high-sensitivity-measurement-transcription-factor-dna-binding

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing ...

Ludwig-Maximilians-Universität München

The complex patterns of gene expression in metazoans are controlled by selective binding of transcription factors (TFs) to regulatory DNA. To improve the quantitative understanding of this process, we have developed a novel method that uses fluorescence anisotropy measurements in a controlled delivery system to determine TF-DNA binding energies in solution with high sensitivity and throughput. Owing to its large dynamic range, the method, named high performance fluorescence anisotropy (HiP-FA), allows for reliable quantification of both weak and strong binding; binding specificities are calculated on the basis of equilibrium constant measurements for mutational DNA variants. We determine the binding preference landscapes for 26 TFs and measure high absolute affinities, but mostly lower binding specificities than reported by other methods. The revised binding preferences give rise to improved predictions of in vivo TF occupancy and enhancer expression. Our approach provides a powerful new tool for the systems-biological analysis of gene regulation.

True equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy.

In the original version of this Article, equation three contained a sign error whereby the term RT was added when it should have been subtracted. This has now been corrected in the PDF and HTML versions of the Article.

Ulrich Unnerstall

Gene Center and Department of Biochemistry,
Center for Protein Science Munich (CIPSM),
Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM)

Ludwig-Maximilians-Universität München

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Ulrich Unnerstall

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Gene Center and Department of Biochemistry,Center for Protein Science Munich (CIPSM),Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM)

Ludwig-Maximilians-Universität München

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Gene Center and Department of Biochemistry,
Center for Protein Science Munich (CIPSM),
Gene Center and Department of Biochemistry, Center for Protein Science Munich (CIPSM)