Quantitative modulation of a spatial enhancer through the biophysical properties of a transcription factor binding site

Fine-tuning of gene expression by transcription factors (TF) is essential for normal development, but how TF binding is converted into precise expression levels is poorly understood. We used a Drosophila enhancer responding to the TF Distal-less (Dll) to investigate this relationship. By combining computational structural analysis and quantifying relative TFBS affinity and enhancer activity we describe how TF concentration is converted into precise expression across developing Drosophila wings. We show that the regulatory function of the enhancer closely follow the classical Hill equation, and we describe the effects of affinity, orientation, sequence and wing-region on the maximum achievable expression and the position of transcriptional activity in a tissue. We also found that Dll relies on two distinct sets of amino acid-DNA contacts for higher affinity sites, but contacts are more evenly distributed at lower affinities. Our work integrates information at molecular and tissue scales ..., , , # Quantitative modulation of a spatial enhancer through the biophysical properties of a transcription factor binding site

## Description of the data and file structure

Fallon_et_al_2025_Zenodo_MIT_Software.zip: Quantitative modulation of a spatial enhancer through the biophysical properties of a transcription factor binding site

The compressed folder Fallon_et_al_2025_Dryad_CCO_Data.zip contains:

Analysis Folders - each contains projected and registered fluorescence channel images and a file named Datasetinformation.xlsx. The Datasetinformation.xlsx file contains info used by the Dll_*Modulation_Analysis*_Annotated.py script to group images by construct name.

Analysis_2020: Contains images created for Xin et al and Le Poul et al 2020 papers. B07 is incorporated into Fallon et al. 2025 analysis as the line A00. predSA is a blank line that is used for background removal. WT is used to normalise the values to images created for Fallon et al. 2025.

Analysis_*2025*_AFR: Contains images...