Abstract
Sequence-specific protein-DNA interactions mediate most regulatory processes underlying gene expression, such as transcriptional regulation by transcription factors (TFs) or chromatin organization. Current knowledge about DNA-binding specificities of TFs is based mostly on low- to medium-throughput methodologies that are time-consuming and often fail to identify DNA motifs recognized by a TF with lower affinity but retaining biological relevance. The use of protein-binding microarrays (PBMs) offers a high-throughput alternative for the identification of protein-DNA specificities. PBM consists in an array of pseudorandomized DNA sequences that are optimized to include all the possible 10- or 11-mer DNA sequences, allowing the determination of binding specificities of most eukaryotic TFs. PBMs that can be synthesized by several manufacturing companies as single-stranded DNA are converted into double-stranded in a simple primer extension reaction. The protein of interest fused to an epitope tag is then incubated onto the PBM, and specific DNA-protein complexes are revealed in a series of immunological reactions coupled to a fluorophore. After scanning and quantifying PBMs, specific DNA motifs recognized by the protein are identified with ready-to-use scripts, generating comprehensive but accessible information about the DNA-binding specificity of the protein. This chapter describes detailed procedures for preparation of double-stranded PBMs, incubation with recombinant protein, and detection of protein-DNA complexes. Finally, we outline some cues for evaluating the biological role of DNA motifs obtained in vitro.
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References
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510
Roulet E et al (2002) High-throughput SELEX SAGE method for quantitative modeling of transcription-factor binding sites. Nat Biotechnol 20:831–835
Jolma A et al (2010) Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome Res 20:861–873
Berger MF et al (2006) Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities. Nat Biotechnol 24:1429–1435
Warren CL et al (2006) Defining the sequence recognition profile of DNA-binding molecules. Proc Natl Acad Sci U S A 103:867–872
Kim MJ et al (2009) Quadruple 9-mer-based protein binding microarray with DsRed fusion protein. BMC Mol Biol 10:91
Berger MF, Bulyk ML (2009) Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors. Nat Protoc 4:393–411
Godoy M et al (2011) Improved protein-binding microarrays for the identification of DNA-binding specificities of transcription factors. Plant J 66:700–711
Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551
Zuo J, Niu QW, Chua NH (2000) An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273
Thomas-Chollier M et al (2011) RSAT 2011: regulatory sequence analysis tools. Nucleic Acids Res 39:W86–W91
Hu TT et al (2011) The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nat Genet 43:476–481
Wang X et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039
Dassanayake M et al (2011) The genome of the extremophile crucifer Thellungiella parvula. Nat Genet 7:913–918
Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674
Dudley AM et al (2002) Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range. Proc Natl Acad Sci U S A 28:7554–7559
Acknowledgements
The authors thank Marta Godoy, Irene López-Vidriero, and Juan A. García for their help in PBM assays. This work was funded by grants to R.S. from the Spanish Ministerio de Ciencia e Innovación [BIO2004-02502, BIO2007-66935, GEN2003-20218-C02-02, and CSD2007-00057-B (TRANSPLANTA)].
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Franco-Zorrilla, J.M., Solano, R. (2014). High-Throughput Analysis of Protein-DNA Binding Affinity. In: Sanchez-Serrano, J., Salinas, J. (eds) Arabidopsis Protocols. Methods in Molecular Biology, vol 1062. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-580-4_36
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DOI: https://doi.org/10.1007/978-1-62703-580-4_36
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