
[22]
Yun JJ, Heisler LE, Hwang IIL, Wilkins O, Lau SK, et al. (2006) Genomic DNA functions as a universal external standard in quantitative real-time PCR. Nucleic Acids Res. 34: e85 doi:10.1093/nar/gkl400.
[23]
Yin W, Xiang P, Li Q (2005) Investigations of the effect of DNA size in transient transfection assay using dual luciferase system. Analytical Biochemistry 346: 289–294.
[24]
Plummer M (2003) JAGS:A program for analysis of Bayesian graphical models using Gibbs sampling. Proceedings of the 3rd International Workshop on Distributed Statistical Computing.
[25]
Plummer M, Best N, Cowles K, Vines K (2006) CODA: Convergence Diagnosis and Output Analysis for MCMC. R News 6: 11–17.
[26]
Karin M, Haslinger A, Holtgreve H, Richards RI, Krauter P, et al. (1984) Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene. Nature 308: 513–519.
[27]
Dhanshinamoorthy S, Long II DJ, Jaiswal AK (2000) Antioxidant Regulation of Genes Encoding Enzymes That Detoxify Xenobiotics and Carcinogens. Curr Top Cell Regul 36: 201–16.
[28]
Prestera T, Holtzclaw D, Zhang Y, Talalay P (1993) Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc Natl Acad Sci USA 90: 2965–2969.
[29]
Strahle U, Klock G, Schutz GA (1987) DNA sequence of 15 base pairs is sufficient to mediate both glucocorticoid and progesterone induction of gene expression. Proc Natl Acad Sci USA 84: 7871–7875.
[30]
Shaulian E, Karin M (2002) AP-1 as a regulator of cell life and death. Nature Cell Biol 4: E131–E136.
[31]
Lee W, Mitchell P, Tjian R (1987) Purified Transcription Factor AP-1 Interacts with TPA-Inducible Enhancer Elements. Cell 49: 741–752.
[32]
Krappmann D, Patke A, Heissmeyer V, Scheidereit C (2001) B-Cell receptor- and phorbol ester-induced NF-κB and c-Jun N-terminal kinase activation in B cells requires novel protein kinase C’s. Mol Cellular Biol 21: 6640–6650.
Conkright MD, Guzman E, Flechner L, Su AI, Hogenesch JB, et al. (2003) Genome-Wide Analysis of CREB Target Short Article Genes Reveals A Core Promoter Requirement for cAMP Responsiveness. Mol Cell 11: 1101–1108.
[35]
Cheng J, Grande JP (2007) Cyclic Nucleotide Phosphodiesterase (PDE) Inhibitors: Novel Therapeutic Agents for Progressive Renal Disease. Exp Biol Med 232: 38–51.
[36]
Titus SA, Li X, Southall N, Lu J, Inglese J, et al. (2008) A Cell-based PDE4 Assay in 1536-well Plate format for High Throughput Screening. J Biomol Screen 13: 609–18.
[37]
Herget S, Lohse MJ, Nikolaev AO (2008) Real-time monitoring of phosphodiesterase inhibition in intact cells. Cellular Signalling 20: 1423–1431.
[38]
Woolson HD, Thomson VS, Rutherford C, Yarwood SJ, Palmer TM (2009) Selective inhibition of cytokine-activated extracellular signal-regulated kinase by cyclic AMP via Epac1-dependent induction of suppressor of cytokine signalling-3. Cellular Signalling 21: 1706–1715.
[39]
Rybalkin SD, Rybalkina IG, Shimizu-Albergine M, Tang X-B, Beavo JA (2003) PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. EMBO J 23: 469–478.
[40]
Yan Y, Dalmasso G, Sitaraman S, Merlin D (2007) Characterization of the human intestinal CD98 promoter and its regulation by interferon-γ. American J. Physiology Gastrointestinal and Liver Physiology 292: G535–G545.
[41]
Karin M, Haslinger A, Heguy A, Dietlin T, Cooke T (1987) Metal responsive elements act as positive modulators of human metallothionein-IIA enhancer activity. Mol Cell Biol 7: 606–613.
