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  • Review Article
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Determining the specificity of protein–DNA interactions

Nature Reviews Genetics volume 11, pages 751–760 (2010)Cite this article

Subjects

Key Points

  • Sequence-specific transcription factors (TFs) control gene expression.

  • New methods allow for the rapid and accurate determination of TF binding specificity.

  • Medium-throughput methods using microfluidic devices or surface plasmon resonance (SPR) can determine binding affinities directly.

  • Microarray methods, such as protein-binding microarray (PBM) and cognate site identifier (CSI), can lead to very high-throughput measurements of TF specificity with binding sites of up to about ten base pairs.

  • High-throughput versions of SELEX, with or without multiple rounds of selection, can provide accurate binding site specificities rapidly.

  • Bacterial one-hybrid methods can also be made high-throughput and give accurate binding site models.

  • Computational models can utilize the high-throughput data to make predictions beyond the data itself, provide information about the interaction and help in the design of factors with novel specificity.

Abstract

Proteins, such as many transcription factors, that bind to specific DNA sequences are essential for the proper regulation of gene expression. Identifying the specific sequences that each factor binds can help to elucidate regulatory networks within cells and how genetic variation can cause disruption of normal gene expression, which is often associated with disease. Traditional methods for determining the specificity of DNA-binding proteins are slow and laborious, but several new high-throughput methods can provide comprehensive binding information much more rapidly. Combined with in vivo determinations of transcription factor binding locations, this information provides more detailed views of the regulatory circuitry of cells and the effects of variation on gene expression.

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Figure 1: Different methods for studying DNA–protein interactions.
Figure 2: Comparison of data and analysis from three methods.

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Acknowledgements

We thank S. Wolfe for providing the plasmids and cells for the B1H experiments and useful comments on the manuscript. We thank members of the Stormo Laboratory for comments on the manuscript and especially R. Christensen, D. Granas, Z. Zuo and L. Schriefer for providing the data from the B1H selections with ZIF268.

Author information

Authors and Affiliations

  1. Department of Genetics, Washington University School of Medicine, 4444 Forest Park Blvd, St. Louis, 63110-8510, Missouri, USA

    Gary D. Stormo & Yue Zhao

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  1. Gary D. Stormo
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Correspondence to Gary D. Stormo.

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FURTHER INFORMATION

Gary D. Stormo's homepage

Berkeley database of Drosophila TF specificities

Database of Drosophila TF DNA-binding specificities

JASPAR database of TF binding specificities

PAZAR database

SELEX-SAGE database

UniProbe database

Glossary

In vivo

In this context we use the term in vivo to refer to any experiments performed on living cells, whether within or outside a whole organism (sometimes referred to as ex vivo).

Chromatin immunoprecipitation

(ChIP). A technique that is used to identify the location of DNA-binding proteins and epigenetic marks in the genome. Genomic sequences containing the mark of interest are enriched by binding soluble DNA chromatin extracts (complexes of DNA and protein) to an antibody that recognizes the mark. ChIP can be followed by analysis of the precipitated DNA through hybridization to a microarray (ChIP–chip) or by sequencing of the precipitated DNA (ChIP–seq).

Motif

In this Review motif refers to a representation of the specificity of a transcription factor. For instance, a position weight matrix is a motif for a transcription factor, but there could be other ways to represent the specificity.

Microfluidic device

A device in which fluids are conveyed to samples in channels with diameters in the order of 1 μm; these chambers can be used to precisely and dynamically control the microenvironment to which cells are exposed.

K d

The dissociation constant between two molecules (here, for a transcription factor and a DNA sequence). It is the ratio of the off-to-on rate for the formation and dissolution of the complex.

Consensus sequence

A single sequence (possibly degenerate) that represents the specificity of a transcription factor. It is usually the highest affinity sequence and would be the most frequent base at each position in a collection of binding sites. It is also possible to use degeneracies, for example, 'R = A or G', if two (or more) bases are equivalent.

Gibbs standard free energy

The difference in free energy between the equilibrium state and the standard state (all reactants and products at 1M concentration).

Gas constant

(Represented by R). The thermodynamic constant relating energy per mole to temperature.

Microarray

A high-density array of DNA molecules, typically with each element of the array containing a different DNA sequence. Single-stranded DNA arrays are used to hybridize to labelled DNA (or RNA) sequences to determine the relative abundances of different sequences. Double-stranded DNA arrays are used in protein-binding microarray and cognate site identifier methods to determine the binding preferences of transcription factors or other DNA-binding molecules.

Gel filtration

A permeable gel, such a polyacrylamide or agarose, is used to separate molecules and molecular complexes based on their size. DNA will migrate faster through a gel than the same DNA that is bound to a protein, so the protein–DNA complex can be separated from the free DNA.

Barcoding

The process of adding the same unique DNA sequence to the ends of DNA molecules from the same experiment, so that the resulting DNA reads can be traced back to the experiment that generated them, allowing several experiments to be sequenced simultaneously (multiplexed).

Multiplexing

The process of mixing several experimental samples together and sequencing them (or some other process) simultaneously. Each sample can be barcoded to recover the information about its experimental origin.

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Stormo, G., Zhao, Y. Determining the specificity of protein–DNA interactions. Nat Rev Genet 11, 751–760 (2010). https://doi.org/10.1038/nrg2845

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