Transcription Factor Binding Affinities and DNA Shape Readout.

An essential event in gene regulation is the binding of a transcription factor (TF) to its target DNA. Models considering the interactions between the TF and the DNA geometry proved to be successful approaches to describe this binding event, while conserving data interpretability. However, a direct characterization of the DNA shape contribution to binding is still missing due to the lack of accurate and large-scale binding affinity data. Here, we use a binding assay we recently established to measure with high sensitivity the binding specificities of 13 Drosophila TFs, including dinucleotide dependencies to capture non-independent amino acid-base interactions. Correlating the binding affinities with all DNA shape features, we find that shape readout is widely used by these factors. A shape readout/TF-DNA complex structure analysis validates our approach while providing biological insights such as positively charged or highly polar amino acids often contact nucleotides that exhibit strong shape readout.

Author Correction: 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.

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

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.