DOI: 10.5061/dryad.mgqnk99c8
title: Quantitative modulation of a spatial enhancer through the biophysical properties of a transcription factor binding site

description: 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
 to describe how the modulation of a single TF binding site determines the
 regulatory function of an enhancer in a complex in vivo environment.
description: # 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 of constructs
 used in A, F and R series, described in figure 1 of Fallon et al. 2025
 Analysis_*2025_I*_Series: Contains images of constructs used in initial
 round of imaging, described in figure S1A of Fallon et al. 2025
 Analysis_TF: Contains images of transcription factor reporter lines used
 to create maps of relative concentrations of Distal-less.
 Construct_Identities.csv: Gives the name of constructs, their affinities,
 sequence info used by the python script to create different plots.
 Dataset_Conversion.csv: Provides the value of average signal intensity of
 reference lines WT and 91-4/F51 in each dataset for the purpose of
 normalisation. Dll-Modulation-Analysis-Annotated.py: The script to
 generate PCAs and all the plots seen in Fallon et al. 2025. This folder
 should contain everything the script needs to work if set as working
 directory. There are directory name variables that need to be set in the
 'main' section. masks: A folder containing different masks used
 in our analysis The compressed folder
 Fallon_et_al_2025_Zenodo_MIT_Software.zip uploaded to Zenodo contains:
 Image-registration-gui-Ceolin: Package used to register projected wing
 images and create the images stored in the analysis folders. ## Access
 information Registration software is also accessible at:
 [https://github.com/UniBonn-GompelLab/Image-registration-gui/tree/updates](https://github.com/UniBonn-GompelLab/Image-registration-gui/tree/updates) Note that as of 22/09/2025 this is not the main branch of the software. The package provided on Zenodo was used for this paper.