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.

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.

The DNA of eukaryotes is wrapped around histone octamers to form nucleosomes. Although it is well established that the DNA sequence significantly influences nucleosome formation, its precise contribution has remained controversial, partially owing to the lack of quantitative affinity data. Here, we present a method to measure DNA-histone binding free energies at medium throughput and with high sensitivity. Competitive nucleosome formation is achieved through automation, and a modified epifluorescence microscope is used to rapidly and accurately measure the fractions of bound/unbound DNA based on fluorescence anisotropy. The procedure allows us to obtain full titration curves with high reproducibility. We applied this technique to measure the histone-DNA affinities for 47 DNA sequences and analyzed how the affinities correlate with relevant DNA sequence features. We found that the GC content has a significant impact on nucleosome-forming preferences, but 10 bp dinucleotide periodicities and the presence of poly(dA:dT) stretches do not.

Accurate quantification of transcription factor (TF)-DNA interactions is essential for understanding the regulation of gene expression. Since existing approaches suffer from significant limitations, we have developed a new method for determining TF-DNA binding affinities with high sensitivity on a large scale. The assay relies on the established fluorescence anisotropy (FA) principle but introduces important technical improvements. First, we measure a full FA competitive titration curve in a single well by incorporating TF and a fluorescently labeled reference DNA in a porous agarose gel matrix. Unlabeled DNA oligomer is loaded on the top as a competitor and, through diffusion, forms a spatiotemporal gradient. The resulting FA gradient is then read out using a customized epifluorescence microscope setup. This improved setup greatly increases the sensitivity of FA signal detection, allowing both weak and strong binding to be reliably quantified, even for molecules of similar molecular weights. In this fashion, we can measure one titration curve per well of a multi-well plate, and through a fitting procedure, we can extract both the absolute dissociation constant (K D ) and active protein concentration. By testing all single-point mutation variants of a given consensus binding sequence, we can survey the entire binding specificity landscape of a TF, typically on a single plate. The resulting position weight matrices (PWMs) outperform those derived from other methods in predicting in vivo TF occupancy. Here, we present a detailed guide for implementing HiP-FA on a conventional automated fluorescent microscope and the data analysis pipeline.

The core promoter, the region immediately surrounding the transcription start site, plays a central role in setting metazoan gene expression levels, but how exactly it computes expression remains poorly understood. To dissect core promoter function, we carried out a comprehensive structure-function analysis to measure synthetic promoters activities, with and without an external stimulus (hormonal activation). By using robotics and a dual-luciferase reporter assay, we tested ~3000 mutational variants representing 19 different Drosophila melanogaster promoter architectures. We explored the impact of different types of mutations, including knockout of individual sequence motifs and motif combinations, variations of motif strength, positioning, and flanking sequences. We observe strong effects of the mutations on activity, and a linear combination of the individual motif features can largely account for the combinatorial effects on core promoter activity. Our findings shed new light on the quantitative assessment of gene expression, a fundamental process in all metazoans.

Hunchback (Hb) is considered a context-dependent transcription factor, able to activate or repress different enhancers during Drosophila Melanogaster embryo segmentation. The mechanism driving the context dependent activity of Hb is however not well understood. Here, we design 20 synthetic enhancers to elucidate the effect of Hb binding sites in Drosophila segmentation and quantitatively measure their activity. We obtain the spatiotemporal activity dynamics of all synthetic enhancers in-vivo, by using a quantitative and sensitive reporter system that we recently developed. Our data reveal a dual role for Hb binding sites in segmentation enhancers: on one hand, Hb act as a typical short range repressor by binding to its cognate sequences; on the other hand, we report a novel effect of a sequence containing multiple Hb binding sites, which is able to increase enhancer activity independently from Hb binding. This sequence, which contains multiple Poly-dA stretches, increases the activity of enhancers driven by different activators, possibly by disfavoring nucleosome occupancy.

In the original version of this Article, equation 3 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.